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Sample records for similarly murine mabs

  1. A targeted complement-dependent strategy to improve the outcome of mAb therapy, and characterization in a murine model of metastatic cancer

    Science.gov (United States)

    Elvington, Michelle; Huang, Yuxiang; Morgan, B. Paul; Qiao, Fei; van Rooijen, Nico; Atkinson, Carl

    2012-01-01

    Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer. PMID:22442351

  2. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-02-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

  3. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-01-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG 1 ), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 μg/100 μCi of 99m Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of 99m Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14±2.50 %ID/g, 5.06±2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy

  4. The anti-(+-methamphetamine monoclonal antibody mAb7F9 attenuates acute (+-methamphetamine effects on intracranial self-stimulation in rats.

    Directory of Open Access Journals (Sweden)

    Andrew C Harris

    Full Text Available Passive immunization with monoclonal antibodies (mAbs against (+-methamphetamine (METH is being evaluated for the treatment of METH addiction. A human/mouse chimeric form of the murine anti-METH mAb7F9 has entered clinical trials. This study examined the effects of murine mAb7F9 on certain addiction-related behavioral effects of METH in rats as measured using intracranial self-stimulation (ICSS. Initial studies indicated that acute METH (0.1-0.56 mg/kg, s.c. lowered the minimal (threshold stimulation intensity that maintained ICSS. METH (0.3 mg/kg, s.c. also blocked elevations in ICSS thresholds (anhedonia-like behavior during spontaneous withdrawal from a chronic METH infusion (10 mg/kg/day x 7 days. In studies examining effects of i.v. pretreatment with mAb7F9 (at 30, 100, or 200 mg/kg, 200 mg/kg blocked the ability of an initial injection of METH (0.3 mg/kg, s.c. to reduce baseline ICSS thresholds, but was less capable of attenuating the effect of subsequent daily injections of METH. MAb7F9 (200 mg/kg also produced a small but significant reduction in the ability of METH (0.3 mg/kg, s.c. to reverse METH withdrawal-induced elevations in ICSS thresholds. These studies demonstrate that mAb7F9 can partially attenuate some addiction-related effects of acute METH in an ICSS model, and provide some support for the therapeutic potential of mAb7F9 for the treatment of METH addiction.

  5. In vitro and in vivo comparison of binding of 99m-Tc-labeled anti-CEA MAb F33-104 with 99m-Tc-labeled anti-CEA MAb BW431/26

    International Nuclear Information System (INIS)

    Watanabe, N.; Gunma Univ. School of Medicine; Oriuchi, N.; Inoue, T.; Sugiyama, S.; Kuroki, M.; Matsuoka, Y.; Tanada, S.; Murata, H.; Sasaki, Y.

    1999-01-01

    Aim: The purpose of this study was to assess the potential for radioimmunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. Methods: The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. Results: The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tc-labeled anti-CEA MAb BW431/261 (31.4 ± 0.95% vs. 11.9 ± 0.55% at 100 ng/mL of soluble CEA, 83.5 ± 2.84% vs. 54.0 ± 2.54% at 10 7 of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 ± 3.50% ID/g vs. 14.4 ± 3.30% ID/g). 99m-Tc-activity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 ± 2.10% ID/g vs. 8.01 ± 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 ± 1.70% ID/g vs. 8.10 ± 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). Conclusion: 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer. (orig.) [de

  6. Novel Monoclonal Antibody LpMab-17 Developed by CasMab Technology Distinguishes Human Podoplanin from Monkey Podoplanin.

    Science.gov (United States)

    Kato, Yukinari; Ogasawara, Satoshi; Oki, Hiroharu; Honma, Ryusuke; Takagi, Michiaki; Fujii, Yuki; Nakamura, Takuro; Saidoh, Noriko; Kanno, Hazuki; Umetsu, Mitsuo; Kamata, Satoshi; Kubo, Hiroshi; Yamada, Mitsuhiro; Sawa, Yoshihiko; Morita, Kei-Ichi; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika Kato

    2016-04-01

    Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain in its N-terminus. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for the binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. Although many anti-PDPN monoclonal antibodies (mAbs) have been established, almost all mAbs bind to PLAG domains. We recently established CasMab technology to produce mAbs against membranous proteins. Using CasMab technology, we produced a novel anti-PDPN mAb, LpMab-17, which binds to non-PLAG domains. LpMab-17 clearly detected endogenous PDPN of cancer cells and normal cells in Western-blot, flow cytometry, and immunohistochemistry. LpMab-17 recognized glycan-deficient PDPN in flow cytometry, indicating that the interaction between LpMab-17 and PDPN is independent of its glycosylation. The minimum epitope of LpMab-17 was identified as Gly77-Asp82 of PDPN using enzyme-linked immunosorbent assay. Of interest, LpMab-17 did not bind to monkey PDPN, whereas the homology is 94% between human PDPN and monkey PDPN, indicating that the epitope of LpMab-17 is unique compared with the other anti-PDPN mAbs. The combination of different epitope-possessing mAbs could be advantageous for the PDPN-targeting diagnosis or therapy.

  7. Acute lethal toxicity following passive immunization for treatment of murine cryptococcosis.

    OpenAIRE

    Savoy, A C; Lupan, D M; Manalo, P B; Roberts, J S; Schlageter, A M; Weinhold, L C; Kozel, T R

    1997-01-01

    Passive immunization with monoclonal antibodies (MAbs) specific for the major capsular polysaccharide of Cryptococcus neoformans alters the course of murine cryptococcosis. During studies of passive immunization for treatment of murine cryptococcosis, we noted the occurrence of an acute, lethal toxicity. Toxicity was characterized by scratching, lethargy, respiratory distress, collapse, and death within 20 to 60 min after injection of antibody. The toxic effect was observed only in mice with ...

  8. Freeze-dried formulation for direct 99mTc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando

    1999-01-01

    Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with 99m Tc at high yield. The resulting 99m Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required

  9. Freeze-dried formulation for direct {sup 99m}Tc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando E-mail: normando@ict.sld.cu

    1999-08-01

    Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with {sup 99m}Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with {sup 99m}Tc at high yield. The resulting {sup 99m}Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required.

  10. Analysis list: mab-5 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available mab-5 Embryo,Larvae + ce10 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/target/m...ab-5.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/target/mab-5.5.tsv http://dbarchive.biosciencedbc....jp/kyushu-u/ce10/target/mab-5.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/colo/mab-5.Embryo.tsv,http://dbarchive.bioscien...cedbc.jp/kyushu-u/ce10/colo/mab-5.Larvae.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/ce10/colo/Embryo.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/colo/Larvae.gml ...

  11. Structural and biochemical characterization of the cell fate determining nucleotidyltransferase fold protein MAB21L1.

    Science.gov (United States)

    de Oliveira Mann, Carina C; Kiefersauer, Reiner; Witte, Gregor; Hopfner, Karl-Peter

    2016-06-08

    The exceptionally conserved metazoan MAB21 proteins are implicated in cell fate decisions and share considerable sequence homology with the cyclic GMP-AMP synthase. cGAS is the major innate immune sensor for cytosolic DNA and produces the second messenger 2'-5', 3'-5' cyclic GMP-AMP. Little is known about the structure and biochemical function of other proteins of the cGAS-MAB21 subfamily, such as MAB21L1, MAB21L2 and MAB21L3. We have determined the crystal structure of human full-length MAB21L1. Our analysis reveals high structural conservation between MAB21L1 and cGAS but also uncovers important differences. Although monomeric in solution, MAB21L1 forms a highly symmetric double-pentameric oligomer in the crystal, raising the possibility that oligomerization could be a feature of MAB21L1. In the crystal, MAB21L1 is in an inactive conformation requiring a conformational change - similar to cGAS - to develop any nucleotidyltransferase activity. Co-crystallization with NTP identified a putative ligand binding site of MAB21 proteins that corresponds to the DNA binding site of cGAS. Finally, we offer a structure-based explanation for the effects of MAB21L2 mutations in patients with eye malformations. The underlying residues participate in fold-stabilizing interaction networks and mutations destabilize the protein. In summary, we provide a first structural framework for MAB21 proteins.

  12. LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin.

    Directory of Open Access Journals (Sweden)

    Yukinari Kato

    Full Text Available Podoplanin (PDPN, also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2 and the platelet-aggregating activity of human PDPN (hPDPN. Although various anti-hPDPN monoclonal antibodies (mAbs have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab, LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49-Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38-54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54, which carries α2-6 sialylated N-acetyl-D-galactosamine (GalNAc on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2.

  13. Mapping of Lol p I allergenic epitopes by using murine monoclonal antibodies.

    Science.gov (United States)

    Mourad, W; Bernier, D; Jobin, M; Hébert, J

    1989-11-01

    Murine monoclonal antibodies (MAbs) against three non-overlapping epitopes of Lol p I allergen were previously produced and subsequently used for purification of the allergen. In the present study, these MAbs were further characterized, and the biological activity of the purified allergen assessed. The three MAbs were of the IgG isotype and carried a kappa light chain. Their affinity constants were in the range of 7.4-15.1 x 10(-9) mol/l. Purified Lol p I kept its biological activity, as shown by its ability to induce histamine release by basophils of Lol p I-sensitive patients. The profiles of histamine release induced by either Lol p I or crude Lolium perenne extracts were comparable. This observation suggests that human IgE bound to basophils are polyspecific which has been confirmed by immunoblot and inhibition assay. Our data indicated also that Lol p I possesses a major allergenic epitope recognized by all human serum IgE tested. This epitope seems to be partially shared by those recognized by the three MAbs. Finally, preincubation of Lol p I with either one of the Mabs did not affect significantly the basophil-histamine release induced by the purified allergen. This suggests that Lol p I possesses allergenic sites other than the one shared by MAbs and IgE Abs.

  14. Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs.

    Directory of Open Access Journals (Sweden)

    Marnie L Fusco

    2015-06-01

    Full Text Available The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston, but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel "wing" feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.

  15. LpMab-23: A Cancer-Specific Monoclonal Antibody Against Human Podoplanin.

    Science.gov (United States)

    Yamada, Shinji; Ogasawara, Satoshi; Kaneko, Mika K; Kato, Yukinari

    2017-04-01

    Human podoplanin (hPDPN), the ligand of C-type lectin-like receptor-2, is involved in cancer metastasis. Until now, many monoclonal antibodies (mAbs) have been established against hPDPN. However, it is still difficult to develop a cancer-specific mAb (CasMab) against hPDPN because the protein sequence of hPDPN expressed in cancer cells is the same as that in normal cells. Herein, we report LpMab-23 of the mouse IgG 1 subclass, a novel CasMab against hPDPN. In an immunohistochemical analysis, LpMab-23 reacted with tumor cells of human oral cancer, but did not react with normal cells such as lymphatic endothelial cells (LECs). In contrast, LpMab-17, another anti-hPDPN mAb, reacted with both tumor cells and LECs. Furthermore, flow cytometric analysis revealed that LpMab-23 reacted with hPDPN-expressing cancer cell lines (LN319, RERF-LC-AI/hPDPN, Y-MESO-14/hPDPN, and HSC3/hPDPN) but showed little reaction with normal cells (LECs and HEK-293T), although another anti-hPDPN mAb, LpMab-7, reacted with both hPDPN-expressing cancer cells and normal cells, indicating that LpMab-23 is a CasMab against hPDPN.

  16. Immobilization of Murine Anti-BMP-2 Monoclonal Antibody on Various Biomaterials for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Sahar Ansari

    2014-01-01

    Full Text Available Biomaterials are widely used as scaffolds for tissue engineering. We have developed a strategy for bone tissue engineering that entails application of immobilized anti-BMP-2 monoclonal antibodies (mAbs to capture endogenous BMPs in vivo and promote antibody-mediated osseous regeneration (AMOR. The purpose of the current study was to compare the efficacy of immobilization of a specific murine anti-BMP-2 mAb on three different types of biomaterials and to evaluate their suitability as scaffolds for AMOR. Anti-BMP-2 mAb or isotype control mAb was immobilized on titanium (Ti microbeads, alginate hydrogel, and ACS. The treated biomaterials were surgically implanted in rat critical-sized calvarial defects. After 8 weeks, de novo bone formation was assessed using micro-CT and histomorphometric analyses. Results showed de novo bone regeneration with all three scaffolds with immobilized anti-BMP-2 mAb, but not isotype control mAb. Ti microbeads showed the highest volume of bone regeneration, followed by ACS. Alginate showed the lowest volume of bone. Localization of BMP-2, -4, and -7 antigens was detected on all 3 scaffolds with immobilized anti-BMP-2 mAb implanted in calvarial defects. Altogether, these data suggested a potential mechanism for bone regeneration through entrapment of endogenous BMP-2, -4, and -7 proteins leading to bone formation using different types of scaffolds via AMOR.

  17. Evaluation of anti-IL-6 monoclonal antibody therapy using murine type II collagen-induced arthritis

    Directory of Open Access Journals (Sweden)

    Shealy David

    2009-04-01

    Full Text Available Abstract Interleukin-6 is a multifunctional cytokine that is critical for T/B-cell differentiation and maturation, immunoglobulin secretion, acute-phase protein production, and macrophage/monocyte functions. Extensive research into the biology of IL-6 has implicated IL-6 in the pathophysiology and pathogenesis of RA. An anti-murine IL-6 mAb that neutralizes mouse IL-6 activities was tested in animal model of collagen-induced arthritis. Prophylactic treatment with anti-IL-6 mAb significantly reduced the incidence and severity of arthritis compared to control mAb treated mice. The mitogenic response of B and T cells isolated from the lymph nodes of anti-IL-6 treated mice was significantly reduced compared to cells isolated from control mAb treated mice. The overall histopathology score for paws from the anti-IL-6 treated mice was significantly reduced when compared to paws from mice treated with control mAb, including both inflammatory (synovitis and pannus and erosive (erosions and architecture parameters. Reduced loss of cartilage matrix components was also observed in the anti-IL-6 treated mice. Collectively, these data suggest that IL-6 plays a major role in the pathophysiology of rheumatoid arthritis, and thus support the potential benefit of anti-IL-6 mAb treatment in rheumatoid arthritis patients.

  18. Germline-specific MATH-BTB substrate adaptor MAB1 regulates spindle length and nuclei identity in maize.

    Science.gov (United States)

    Juranič, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanic, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

    2012-12-01

    Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).

  19. Acute lethal toxicity following passive immunization for treatment of murine cryptococcosis.

    Science.gov (United States)

    Savoy, A C; Lupan, D M; Manalo, P B; Roberts, J S; Schlageter, A M; Weinhold, L C; Kozel, T R

    1997-01-01

    Passive immunization with monoclonal antibodies (MAbs) specific for the major capsular polysaccharide of Cryptococcus neoformans alters the course of murine cryptococcosis. During studies of passive immunization for treatment of murine cryptococcosis, we noted the occurrence of an acute, lethal toxicity. Toxicity was characterized by scratching, lethargy, respiratory distress, collapse, and death within 20 to 60 min after injection of antibody. The toxic effect was observed only in mice with a cryptococcal infection and was reduced or absent in the early and late stages of disease. The clinical course and histopathology were consistent with those for shock. There was considerable variation between mouse strains in susceptibility to toxicity. Swiss Webster mice from the Charles River colony were most susceptible, followed by C3H/He, BALB/c, and C57BL/6 mice. DBA/2 mice and Swiss Webster mice from the Simonsen colony were resistant. Acute toxicity was mimicked by injection of preformed complexes of MAb and purified polysaccharide. The toxic effect was also produced by injection of MAbs into mice that were preloaded with polysaccharide. The toxic effect was not blocked by treatment of mice with chloropheniramine or anti-tumor necrosis factor alpha antibodies or by depletion of complement components via pretreatment with cobra venom factor. Toxicity was reduced by treatment of mice with high doses of epinephrine, dexamethasone, or chlorpromazine. Finally, the toxic effect was completely blocked by treatment of mice with the platelet-activating factor antagonist WEB 2170 BS or by pretreatment of mice with the liposome-encapsulated drug dichloromethylene diphosphonate, a procedure which depletes macrophages from the spleen and liver. PMID:9125564

  20. LpMab-19 Recognizes Sialylated O-Glycan on Thr76 of Human Podoplanin.

    Science.gov (United States)

    Ogasawara, Satoshi; Kaneko, Mika K; Kato, Yukinari

    2016-08-26

    Human podoplanin (hPDPN) is expressed in lymphatic vessels, pulmonary type-I alveolar cells, and renal glomerulus. The hPDPN/C-type lectin-like receptor-2 (CLEC-2) interaction is involved in platelet aggregation and cancer metastasis. High expression of hPDPN in cancer cells or cancer-associated fibroblasts (CAFs) leads to a poor prognosis for cancer patients. In our previous research, we reported on several anti-hPDPN monoclonal antibodies (mAbs), including LpMab-2, LpMab-3, LpMab-7, LpMab-9, LpMab-12, LpMab-13, and LpMab-17 of mouse IgG 1 subclass, which were produced using CasMab technology. Here we produced a novel anti-hPDPN mAb LpMab-19 of mouse IgG 2b subclass. Flow cytometry revealed that the epitope of LpMab-19 includes O-glycan, which is attached to Thr76 of hPDPN. We further identified the minimum epitope of LpMab-19 as Thr76-Arg79 of hPDPN. Immunohistochemistry revealed that LpMab-19 is useful for detecting not only normal cells, including lymphatic vessels, but also glioblastoma and oral squamous cell carcinoma cells. LpMab-19 could be useful for investigating the physiological function of O-glycosylated hPDPN.

  1. The proximal first exon architecture of the murine ghrelin gene is highly similar to its human orthologue

    Directory of Open Access Journals (Sweden)

    Seim Inge

    2009-05-01

    Full Text Available Abstract Background The murine ghrelin gene (Ghrl, originally sequenced from stomach tissue, contains five exons and a single transcription start site in a short, 19 bp first exon (exon 0. We recently isolated several novel first exons of the human ghrelin gene and found evidence of a complex transcriptional repertoire. In this report, we examined the 5' exons of the murine ghrelin orthologue in a range of tissues using 5' RACE. Findings 5' RACE revealed two transcription start sites (TSSs in exon 0 and four TSSs in intron 0, which correspond to 5' extensions of exon 1. Using quantitative, real-time RT-PCR (qRT-PCR, we demonstrated that extended exon 1 containing Ghrl transcripts are largely confined to the spleen, adrenal gland, stomach, and skin. Conclusion We demonstrate that multiple transcription start sites are present in exon 0 and an extended exon 1 of the murine ghrelin gene, similar to the proximal first exon organisation of its human orthologue. The identification of several transcription start sites in intron 0 of mouse ghrelin (resulting in an extension of exon 1 raises the possibility that developmental-, cell- and tissue-specific Ghrl mRNA species are created by employing alternative promoters and further studies of the murine ghrelin gene are warranted.

  2. Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize[W

    Science.gov (United States)

    Juranić, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanić, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

    2012-01-01

    Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle–dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s). PMID:23250449

  3. Sequence-specific interaction between the disintegrin domain of mouse ADAM 3 and murine eggs: role of beta1 integrin-associated proteins CD9, CD81, and CD98.

    Science.gov (United States)

    Takahashi, Y; Bigler, D; Ito, Y; White, J M

    2001-04-01

    ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-alpha6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-alpha6 mAb, or by mAbs against either the alphav or beta3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other beta1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg beta1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface "tetraspan web" facilitates fertilization and that it may do so by fostering ADAM-integrin interactions.

  4. Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

    Science.gov (United States)

    Takahashi, Yuji; Bigler, Dora; Ito, Yasuhiko; White, Judith M.

    2001-01-01

    ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions. PMID:11294888

  5. Determination of critical epitope of PcMab-47 against human podocalyxin

    Directory of Open Access Journals (Sweden)

    Shunsuke Itai

    2018-07-01

    Full Text Available Podocalyxin (PODXL is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA, flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

  6. Determination of critical epitope of PcMab-47 against human podocalyxin.

    Science.gov (United States)

    Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

    2018-07-01

    Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

  7. Role of polymorphic Fc receptor Fc gammaRIIa in cytokine release and adverse effects of murine IgG1 anti-CD3/T cell receptor antibody (WT31).

    Science.gov (United States)

    Tax, W J; Tamboer, W P; Jacobs, C W; Frenken, L A; Koene, R A

    1997-01-15

    Anti-CD3 monoclonal antibody (mAb) OKT3 is immunosuppressive, but causes severe adverse effects during the first administration ("first-dose reaction"). These adverse effects are presumably caused by cytokine release that results from T-cell activation. In vitro, T-cell activation by anti-CD3 mAb requires interaction with monocyte Fc receptors. The Fc receptor for murine IgG1, Fc gammaRIIa, is polymorphic. In some individuals, murine IgG1 anti-CD3 mAb causes T-cell proliferation and cytokine release in vitro (high responders [HR]), whereas in individuals with the low-responder (LR) phenotype it does not. We have now investigated the role of this Fc gammaRIIa polymorphism in the release of cytokines in vivo and the occurrence of adverse effects after the administration of WT31, a murine IgG1 anti-CD3/T cell receptor mAb. WT31 caused an increase of plasma tumor necrosis factor-alpha in all four HR patients and none of the five LR patients. In all HR patients except one, plasma gamma-interferon and interleukin 6 also increased, and a first-dose response was observed, whereas no cytokine release or adverse effects occurred in any of the LR patients. WT31 caused lymphopenia in all HR and none of the LR patients. FACS analysis demonstrated that in HR patients, after the initial disappearance of CD3+ cells from peripheral blood, modulation of CD3 occurred, whereas in LR patients a high degree of coating of the lymphocytes was observed. Surprisingly, WT31 also induced a marked granulocytopenia, as well as a decrease of thrombocytes, in three of the four HR patients (and in none of the LR patients). These data provide direct clinical evidence that Fc receptor interaction determines the release of cytokines and the occurrence of adverse effects after administration of anti-CD3/T cell receptor mAb. Furthermore, these data suggest that tumor necrosis factor-alpha by itself is not sufficient to induce the first-dose reaction.

  8. Development of murine monoclonal antibodies for the immunohistochemical diagnosis of systemic bovine aspergillosis

    DEFF Research Database (Denmark)

    Jensen, H.E.; Aalbaek, B.; Lind, Peter

    1996-01-01

    ) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also......), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i...

  9. First experiences with the AMERLEX-MAB FREE T4 assay

    International Nuclear Information System (INIS)

    Nijhof, W.A.; Penders, T.J.

    1989-01-01

    The new Amerlex-MAB FT 4 is a quick direct free T 4 assay with good reproducability. The correlation between the Amerlex-MAB FT 4 and the free T 4 of Byk is good. In the non-thyreoidal illness patient group no deviation for the values were found. Amerlex-MAB FT 4 is cheaper, because no total T4 has to be measured. More research has to be done for special patient sera. Disturbing influences as free fatty acids, heparin and auto-antibodies have to be checked. (R.B.). 3 refs.; 3 figs.; 4 tabs

  10. Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

    International Nuclear Information System (INIS)

    Qian Weizhu; Wang Ling; Li Bohua; Wang Hao; Hou Sheng; Hong Xueyu; Zhang Dapeng; Guo Yajun

    2008-01-01

    Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application

  11. TGF-beta Sma/Mab signaling mutations uncouple reproductive aging from somatic aging.

    Directory of Open Access Journals (Sweden)

    Shijing Luo

    2009-12-01

    Full Text Available Female reproductive cessation is one of the earliest age-related declines humans experience, occurring in mid-adulthood. Similarly, Caenorhabditis elegans' reproductive span is short relative to its total life span, with reproduction ceasing about a third into its 15-20 day adulthood. All of the known mutations and treatments that extend C. elegans' reproductive period also regulate longevity, suggesting that reproductive span is normally linked to life span. C. elegans has two canonical TGF-beta signaling pathways. We recently found that the TGF-beta Dauer pathway regulates longevity through the Insulin/IGF-1 Signaling (IIS pathway; here we show that this pathway has a moderate effect on reproductive span. By contrast, TGF-beta Sma/Mab signaling mutants exhibit a substantially extended reproductive period, more than doubling reproductive span in some cases. Sma/Mab mutations extend reproductive span disproportionately to life span and act independently of known regulators of somatic aging, such as Insulin/IGF-1 Signaling and Dietary Restriction. This is the first discovery of a pathway that regulates reproductive span independently of longevity and the first identification of the TGF-beta Sma/Mab pathway as a regulator of reproductive aging. Our results suggest that longevity and reproductive span regulation can be uncoupled, although they appear to normally be linked through regulatory pathways.

  12. Production and characterization of monoclonal antibodies (mAbs) against human serum albumin (HSA) for the development of an immunoaffinity system with oriented anti-HSA mAbs as immobilized ligand.

    Science.gov (United States)

    Rajak, Poonam; Vijayalakshmi, M A; Jayaprakash, N S

    2013-05-05

    Proteins present in human serum are of immense importance in the field of biomarker discovery. But, the presence of high-abundant proteins like albumin makes the analysis more challenging because of masking effect on low-abundant proteins. Therefore, removal of albumin using highly specific monoclonal antibodies (mAbs) can potentiate the discovery of low-abundant proteins. In the present study, mAbs against human serum albumin (HSA) were developed and integrated in to an immunoaffinity based system for specific removal of albumin from the serum. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells with spleen cells from the mouse immunized with HSA. Five clones (AHSA1-5) producing mAbs specific to HSA were established and characterized by enzyme linked immunosorbent assay (ELISA) and immunoblotting for specificity, sensitivity and affinity in terms of antigen binding. The mAbs were able to bind to both native albumin as well as its glycated isoform. Reactivity of mAbs with different mammalian sera was tested. The affinity constant of the mAbs ranged from 10(8) to 10(9)M(-1). An approach based on oriented immobilization was followed to immobilize purified anti-HSA mAbs on hydrazine activated agarose gel and the dynamic binding capacity of the column was determined. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Sorbitol crystallization-induced aggregation in frozen mAb formulations.

    Science.gov (United States)

    Piedmonte, Deirdre Murphy; Hair, Alison; Baker, Priti; Brych, Lejla; Nagapudi, Karthik; Lin, Hong; Cao, Wenjin; Hershenson, Susan; Ratnaswamy, Gayathri

    2015-02-01

    Sorbitol crystallization-induced aggregation of mAbs in the frozen state was evaluated. The effect of protein aggregation resulting from sorbitol crystallization was measured as a function of formulation variables such as protein concentration and pH. Long-term studies were performed on both IgG1 and IgG2 mAbs over the protein concentration range of 0.1-120 mg/mL. Protein aggregation was measured by size-exclusion HPLC (SE-HPLC) and further characterized by capillary-electrophoresis SDS. Sorbitol crystallization was monitored and characterized by subambient differential scanning calorimetry and X-ray diffraction. Aggregation due to sorbitol crystallization is inversely proportional to both protein concentration and formulation pH. At high protein concentrations, sorbitol crystallization was suppressed, and minimal aggregation by SE-HPLC resulted, presumably because of self-stabilization of the mAbs. The glass transition temperature (Tg ') and fragility index measurements were made to assess the influence of molecular mobility on the crystallization of sorbitol. Tg ' increased with increasing protein concentration for both mAbs. The fragility index decreased with increasing protein concentration, suggesting that it is increasingly difficult for sorbitol to crystallize at high protein concentrations. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  14. Multicapillary SDS-gel electrophoresis for the analysis of fluorescently labeled mAb preparations: a high throughput quality control process for the production of QuantiPlasma and PlasmaScan mAb libraries.

    Science.gov (United States)

    Székely, Andrea; Szekrényes, Akos; Kerékgyártó, Márta; Balogh, Attila; Kádas, János; Lázár, József; Guttman, András; Kurucz, István; Takács, László

    2014-08-01

    Molecular heterogeneity of mAb preparations is the result of various co- and post-translational modifications and to contaminants related to the production process. Changes in molecular composition results in alterations of functional performance, therefore quality control and validation of therapeutic or diagnostic protein products is essential. A special case is the consistent production of mAb libraries (QuantiPlasma™ and PlasmaScan™) for proteome profiling, quality control of which represents a challenge because of high number of mAbs (>1000). Here, we devise a generally applicable multicapillary SDS-gel electrophoresis process for the analysis of fluorescently labeled mAb preparations for the high throughput quality control of mAbs of the QuantiPlasma™ and PlasmaScan™ libraries. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. The UC Davis/NIH NeuroMab Facility

    Data.gov (United States)

    Federal Laboratory Consortium — The mission of the UC Davis/NIH NeuroMab facility is to generate and distribute high quality, validated mouse monoclonal antibodies against molecular targets found...

  16. Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

    Science.gov (United States)

    Li, Zongyi; Li, Ruiheng; Smith, Charles; Pan, Fang; Campana, Mario; Webster, John R P; van der Walle, Christopher F; Uddin, Shahid; Bishop, Steve M; Narwal, Rojaramani; Warwicker, Jim; Lu, Jian Ren

    2017-07-12

    Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

  17. Analysis of Japanese Municipalities With Geopark, MAB, and GIAHS Certification

    Directory of Open Access Journals (Sweden)

    Ryo Kohsaka

    2015-11-01

    Full Text Available We analyzed the discussions of Japanese municipalities in their process for obtaining certifications for the Geoparks by the United Nations Educational, Scientific and Cultural Organization (UNESCO, the Man and the Biosphere Programme (MAB by the UNESCO, and the Globally Important Agricultural Heritage systems (GIAHS by the Food and Agriculture Organization (FAO of the United Nations. The official records at the municipality diet were analyzed in a quantitative manner from 2011 to 2013. As the first step, we analyzed the eight municipalities of Noto and Sado for the GIAHS, the cities Itoigawa and Hakusan for the Geopark, and Katsuyama Yamanouchi village from Nagano for the MAB. As individual examples, we analyzed City of Suzu with GIAHS, Itoigawa (Geopark, and Yamanouchi town (MAB with the text-mining approach. For the GIAHS, it was clear that the larger municipalities with city status tended to discuss certification issues more frequently than the smaller towns and villages. Terms such as conservation and certification tended to be used with GIAHS at the Suzu City. The term brand was used with GIAHS and MAB but not for the Geopark. The findings using quantitative methods are at initial stage for analysis of municipality strategies and require further future research.

  18. Demonstration of Functional Similarity of Proposed Biosimilar ABP 501 to Adalimumab.

    Science.gov (United States)

    Velayudhan, Jyoti; Chen, Yuh-Feng; Rohrbach, Amanda; Pastula, Christina; Maher, Gwen; Thomas, Heather; Brown, Ryan; Born, Teresa L

    2016-08-01

    Due to the complex molecular structure and proprietary manufacturing processes of monoclonal antibodies (mAbs), differences in structure and function may be expected during development of biosimilar mAbs. Important regulatory requirements for approval of biosimilar products involve comprehensive assessments of any potential differences between proposed biosimilars and reference mAbs, including differences in all known mechanisms of action, using sensitive and relevant methods. Any identified structural differences should not result in differences in biofunctional or clinical activity. A comprehensive assessment comparing the Amgen biosimilar candidate ABP 501 with FDA-licensed adalimumab (adalimumab [US]) and EU-authorized adalimumab (adalimumab [EU]) was conducted to demonstrate similarity in biofunctional activity. The functional similarity assessment included testing of binding kinetics to soluble tumor necrosis factor α (TNFα) and relative binding to transmembrane TNFα. The neutralization of TNFα-induced caspase activation, TNFα- and lymphotoxin-α (LTα)-induced chemokine production, and cytotoxicity was also tested. Binding to Fc-gamma receptors FcγRIa, FcγRIIa (131H), FcγRIIIa (158V and 158F), and neonatal Fc receptor (FcRn) was compared with the reference mAbs, as was antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The data demonstrate that ABP 501 is similar to both adalimumab (US) and adalimumab (EU) with respect to evaluated biofunctional activities. Similarity in biofunctional activity is a critical component of the totality of evidence required for demonstration of biosimilarity. The functional similarity demonstrated for ABP 501 comprehensively assesses the known mechanisms of action of adalimumab, supporting the conclusion that ABP 501, adalimumab (US), and adalimumab (EU) are likely to be clinically similar.

  19. ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via Antibody-Dependent Cellular Cytotoxicity.

    Science.gov (United States)

    Kaneko, Mika K; Nakamura, Takuro; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Yamada, Shinji; Yanaka, Miyuki; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

    2017-06-01

    Podoplanin is expressed in many cancers, including oral cancers and brain tumors. The interaction between podoplanin and its receptor C-type lectin-like receptor 2 (CLEC-2) has been reported to be involved in cancer metastasis and tumor malignancy. We previously established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-23 (IgG 1 , kappa), one of the mouse anti-podoplanin mAbs, was shown to be a CasMab. However, we have not shown the usefulness of LpMab-23 for antibody therapy against podoplanin-expressing cancers. In this study, we first determined the minimum epitope of LpMab-23 and revealed that Gly54-Leu64 peptide, especially Gly54, Thr55, Ser56, Glu57, Asp58, Arg59, Tyr60, and Leu64 of podoplanin, is a critical epitope of LpMab-23. We further produced human-mouse chimeric LpMab-23 (chLpMab-23) and investigated whether chLpMab-23 exerts antibody-dependent cellular cytotoxicity (ADCC) and antitumor activity. In flow cytometry, chLpMab-23 showed high sensitivity against a podoplanin-expressing glioblastoma cell line, LN319, and an oral cancer cell line, HSC-2. chLpMab-23 also showed ADCC activity against podoplanin-expressing CHO cells (CHO/podoplanin). In xenograft models with HSC-2 and CHO/podoplanin, chLpMab-23 exerts antitumor activity using human natural killer cells, indicating that chLpMab-23 could be useful for antibody therapy against podoplanin-expressing cancers.

  20. Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

    Science.gov (United States)

    Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

    2018-04-01

    Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

  1. Dynamics of immature mAb glycoform secretion during CHO cell culture

    DEFF Research Database (Denmark)

    Jimenez del Val, Ioscani; Fan, Yuzhou; Weilguny, Dietmar

    2016-01-01

    Ensuring consistent glycosylation-associated quality of therapeutic monoclonal antibodies (mAbs) has become a priority in pharmaceutical bioprocessing given that the distribution and composition of the carbohydrates (glycans) bound to these molecules determines their therapeutic efficacy and immu......Ensuring consistent glycosylation-associated quality of therapeutic monoclonal antibodies (mAbs) has become a priority in pharmaceutical bioprocessing given that the distribution and composition of the carbohydrates (glycans) bound to these molecules determines their therapeutic efficacy...

  2. Development of at-line assay to monitor charge variants of MAbs during production.

    Science.gov (United States)

    St Amand, M M; Ogunnaike, B A; Robinson, A S

    2014-01-01

    One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro-heterogeneity can result from post-translational modifications such as sialylation, galactosylation, C-terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on-line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post-translational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on-line or at-line measurements of these attributes. In this work, we describe the development of an at-line assay to separate MAb charge variants in near real-time, which could ultimately be used to implement on-line quality control strategies for MAb production. The assay consists of a 2D-HPLC method with sequential in-line Protein A and WCX-10 HPLC column steps. To perform the 2D-HPLC assay at-line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered to the 2D-HPLC for analysis. With this at-line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions. © 2013 American Institute of Chemical Engineers.

  3. Development of an Anti-HER2 Monoclonal Antibody H2Mab-139 Against Colon Cancer.

    Science.gov (United States)

    Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Kato, Yukinari

    2018-02-01

    Human epidermal growth factor receptor 2 (HER2) expression has been reported in several cancers, such as breast, gastric, lung, pancreatic, and colorectal cancers. HER2 is overexpressed in those cancers and is associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-139 (IgG 1 , kappa) and investigated it against colon cancers using flow cytometry, western blot, and immunohistochemical analyses. Flow cytometry analysis revealed that H 2 Mab-139 reacted with colon cancer cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS 174T, COLO 201, COLO 205, HCT-8, SW1116, and DLD-1. Although H 2 Mab-139 strongly reacted with LN229/HER2 cells on the western blot, we did not observe a specific signal for HER2 in colon cancer cell lines. Immunohistochemical analyses revealed sensitive and specific reactions of H 2 Mab-139 against colon cancers, indicating that H 2 Mab-139 is useful in detecting HER2 overexpression in colon cancers using flow cytometry and immunohistochemical analyses.

  4. H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

    Science.gov (United States)

    Itai, Shunsuke; Fujii, Yuki; Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Takahashi, Maki; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

    2017-08-01

    Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H 2 Mab-77 (IgG 1 , kappa) was selected. Finally, immunohistochemical analyses with H 2 Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H 2 Mab-77 to detect HER2 in pathological analyses of breast cancers.

  5. Ecotropic murine leukemia virus-induced fusion of murine cells

    International Nuclear Information System (INIS)

    Pinter, A.; Chen, T.; Lowy, A.; Cortez, N.G.; Silagi, S.

    1986-01-01

    Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with [ 3 H]thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses. Mouse cells infected with nonecotropic viruses retain their sensitivity toward fusion, whereas infection with ecotropic viruses abrogates the fusion of these cells upon cocultivation with other ecotropic MuLV-producing cells. Nonmurine cells lacking the ecotropic gp70 receptor are not fused under similar conditions. Fusion is effectively inhibited by monospecific antisera to gp70, but not by antisera to p15(E), and studies with monoclonal antibodies identify distinct amino- and carboxy-terminal gp70 regions which play a role in the fusion reaction. The enhanced fusion which occurs in the presence of amphotericin B provides a rapid and sensitive assay for the expression of ecotropic MuLVs and should facilitate further mechanistic studies of MuLV-induced fusion of murine cells

  6. Antiglycopeptide Mouse Monoclonal Antibody LpMab-21 Exerts Antitumor Activity Against Human Podoplanin Through Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity.

    Science.gov (United States)

    Kato, Yukinari; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Uchida, Hiroaki; Tahara, Hideaki; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Honma, Ryusuke; Takagi, Michiaki; Ogasawara, Satoshi; Murata, Takeshi; Kaneko, Mika K

    2017-02-01

    The interaction between podoplanin (PDPN) and C-type lectin-like receptor 2 (CLEC-2) is involved in tumor malignancy. We have established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-21, one of the mouse antipodoplanin mAbs, is of the IgG 2a subclass, and its minimum epitope was determined to be Thr76-Arg79 of the human podoplanin. Importantly, sialic acid is linked to Thr76; therefore, LpMab-21 is an antiglycopeptide mAb (GpMab). In this study, we investigated whether LpMab-21 shows antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cell lines in vitro and also studied its antitumor activities using a xenograft model. LpMab-21 showed high ADCC and CDC activities against not only podoplanin-expressing Chinese hamster ovary cells but also LN319 glioblastoma cells and PC-10 lung cancer cells, both of which endogenously express podoplanin. Furthermore, LpMab-21 decreased tumor growth in vivo, indicating that LpMab-21 could be useful for antibody therapy against human podoplanin-expressing cancers.

  7. SPECT scintigraphy with HDP and Mab BW 250/183 of loosened hip endoprothesis

    Energy Technology Data Exchange (ETDEWEB)

    Predic, P [Hospital Celje, Celje (Sierra Leone); Gregoric, E [Hospital Izola, Izola (Sierra Leone); Dodig, D [Clinical Hospital Centre, Zagreb (Croatia). Dept. of Nuclear Medicine and Radiation Protection

    1994-10-01

    Main problem of the loosened hip endoprothesis is in distinguishing between the aseptic and septic loosening of endoprothesis. The study involved 27 pts with a loosened hip; 15 pts with aseptic and 12 pts with septic loosening. The patients were injected 550-770 MBq Tc-99m-HDP and underwent SPECT scintigraphy of the hips to repeat then the examination with only 370 MBq Tc-99m-Mab Bw 230/183. HDP application evidenced positive accumulation at the endoprothesis in all patients with a loosened hip while Mab Bw 250/183 only in the patients with septic loosening. Conclusion: SPECT scintigraphy of hip endoprothesis with HDP and Mab BW 250/183 allows differential diagnosing between septic and aseptic hip loosening and hereby a correct therapeutical approach. (author).

  8. Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity.

    Science.gov (United States)

    Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Abe, Shinji; Nishioka, Yasuhiko; Kunita, Akiko; Fukayama, Masashi; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

    2017-04-01

    Human podoplanin (hPDPN), a platelet aggregation-inducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to C-type lectin-like receptor 2 (CLEC-2). The overexpression of hPDPN is involved in invasion and metastasis. Anti-hPDPN monoclonal antibodies (mAbs) such as NZ-1 have shown antitumor and antimetastatic activities by binding to the platelet aggregation-stimulating (PLAG) domain of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-2, using the cancer-specific mAb (CasMab) technology. In this study we developed chLpMab-2, a human-mouse chimeric anti-hPDPN antibody, derived from LpMab-2. chLpMab-2 was produced using fucosyltransferase 8-knockout (KO) Chinese hamster ovary (CHO)-S cell lines. By flow cytometry, chLpMab-2 reacted with hPDPN-expressing cancer cell lines including glioblastomas, mesotheliomas, and lung cancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMab-2 exhibited high antibody-dependent cellular cytotoxicity (ADCC) against PDPN-expressing cells, despite its low complement-dependent cytotoxicity. Furthermore, treatment with chLpMab-2 abolished tumor growth in xenograft models of CHO/hPDPN, indicating that chLpMab-2 suppressed tumor development via ADCC. In conclusion, chLpMab-2 could be useful as a novel antibody-based therapy against hPDPN-expressing tumors. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  9. Experimental radioimmunotherapy of a xenografted human glioma using [sup 131]I-labeled monoclonal antibody to epidermal growth factor receptor

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Hiroshi; Nakazawa, Shozo [Nippon Medical School, Tokyo (Japan); Herlyn, D

    1993-09-01

    [sup 131]I-labeled F (ab')[sub 2] fragments of murine monoclonal antibodies (MAb) 425 specific to the epidermal growth factor receptor expressed on human gliomas were used in experimental human malignant glioma immunotherapy. Two injections of 150 [mu]Ci [sup 131]I-labeled 425 F(ab')[sub 2] achieved growth inhibition of U-87MG human malignant glioma xenografts in nude mice. This radiolabeled specific MAb F(ab')[sub 2] was significantly superior to radiolabeled fragments of an anti-hepatitis virus control MAb A5C3 in influencing tumor growth. However, similar treatment of established human malignant glioma xenografts did not inhibit progressive tumor growth significantly. No clear tumor inhibition was produced by unlabeled MAb 425F(ab')[sub 2]. These studies suggest that [sup 131]I-labeled MAbs have a significant antitumor effect where unmodified antibody is ineffective. Multiple doses of antibody may achieve an increase in labeled MAb concentration in tumors. (author).

  10. Application of murine monoclonal antibodies to the serodiagnosis of tuberculosis

    International Nuclear Information System (INIS)

    Ivanyl, J.; Coates, A.R.M.; Krambovitis, E.

    1982-01-01

    The immune response during infectious diseases leads to a rise in antibody titre to the various different antigenic determinants of the causative organism. The response is further complicated by the fact that it is relatively unusual for one individual to respond to all antigenic components of an organism. Demonstration of the specific immune response of an infected host by serological tests is often hampered by the broad cross-reactivity between several bacterial antigens. The authors report on a serodiagnostic application of murine monoclonal antibodies (MAB), specific for a human pathogen, M. tuberculosis by a technique which is applicable in principle to the serodiagnosis of many other infectious diseases. The serum diagnostic test is based on the competitive inhibition by human sera of the binding of 125 I-labelled murine monoclonal antibodies to M. tuberculosis-coated polyvinyl plates. Five monoclonal antibodies binding to distinct antigenic determinants of the organism were used as structural probes which conferred their stringent combining site specificities to the polyclonal mixture of antibodies from patients' sera. When compared with healthy controls, increased titres of inhibitory antibodies were found in about 70% of patients with active tuberculosis. The diagnostic value of the individual monoclonal antibodies as well as the benefit from the use of multiple specificity probes has been qualified

  11. Conservation Compromises: The MAB and the Legacy of the International Biological Program, 1964-1974.

    Science.gov (United States)

    Schleper, Simone

    2017-02-01

    This article looks at the International Biological Program (IBP) as the predecessor of UNESCO's well-known and highly successful Man and the Biosphere Programme (MAB). It argues that international conservation efforts of the 1970s, such as the MAB, must in fact be understood as a compound of two opposing attempts to reform international conservation in the 1960s. The scientific framework of the MAB has its origins in disputes between high-level conservationists affiliated with the International Union for the Conservation of Nature and Natural Resources (IUCN) about what the IBP meant for the future of conservation. Their respective visions entailed different ecological philosophies as much as diverging sets of political ideologies regarding the global implementation of conservation. Within the IBP's Conservation Section, one group propagated a universal systems approach to conservation with a centralized, technocratic management of nature and society by an elite group of independent scientific experts. Within IUCN, a second group based their notion of environmental expert roles on a more descriptive and local ecology of resource mapping as practiced by UNESCO. When the IBP came to an end in 1974, both groups' ecological philosophies played into the scientific framework underlying the MAB's World Network or Biosphere Reserves. The article argues that it is impossible to understand the course of conservation within the MAB without studying the dynamics and discourses between the two underlying expert groups and their respective visions for reforming conservation.

  12. Generation, characterization and therapeutic potential of anti-feline TNF-alpha MAbs for feline infectious peritonitis.

    Science.gov (United States)

    Doki, Tomoyoshi; Takano, Tomomi; Nishiyama, Yuri; Nakamura, Michiyo; Hohdatsu, Tsutomu

    2013-12-01

    Feline infectious peritonitis (FIP) is a lethal infectious disease affecting domestic and wild cats. Several reports suggested that TNF-alpha is related to the progression of FIP. Thus, the administration of a feline TNF-alpha-neutralizing antibody to cats with FIP may reduce the disease progression. In this study, we have prepared nine monoclonal antibodies (MAbs) that recognize feline TNF-alpha. All MAbs neutralized recombinant TNF-alpha. The 50% inhibitory concentrations (IC50) of the MAbs for the cytotoxicity of recombinant TNF-alpha were 5-684 ng/ml. MAb 2-4 exhibited high neutralizing activity against natural TNF-alpha derived from FIPV-infected macrophages, and was confirmed to inhibit the following feline TNF-alpha-induced conditions in vitro: (i) an increase in the survival rate of neutrophils from cats with FIP, (ii) aminopeptidase N (APN) mRNA expression in macrophages, and (iii) apoptosis of a feline T-lymphocyte cell line. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Immunohistochemical Analysis Using Antipodocalyxin Monoclonal Antibody PcMab-47 Demonstrates Podocalyxin Expression in Oral Squamous Cell Carcinomas.

    Science.gov (United States)

    Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Harada, Hiroyuki; Kato, Yukinari

    2017-10-01

    Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan, and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed in many types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced a novel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry, and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern and detected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.

  14. Anti-CD25 mAb administration prevents spontaneous liver transplant tolerance.

    Science.gov (United States)

    Li, W; Carper, K; Liang, Y; Zheng, X X; Kuhr, C S; Reyes, J D; Perkins, D L; Thomson, A W; Perkins, J D

    2006-12-01

    Liver allografts are accepted spontaneously in all mouse strain combinations without immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we examined the effect of CD4+ CD25+ T regulatory cells (Treg) on the induction of mouse liver transplant tolerance. Orthotopic liver transplantation was performed from B10 (H2b) to C3H (H2k) mice. Depleting rat anti-mouse CD25 mAb (PC61) was given to the donors or recipients (250 microg/d IP) pretransplant or to the recipients postoperatively. At day 5 posttransplantation, both effector T cells (mainly CD8) and CD4+ CD25+ Treg were increased in the liver allografts and host spleens compared to naïve mice. Anti-CD25 mAb administration, either pretransplantation or posttransplantation, reduced the ratio of CD4+ CD25+ Treg to the CD3 T cells of liver grafts and recipient spleens and induced liver allograft acute rejection compared to IgG treatment. Anti-CD25 mAb administration elevated anti-donor T-cell proliferative responses and CTL and NK activities of graft infiltrates and host splenocytes; reduced CTLA4, Foxp3, and IDO mRNA levels; increased IL-10 and IFN-gamma; and decreased IL-4 mRNA levels in the livers or host spleens. The number of apoptotic T cells was reduced significantly in the liver grafts and treated host spleens. Therefore, anti-CD25 mAb administration changed the balance of CD4+ CD25+ Treg to activated T cells of liver graft recipients, preventing liver transplant tolerance. This was associated with enhanced anti-donor immune reactivity, downregulated Treg gene expression, and reduced T cell apoptosis in the grafts and host spleens.

  15. The UNC-4 homeobox protein represses mab-9 expression in DA motor neurons in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Jafari, Gholamali; Appleford, Peter J; Seago, Julian

    2011-01-01

    , an RNAi screen designed to identify upstream transcriptional regulators of mab-9 showed that silencing of unc-4 (encoding a paired-class homeodomain protein) increases mab-9::gfp expression in the nervous system, specifically in posterior DA motor neurons. Over-expression of unc-4 from a heat...

  16. Identification and quantification of predominant metabolites of synthetic cannabinoid MAB-CHMINACA in an authentic human urine specimen.

    Science.gov (United States)

    Hasegawa, Koutaro; Minakata, Kayoko; Gonmori, Kunio; Nozawa, Hideki; Yamagishi, Itaru; Watanabe, Kanako; Suzuki, Osamu

    2018-02-01

    An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB-CHMINACA, was investigated. Although unchanged MAB-CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from a urine specimen. We obtained six kinds of reference standards of MAB-CHMINACA metabolites from a commercial source. The MAB-CHMINACA metabolites from the urine specimen of the abuser were extracted using a QuEChERS method including dispersive solid-phase extraction, and analyzed by liquid chromatography-tandem mass spectrometry with or without hydrolysis with β-glucuronidase. Among the six MAB-CHMINACA metabolites tested, two predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with β-glucuronidase, an increase of the two metabolites was not observed. The metabolites detected were a 4-monohydroxycyclohexylmethyl metabolite M1 (N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide) and a dihydroxyl (4-hydroxycyclohexylmethyl and tert-butylhydroxyl) metabolite M11 (N-(1-amino-4-hydroxy-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide). Their concentrations were 2.17 ± 0.15 and 10.2 ± 0.3 ng/mL (n = 3, each) for M1 and M11, respectively. Although there is one previous in vitro study showing the estimation of metabolism of MAB-CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and quantification of MAB-CHMINACA metabolites in an authentic human urine specimen. Copyright © 2017 John Wiley & Sons, Ltd.

  17. Detection of high PD-L1 expression in oral cancers by a novel monoclonal antibody L1Mab-4.

    Science.gov (United States)

    Yamada, Shinji; Itai, Shunsuke; Kaneko, Mika K; Kato, Yukinari

    2018-03-01

    Programmed cell death-ligand 1 (PD-L1), which is a ligand of programmed cell death-1 (PD-1), is a type I transmembrane glycoprotein that is expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. There is a strong correlation between human PD-L1 (hPD-L1) expression on tumor cells and negative prognosis in cancer patients. In this study, we produced a novel anti-hPD-L1 monoclonal antibody (mAb), L 1 Mab-4 (IgG 2b , kappa), using cell-based immunization and screening (CBIS) method and investigated hPD-L1 expression in oral cancers. L 1 Mab-4 reacted with oral cancer cell lines (Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4) in flow cytometry and stained oral cancers in a membrane-staining pattern. L 1 Mab-4 stained 106/150 (70.7%) of oral squamous cell carcinomas, indicating the very high sensitivity of L 1 Mab-4. These results indicate that L 1 Mab-4 could be useful for investigating the function of hPD-L1 in oral cancers.

  18. Anatomy and Histology of the Human and Murine Prostate.

    Science.gov (United States)

    Ittmann, Michael

    2018-05-01

    The human and murine prostate glands have similar functional roles in the generation of seminal fluid to assist in reproduction. There are significant differences in the anatomy and histology of murine and human prostate and knowledge of the normal anatomy and histology of the murine prostate is essential to interpreting changes in genetically engineered mouse models. In this review, the normal anatomy and histology of both human and mouse prostate will be described. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

  19. Detection of high PD-L1 expression in oral cancers by a novel monoclonal antibody L1Mab-4

    Directory of Open Access Journals (Sweden)

    Shinji Yamada

    2018-03-01

    Full Text Available Programmed cell death-ligand 1 (PD-L1, which is a ligand of programmed cell death-1 (PD-1, is a type I transmembrane glycoprotein that is expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. There is a strong correlation between human PD-L1 (hPD-L1 expression on tumor cells and negative prognosis in cancer patients. In this study, we produced a novel anti-hPD-L1 monoclonal antibody (mAb, L1Mab-4 (IgG2b, kappa, using cell-based immunization and screening (CBIS method and investigated hPD-L1 expression in oral cancers. L1Mab-4 reacted with oral cancer cell lines (Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 in flow cytometry and stained oral cancers in a membrane-staining pattern. L1Mab-4 stained 106/150 (70.7% of oral squamous cell carcinomas, indicating the very high sensitivity of L1Mab-4. These results indicate that L1Mab-4 could be useful for investigating the function of hPD-L1 in oral cancers. Keywords: Programmed cell death-ligand 1, Monoclonal antibody, Oral cancer

  20. Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

    Science.gov (United States)

    Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

    2017-12-01

    Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

  1. Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture

    Energy Technology Data Exchange (ETDEWEB)

    Gu, Angel [Department of Immunology, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010 (United States); Tsark, Walter [Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010 (United States); Holmes, Kathryn V. [Department of Microbiology, University of Colorado Health Sciences, Aurora, CO 80045 (United States); Shively, John E., E-mail: jshively@coh.org [Department of Immunology, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010 (United States)

    2009-06-10

    CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion has been shown to act as an angiogenic factor for mouse and human endothelial cells. Based on the ability of CEACAM1 to initiate lumen formation in human mammary epithelial cells grown in 3D culture (Matrigel), we hypothesized that murine CEACAM1 may play a similar role in vasculogenesis. In order to test this hypothesis, murine embryonic stem (ES) cells stimulated with VEGF were differentiated into embryoid bodies (EB) for 8 days (- 8-0 d) and transferred to Matrigel in the presence or absence of anti-CEACAM1 antibody for an additional 12 days (0-12 d). In the absence of anti-CEACAM1 antibody or in the presence of an isotype control antibody, the EB in Matrigel underwent extensive sprouting, generating lengthy vascular structures with well-defined lumina as demonstrated by confocal microscopy, electron microscopy, and immunohistochemical analysis. Both the length and architecture of the vascular tubes were inhibited by anti-CEACAM1 mAb CC1, a mAb that blocks the cell-cell adhesion functions of CEACAM1, thus demonstrating a critical role for this cell-cell adhesion molecule in generating and maintaining vasculogenesis. QRT-PCR analysis of the VEGF treated ES cells grown under conditions that convert them to EB revealed expression of Ceacam1 as early as - 5 to - 3 d reaching a maximum at day 0 at which time EBs were transferred to Matrigel, thereafter levels at first declined and then increased over time. Other markers of vasculogenesis including Pecam1, VE-Cad, and Tie-1 were not detected until day 0 when EBs were transferred to Matrigel followed by a steady increase in levels, indicating later roles in vasculogenesis. In contrast, Tie-2 and Flk-1 (VEGFR2) were detected on day five of EB formation reaching a maximum at day 0 on transfer to Matrigel, similar to Ceacam1, but after which Tie-2 declined over time, while Flk-1 increased

  2. Combined roles of human IgG subclass, alternative complement pathway activation, and epitope density in the bactericidal activity of antibodies to meningococcal factor h binding protein.

    Science.gov (United States)

    Giuntini, Serena; Reason, Donald C; Granoff, Dan M

    2012-01-01

    Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.

  3. Physico-chemical Stability of MabThera Drug-product Solution for Subcutaneous Injection under in-use Conditions with Different Administration Materials.

    Science.gov (United States)

    Mueller, Claudia; Dietel, Elke; Heynen, Severin R; Nalenz, Heiko; Goldbach, Pierre; Mahler, Hanns-Christian; Schmidt, Johannes; Grauschopf, Ulla; Schoenhamnmer, Karin

    2015-01-01

    MabThera is an essential component of the standard-of-care regimens in the treatment of non-Hodgkin lymphoma and Chronic Lymphatic Leukemia. MabThera for subcutaneous injection is a novel line extension that has been approved by the European Medicines Agency for the treatment of patients with follicular lymphoma and diffuse large B-cell lymphoma. This study aimed to evaluate in-use stability data of MabThera subcutaneous drug-product solution in single-use syringes for subcutaneous administration according to the European Medicines Agency guideline. The drug-product solution was exposed to material contact surfaces of five different administration setups commonly used in subcutaneous drug delivery. MabThera subcutaneous was transferred under aseptic conditions into polypropylene and polycarbonate syringes and stored for 1, 2, and 4 weeks at 2°C to 8°C followed by 24 hours at 30°C. After storage, subcutaneous administration was simulated and MabThera subcutaneous drug-product solution quality attributes were evaluated by using compendial physico-chemical tests, as well as suitable and validated molecule- and formulation-specific analytical methods. MabThera subcutaneous vials were treated and analyzed in parallel. The physico-chemical results of MabThera subcutaneous in the different setups were comparable to the control for all timepoints. No change in drug-product quality after storage and simulated administration was found compared to the control. However, since single-dose products do not contain preservatives, microbial contamination and growth needs to be avoided and product sterility needs to be ensured. The results showed that MabThera subcutaneous remains compatible and stable, from a physico-chemical perspective, for up to 4 weeks at 2°C to 8°C followed by 24 hours at 30°C with the contact materials tested in this study. In order to avoid and minimize microbial growth, MabThera subcutaneous should be used immediately after removal from the original

  4. Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA.

    Science.gov (United States)

    Küssau, Tanja; Flipo, Marion; Van Wyk, Niel; Viljoen, Albertus; Olieric, Vincent; Kremer, Laurent; Blaise, Mickaël

    2018-05-01

    In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP + -bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP + -bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

  5. Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

    Science.gov (United States)

    Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

    2014-08-01

    Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Detection of high CD44 expression in oral cancers using the novel monoclonal antibody, C44Mab-5

    Directory of Open Access Journals (Sweden)

    Shinji Yamada

    2018-07-01

    Full Text Available CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s, which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C44Mab-5 (IgG1, kappa, recognized both CD44s and CD44v3-10. C44Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that C44Mab-5 detected 166/182 (91.2% of oral cancers. These results suggest that the C44Mab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.

  7. Detection of high CD44 expression in oral cancers using the novel monoclonal antibody, C44Mab-5.

    Science.gov (United States)

    Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Kaneko, Mika K; Kato, Yukinari

    2018-07-01

    CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s), which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C 44 Mab-5 (IgG 1 , kappa), recognized both CD44s and CD44v3-10. C 44 Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that C 44 Mab-5 detected 166/182 (91.2%) of oral cancers. These results suggest that the C 44 Mab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.

  8. Sequential use of indium-111 labeled monoclonal antibodies 96.5 and ZME-018 does not increase detection sensitivity for metastatic melanoma

    International Nuclear Information System (INIS)

    Frontiera, M.; Murray, J.L.; Lamki, L.

    1989-01-01

    Two indium-111 labeled anti-melanoma murine monoclonal antibodies (MAb), 96.5 and ZME-018, each recognizing separate antigens on melanoma cells, were administered intravenously to 17 patients with melanoma in a sequential fashion to determine whether: (1) additional tumor sites could be imaged with the combination compared to a single Mab; (2) the first MAb influenced the biodistribution and tumor localization of the second; and (3) significantly toxicity occurred with the combination. Patients were randomized to receive either 96.5, followed by ZME-018, ZME-018 followed by 96.5, or each MAb followed by itself (controls). Infusions of the second MAb occurred 10 days after the first infusion. Gamma camera images were obtained 72 hours after each antibody infusion. There were 139 known metastatic sites of which 72 lesions were localized by either MAb for an overall sensitivity of 52%. The detection rate was higher when lesions only greater than 1.5 cm were considered. Imaging results were independent of MAb administration sequence. When ZME-018 was given as the first infusion, when ZME-018 was given as a second infusion (p = NS). However, mean sensitivities using 96.5 as the first or second infusion were 48% and 66% respectively (p = NS). There was not a significant number of sites detected by MAb 2 that were missed by MAb 1. Human anti-murine antibody (HAMA) response occurred in seven of eight patients studied; two patients who experienced toxicity had levels of HAMA greater than 2000 ng/ml. We conclude that the use of these two murine anti-melanoma monoclonal antibodies given in sequential fashion did not significantly change the imaging sensitivity from that seen with each individual antibody

  9. Structures and Stability of Metal Amidoboranes (MAB): Density Functional Calculations

    International Nuclear Information System (INIS)

    Li Cailin; Wu Chaoling; Chen Yungui; Zhou Jingjing; Zheng Xin; Pang Lijuan; Deng Gang

    2010-01-01

    Molecule geometry structures, frequencies, and energetic stabilities of ammonia borane (AB, NH 3 BH 3 ) and metal amidoboranes (MAB, MNH 2 BH 3 ), formed by substituting H atom in AB with one of main group metal atoms, have been investigated by density-functional theory and optimized at the B3LYP levels with 6-311G++ (3df, 3pd) basic set. Their structural parameters and infrared spectrum characteristic peaks have been predicted, which should be the criterion of a successfully synthesized material. Several parameters such as binding energies, vibrational frequencies, and the energy gaps between the HOMO and the LUMO have been adopted to characterize and evaluate their structure stabilities. It is also found that the binding energies and HOMO-LUMO energy gaps of the MAB obviously change with the substitution of the atoms. MgAB has the lowest binding energy and is easier to decompose than any other substitutional structures under same conditions, while CaAB has the highest chemical activity. (condensed matter: electronic structure, electrical, magnetic, and optical properties)

  10. Characterization of the binding of radioiodinated hybrid recombinant IFN-alpha A/D to murine and human lymphoid cell lines

    International Nuclear Information System (INIS)

    Faltynek, C.R.; Princler, G.L.; Schwabe, M.; Shata, M.T.; Lewis, G.K.; Kamin-Lewis, R.M.

    1990-01-01

    The hybrid recombinant human interferon (IFN) rIFN-alpha A/D was radioiodinated. Specific binding of [125I]rIFN-alpha A/D was observed with both human and murine cell lines. The binding of [125I]rIFN-alpha A/D to human Daudi cells had similar characteristics to the previously described binding of [125I]rIFN-alpha A or -alpha 2. The following lines of evidence demonstrated that [125I]rIFN-alpha A/D bound with high affinity to the same receptor on murine cells as murine IFN-alpha and -beta: (i) the binding of [125I]rIFN-alpha A/D to murine LBRM cells was inhibited to a similar extent by natural murine IFN-alpha, natural murine IFN-beta, and rIFN-A/D; (ii) the Kd (approximately 2 X 10(-10) M) obtained from both competition experiments and saturation binding experiments with [125I]rIFN-alpha A/D was comparable to the previously reported Kd for the binding of natural murine IFN-alpha and -beta to other murine cell lines; (iii) the size of the cross-linked [125I]rIFN-alpha A/D receptor complex formed on murine LBRM cells was similar to the previously reported cross-linked complex formed after binding radioiodinated natural murine IFN-beta to other murine cell lines. Due to the current lack of readily available recombinant murine IFN-alpha or -beta for radiolabeling and the previously demonstrated biological activity of rIFN-alpha A/D on murine cells, [125I]rIFN-alpha A/D should prove to be a useful reagent for further studies of murine IFN receptors

  11. Immuno-PET of undifferentiated thyroid carcinoma with radioiodine-labelled antibody cMAb U36: application to antibody tumour uptake studies

    Energy Technology Data Exchange (ETDEWEB)

    Fortin, Marc-Andre [Centre Hospitalier Universitaire de Quebec and Laval University, Laboratory for Biomaterials and Bioengineering, Quebec City (Canada); Uppsala University, Biomedical Radiation Sciences, Department of Oncology, Radiology, and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Salnikov, Alexei V. [Uppsala University, BMC, Department of Medical Biochemistry and Microbiology, Uppsala (Sweden); German Cancer Research Center, Division of Molecular Immunology, Heidelberg (Germany); Nestor, Marika [Uppsala University, Division of Otolaryngology and Head and Neck Surgery, Department of Surgical Sciences, Uppsala (Sweden); Heldin, Nils-Erik [Uppsala University, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala (Sweden); Rubin, Kristofer [Uppsala University, BMC, Department of Medical Biochemistry and Microbiology, Uppsala (Sweden); Lundqvist, Hans [Uppsala University, Biomedical Radiation Sciences, Department of Oncology, Radiology, and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden)

    2007-09-15

    We tested the suitability of the chimeric monoclonal anti-human CD44 splice version 6 antibody (cMAb U36) for targeting and visualising human anaplastic thyroid carcinoma with PET. We also performed experiments aimed at elucidating the relation between tumour interstitial fluid pressure (TIFP) and the tumour uptake of antibodies. The affinity and specificity of the cMAb U36 for KAT-4 cells were evaluated in vitro, as was the Na{sup +}/I{sup -} symporter (NIS) expression. Biodistribution studies were performed on KAT-4 carcinoma-bearing mice injected with {sup 124}I-cMAb U36 or free iodine. Biodistribution studies were also performed in animals treated with the specific TGF-{beta}1 and -{beta}3 inhibitor Fc:T{beta}RII, which lowers TIFP. Treated and non-treated animals were scanned by microPET. Cultured human undifferentiated/anaplastic thyroid carcinoma KAT-4 cells expressed low levels of NIS and uptake of free iodine was insignificant. The cMAb U36 expressed an affinity (K{sub D}) of 11 {+-} 2 nM. Tumour radioactivity uptake reached maximum values 48 h after injection of {sup 124}I-cMAb U36 ({proportional_to}22%IA/g). KAT-4 carcinomas were readily identified in all {sup 124}I-immuno-PET images. Radioactivity tumour uptake in Fc:T{beta}RII-treated animals was significantly lower at 24 and 48 h after injection, and five times higher thyroid uptake was also noted. We successfully used {sup 124}I-cMAb U36 to visualise CD44v6-expressing human anaplastic thyroid carcinoma. Given the lack of NIS expression in KAT-4, tumour visualisation is not due to free iodine uptake. Lowering the TIFP in KAT-4 carcinomas did not increase the uptake of mAbs into tumour tissue. (orig.)

  12. Preparation for gluing of Carbon prototype MAB at INEGI, Porto

    CERN Multimedia

    Miguel Moreira, Porto, INEGI

    2001-01-01

    MAB's will assure the alignment of the CMS detector. It is equipped with muon cameras, measuring the position of the barrel muon stations and at the same time linking via the link elements, connecting the barrel muon detectors with the Tracker. In addition there is a connection with the endcap. More details can be found on the muon/alignment homepage on the web

  13. Treatment with anti-C5aR mAb leads to early-onset clinical and mechanistic effects in the murine delayed-type hypersensitivity arthritis model

    DEFF Research Database (Denmark)

    Atkinson, Sara Marie; Nansen, Anneline; Usher, Pernille A.

    2015-01-01

    Blockade of the complement cascade at the C5a/C5a receptor (C5aR)-axis is believed to be an attractive treatment avenue in rheumatoid arthritis (RA). However, the effects of such interventions during the early phases of arthritis remain to be clarified. In this study we use the murine delayed-typ...

  14. Inhibition of the β-Lactamase BlaMab by Avibactam Improves the In Vitro and In Vivo Efficacy of Imipenem against Mycobacterium abscessus.

    Science.gov (United States)

    Lefebvre, Anne-Laure; Le Moigne, Vincent; Bernut, Audrey; Veckerlé, Carole; Compain, Fabrice; Herrmann, Jean-Louis; Kremer, Laurent; Arthur, Michel; Mainardi, Jean-Luc

    2017-04-01

    Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a β-lactam (cefoxitin or imipenem). The triple combination is used without any β-lactamase inhibitor, even though M abscessus produces the broad-spectrum β-lactamase Bla Mab We determine whether inhibition of Bla Mab by avibactam improves the activity of imipenem against M. abscessus The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of Bla Mab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 μg/ml), amikacin (32 μg/ml), and avibactam (4 μg/ml) was bactericidal in broth (imipenem was used at 8 and 32 μg/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 μg/ml) and high (32 μg/ml) dosages of imipenem, respectively. In vivo inhibition of Bla Mab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding Bla Mab was induced (20-fold) in the infected macrophages. Inhibition of Bla Mab by avibactam improved the efficacy of imipenem against M. abscessus in vitro , in macrophages, and in zebrafish embryos, indicating that this β-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of Bla Mab , since the production of the β-lactamase is inducible in macrophages. Copyright © 2017 American Society for Microbiology.

  15. A soluble form of IL-13 receptor alpha 1 promotes IgG2a and IgG2b production by murine germinal center B cells.

    Science.gov (United States)

    Poudrier, J; Graber, P; Herren, S; Gretener, D; Elson, G; Berney, C; Gauchat, J F; Kosco-Vilbois, M H

    1999-08-01

    A functional IL-13R involves at least two cell surface proteins, the IL-13R alpha 1 and IL-4R alpha. Using a soluble form of the murine IL-13R alpha 1 (sIL-13R), we reveal several novel features of this system. The sIL-13R promotes proliferation and augmentation of Ag-specific IgM, IgG2a, and IgG2b production by murine germinal center (GC) B cells in vitro. These effects were enhanced by CD40 signaling and were not inhibited by an anti-IL4R alpha mAb, a result suggesting other ligands. In GC cell cultures, sIL-13R also promoted IL-6 production, and interestingly, sIL-13R-induced IgG2a and IgG2b augmentation was absent in GC cells isolated from IL-6-deficient mice. Furthermore, the effects of the sIL-13R molecule were inhibited in the presence of an anti-IL-13 mAb, and preincubation of GC cells with IL-13 enhanced the sIL-13R-mediated effects. When sIL-13R was injected into mice, it served as an adjuvant-promoting production to varying degrees of IgM and IgG isotypes. We thus propose that IL-13R alpha 1 is a molecule involved in B cell differentiation, using a mechanism that may involve regulation of IL-6-responsive elements. Taken together, our data reveal previously unknown activities as well as suggest that the ligand for the sIL-13R might be a component of the IL-13R complex or a counterstructure yet to be defined.

  16. Aree Protette del Po e della Collina Torinese: studi propedeutici alla Candidatura MAB - Man and the Biosphere

    Directory of Open Access Journals (Sweden)

    Cimnaghi Elisabetta

    2014-03-01

    Full Text Available Il programma MAB fu lanciato dall’UNESCO negli anni ’70 al fine di migliorare il rapporto tra uomo e ambiente. Negli anni il programma ha portato al riconoscimento di Riserve della Biosfera che gli Stati Membri si impegnano a gestire nella prospettiva della conservazione delle risorse. L’Organismo Gestore dell’Area Protetta, partendo, da un lato, dalla consapevolezza della ricchezza ambientale che caratterizza il suo territorio e, dall’altro, del contesto di sviluppo antropico elevato in cui essa è situata, ha riconosciuto nel programma MAB un’opportunità per incrementare le sue attività di protezione e potenziamento delle risorse. Questo articolo descrive i primi risultati ottenuti negli studi preliminari per la Designazione al MAB, con particolare attenzione sia alle opportunità di implementazione di progetti legati al marchio “Collina Po”, creato dall’Organo di Gestione del Parco, si ain termini di scambio di ‘best practices’ con altre Riserve di Biosfera.

  17. Altered tumor growth in vivo after immunization of mice with antitumor antibodies

    International Nuclear Information System (INIS)

    Gorczynski, R.M.; Kennedy, M.; Polidoulis, I.; Price, G.B.

    1984-01-01

    A comparison has been made between the growth patterns of two spontaneously appearing mammary adenocarcinomas in murine bone marrow radiation chimeras and in mice preimmunized with monoclonal antibodies (MAb) detecting embryo-associated antigenic determinants. A correlation was seen between the ability of the embryo-immunized chimeras to produce cytotoxic antibody to the tumors, as assessed by an antibody-dependent cellular cytotoxic assay, and the permissiveness of the mice for growth of a tumor transplant. In addition, mice deliberately preimmunized with cytotoxic MAb (antibody-dependent cellular cytotoxic assay) allowed more rapid growth specifically of that tumor earlier found to be most sensitive to the MAb used for immunization. By comparing the changing antigenic phenotype of tumor cells serially passaged through different immunized, nonimmunized mice, evidence was found suggesting that immunization could cause either antigen modulation of transferred tumor cells or a (transient) selective advantage to antigenically discrete subpopulations within the heterogeneous tumor population. Finally, a study has been made of the growth pattern of tumor cells transplanted into mice immunized with rabbit antibodies directed against the murine MAb. In this case, tumor growth was slowed preferentially for the tumor reactive with the specific MAb, and again, predictable changes in the antigenic spectrum of tumor cells harvested from these animals were observed. Our overall findings are interpreted in terms of the involvement of networks of antibodies reacting with embryo-associated antigens in the regulation of growth of the murine mammary adenocarcinomas studied

  18. Anti-GD2 mAb and Vorinostat synergize in the treatment of neuroblastoma

    NARCIS (Netherlands)

    Kroesen, M.; Bull, C.; Gielen, P.R.; Brok, I.C.; Armandari, I.; Wassink, M.; Looman, M.W.G.; Boon, L.; Brok, M.H.M.G.M. den; Hoogerbrugge, P.M.; Adema, G.J.

    2016-01-01

    Neuroblastoma (NBL) is a childhood malignancy of the sympathetic nervous system. For high-risk NBL patients, the mortality rate is still over 50%, despite intensive multimodal treatment. Anti-GD2 monoclonal antibody (mAB) in combination with systemic cytokine immunotherapy has shown clinical

  19. Experimental Treatment of Bladder Cancer with Bi-213-anti-EGFR MAb

    International Nuclear Information System (INIS)

    Seidl, Christof; Pfost, Birgit; Müller, Felix

    2013-01-01

    Therapy of non-muscle-invasive bladder cancer (carcinoma in situ) comprises transurethral resection of the tumour and subsequent instillation of the chemotherapeutic drug mitomycin C in order to eradicate remaining tumour cells. Yet 15 – 40% of treated patients relapse within 5 years. Therefore, new therapeutic strategies to combat tumour recurrence are needed. Alpha-particle emitting radionuclides efficiently kill single tumour cells or small tumour cell clusters. Because the epidermal growth factor receptor (EGFR) is overexpressed on bladder cancer cells, conjugates composed of the alpha-emitter Bi-213 and the anti-EGFR antibody matuzumab should provide a powerful drug to eliminate disseminated bladder cancer cells. Therefore, the aims of our study were (i) to analyse the cytotoxic effects of Bi-213-anti-EGFR radioimmunoconjugates at the cellular level, (ii) to evaluate therapeutic efficacy of intravesically applied Bi-213- anti-EGFR-Mab in a nude mouse model with intravesical human bladder cancer xenografts, (iii) to compare Bi- 213-anti-EGFR-Mab efficacy with chemotherapy using mitomycin C and (iv) to demonstrate that radioimmunotherapy is not toxic to cells of the bladder wall and of the kidneys

  20. Construction and characterization of an anti-CD20 mAb nanocomb with exceptionally excellent lymphoma-suppressing activity

    Directory of Open Access Journals (Sweden)

    Li H

    2015-07-01

    Full Text Available Hua-Fei Li,1–3,* Cong Wu,4,* Ting Chen,5,* Ge Zhang,1 He Zhao,1 Chang-Hong Ke,1 Zheng Xu21International Joint Cancer Institute, Translation Medicine Institute, 2Planning Division, Scientific Research Department, 3Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, 4Department of Laboratory Diagnosis, Changhai Hospital, 5Department of Cardiology, Changhai Hospital, the Second Military Medical University, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: The CD20-directed monoclonal antibody rituximab (RTX established a new era in the treatment of non-Hodgkin lymphoma (NHL; however, suboptimal response and/or resistance to RTX still limit its clinical merits. Although four effector mechanisms are validated to participate in CD20-based immunotherapy, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, caspase-dependent apoptosis, and lysosome-mediated programmed cell death (PCD, they could hardly be synchronously activated by any anti-CD20 mAb or mAb derivative until now. Herein, a novel mAb nanocomb (polyethylenimine polymer–RTX–tositumomab [PPRT nanocomb] was firstly constructed through mass arming two different anti-CD20 mAbs (RTX and tositumomab to one polymer by nanotechnology. Comparing with free mAbs, PPRT nanocomb possesses a comparable binding ability and reduced “off-rate” to surface CD20 of NHL cells. When treated by PPRT nanocomb, the caspase-dependent apoptosis was remarkably enhanced except for concurrently eliciting complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and lysosome-mediated PCD. Besides, “cross-cell link”-assisted homotypic adhesion by PPRT nanocomb further enhanced the susceptibility to PCD of lymphoma cells. Pharmacokinetic assays revealed that PPRT nanocomb experienced a relatively reduced clearance from peripheral blood compared with free antibodies. With

  1. Technical Note: PLASTIMATCH MABS, an open source tool for automatic image segmentation

    International Nuclear Information System (INIS)

    Zaffino, Paolo; Spadea, Maria Francesca; Raudaschl, Patrik; Fritscher, Karl; Sharp, Gregory C.

    2016-01-01

    Purpose: Multiatlas based segmentation is largely used in many clinical and research applications. Due to its good performances, it has recently been included in some commercial platforms for radiotherapy planning and surgery guidance. Anyway, to date, a software with no restrictions about the anatomical district and image modality is still missing. In this paper we introduce PLASTIMATCH MABS, an open source software that can be used with any image modality for automatic segmentation. Methods: PLASTIMATCH MABS workflow consists of two main parts: (1) an offline phase, where optimal registration and voting parameters are tuned and (2) an online phase, where a new patient is labeled from scratch by using the same parameters as identified in the former phase. Several registration strategies, as well as different voting criteria can be selected. A flexible atlas selection scheme is also available. To prove the effectiveness of the proposed software across anatomical districts and image modalities, it was tested on two very different scenarios: head and neck (H&N) CT segmentation for radiotherapy application, and magnetic resonance image brain labeling for neuroscience investigation. Results: For the neurological study, minimum dice was equal to 0.76 (investigated structures: left and right caudate, putamen, thalamus, and hippocampus). For head and neck case, minimum dice was 0.42 for the most challenging structures (optic nerves and submandibular glands) and 0.62 for the other ones (mandible, brainstem, and parotid glands). Time required to obtain the labels was compatible with a real clinical workflow (35 and 120 min). Conclusions: The proposed software fills a gap in the multiatlas based segmentation field, since all currently available tools (both for commercial and for research purposes) are restricted to a well specified application. Furthermore, it can be adopted as a platform for exploring MABS parameters and as a reference implementation for comparing against

  2. Technical Note: PLASTIMATCH MABS, an open source tool for automatic image segmentation

    Energy Technology Data Exchange (ETDEWEB)

    Zaffino, Paolo; Spadea, Maria Francesca [Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Catanzaro 88100 (Italy); Raudaschl, Patrik; Fritscher, Karl [Institute for Biomedical Image Analysis, Private University of Health Sciences, Medical Informatics and Technology, Hall in Tirol 6060 (Austria); Sharp, Gregory C. [Department for Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts 02114 (United States)

    2016-09-15

    Purpose: Multiatlas based segmentation is largely used in many clinical and research applications. Due to its good performances, it has recently been included in some commercial platforms for radiotherapy planning and surgery guidance. Anyway, to date, a software with no restrictions about the anatomical district and image modality is still missing. In this paper we introduce PLASTIMATCH MABS, an open source software that can be used with any image modality for automatic segmentation. Methods: PLASTIMATCH MABS workflow consists of two main parts: (1) an offline phase, where optimal registration and voting parameters are tuned and (2) an online phase, where a new patient is labeled from scratch by using the same parameters as identified in the former phase. Several registration strategies, as well as different voting criteria can be selected. A flexible atlas selection scheme is also available. To prove the effectiveness of the proposed software across anatomical districts and image modalities, it was tested on two very different scenarios: head and neck (H&N) CT segmentation for radiotherapy application, and magnetic resonance image brain labeling for neuroscience investigation. Results: For the neurological study, minimum dice was equal to 0.76 (investigated structures: left and right caudate, putamen, thalamus, and hippocampus). For head and neck case, minimum dice was 0.42 for the most challenging structures (optic nerves and submandibular glands) and 0.62 for the other ones (mandible, brainstem, and parotid glands). Time required to obtain the labels was compatible with a real clinical workflow (35 and 120 min). Conclusions: The proposed software fills a gap in the multiatlas based segmentation field, since all currently available tools (both for commercial and for research purposes) are restricted to a well specified application. Furthermore, it can be adopted as a platform for exploring MABS parameters and as a reference implementation for comparing against

  3. Immunoreactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Primary Lymphoid Tumors and Lymph Node Metastasis

    Science.gov (United States)

    Blanco, Rancés; Blanco, Damián; Quintana, Yisel; Escobar, Xiomara; Rengifo, Charles E.; Osorio, Marta; Gutiérrez, Zailí; Lamadrid, Janet; Cedeño, Mercedes; Frómeta, Milagros; Carr, Adriana; Rengifo, Enrique

    2013-01-01

    The reactivity of the 14F7 Mab, a highly specific IgG1 against N-glycolyl GM3 ganglioside (NeuGcGM3) in normal tissues, lymphomas, lymph node metastasis, and other metastatic sites was assessed by immunohistochemistry. In addition, the effect of chemical fixation on the 14F7 Mab staining using monolayers of P3X63Ag.653 cells was also evaluated. Moreover, the ability of 14F7 to bind NeuGcGM3 ganglioside inducing complement-independent cytotoxicity by a flow cytometry-based assay was measured. The 14F7 Mab was reactive in unfixed, 4% paraformaldehyde, 4% formaldehyde, and acetone fixed cells. Postfixation with acetone did not alter the localization of NeuGcGM3, while the staining with 14F7 Mab was significantly eliminated in both cells fixed and postfixed with methanol but only partially reduced with ethanol. The staining with 14F7 Mab was evidenced in the 89.2%, 89.4%, and 88.9% of lymphomas, lymph node metastasis, and other metastatic sites, respectively, but not in normal tissues. The treatment with 14F7 Mab affected both morphology and membrane integrity of P3X63Ag.653 cells. This cytotoxic activity was dose-dependent and ranged from 24.0 to 84.7% (10–1000 μg/mL) as compared to the negative control. Our data could support the possible use of NeuGcGM3 as target for both active and passive immunotherapy against malignancies expressing this molecule. PMID:24381785

  4. A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.

    Science.gov (United States)

    Carbonetti, Sara; Oliver, Brian G; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Sack, Brandon; Bergl, Emilee; Kappe, Stefan H I; Sather, D Noah

    2017-09-01

    Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. In vivo targeting of dead tumor cells in a murine tumor model using a monoclonal antibody specific for the La autoantigen.

    Science.gov (United States)

    Al-Ejeh, Fares; Darby, Jocelyn M; Pensa, Katherine; Diener, Kerrilyn R; Hayball, John D; Brown, Michael P

    2007-09-15

    To investigate the potential of the La-specific monoclonal antibody (mAb) 3B9 as an in vivo tumor-targeting agent. The murine EL4 lymphoma cell line was used for in vitro studies and the EL4 model in which apoptosis was induced with cyclophosphamide and etoposide was used for in vivo studies. In vitro studies compared 3B9 binding in the EL4 cell with that in its counterpart primary cell type of the thymocyte. For in vivo studies, 3B9 was intrinsically or extrinsically labeled with carbon-14 or 1,4,7,10-tetra-azacylododecane-N,N',N'',N''''-tetraacetic acid-indium-111, respectively, and biodistribution of the radiotracers was investigated in EL4 tumor-bearing mice, which were treated or not with chemotherapy. La-specific 3B9 mAb bound EL4 cells rather than thymocytes, and binding was detergent resistant. 3B9 binding to dead EL4 cells in vitro was specific, rapid, and saturable. Significantly, more 3B9 bound dead EL4 tumor explant cells after host mice were treated with chemotherapy, which suggested that DNA damage induced 3B9 binding. Tumor binding of 3B9 in vivo was antigen specific and increased significantly after chemotherapy. Tumor accumulation of 3B9 peaked at approximately 50% of the injected dose per gram of tumor 72 h after chemotherapy and correlated with increased tumor cell death. Tumor/organ ratios of 3B9 biodistribution, which included the tumor/blood ratio, exceeded unity 48 or more hours after chemotherapy. La-specific mAb selectively targeted dead tumor cells in vivo, and targeting was augmented by cytotoxic chemotherapy. This novel cell death radioligand may be useful both for radioimmunoscintigraphy and radioimmunotherapy.

  6. Study on in vivo examination of tumors of epithelial origin for the monoclonales antibodies ior c5, ior egf/r3 and humanized hr3, by using inmunogammagraphy technique

    International Nuclear Information System (INIS)

    Ramos Suzarte, Mayra; Iznaga Escobar, Normando; Torres Gemil, Olga; Rodriguez Obaya, Teresita; Perez Rodriguez, Rolando; Rodriguez Mesa, Nelson; Oliva Gonzalez, Juan P.; Perera Pintado, Alejandro; Torres Aroche, Leonel; Batista Cuellar, Juan; Gonzalez Lazo, Nery; Cruz, Teresa; Leonard, Ibrahim; Neninger, Elia

    2003-01-01

    The present work is aimed at demonstrating that humanized hR3 and ior egf/r3, ior c5, ior cea-1 murine monoclonal antibodies (mAbs) can identify primary neoplasia lesions, Their post-surgical received and metastasis by using immunography, Results showed that above mentioned mAbs are useful for the diagnosis of epithelial tumors, their metastasis and post-surgical received. this technique becomes a concept prove for further use of mAbs in cancer therapy

  7. Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukemia-inhibitory factor

    International Nuclear Information System (INIS)

    Gough, N.M.; Gearing, D.P.; King, J.A.; Willson, T.A.; Hilton, D.J.; Nicola, N.A.; Metcalf, D.

    1988-01-01

    A human homologue of the recently cloned murine leukemia-inhibitory factor (LIF) gene was isolated from a genomic library by using the marine cDNA as a hybridization probe. The nucleotide sequence of the human gene indicated that human LIF has 78% amino acid sequence identity with murine LIF, with no insertions or deletions, and that the region of the human gene encoding the mature protein has one intervening sequence. After oligonucleotide-mediated mutagenesis, the mature protein-coding region of the LIF gene was introduced into the yeast expression vector YEpsec1. Yeast cells transformed with the resulting recombinant could be induced with galactose to produce high levels of a factor that induced the differentiation of murine M1 leukemic cells in a manner analogous to murine LIF. This factor competed with 125 I-labeled native murine LIF for binding to specific cellular receptors on murine cells, compatible with a high degree of structural similarity between the murine and human factors

  8. Dose-effect relationship of apoptosis induced by fission-neutron in murine thymocytes

    International Nuclear Information System (INIS)

    Yuan Bin; Li Liang; Xue Wencheng; Sun Jianmin; Wang Baoqin

    2000-01-01

    Objective: To investigate the effectiveness of high LET fission-neutron to induce apoptosis in murine thymocytes and to compare it with that of low LET 60 Co γ-ray. Methods: Apoptosis induction was studied qualitatively by light and transmission electron microscopy and DNA gel electrophoresis,also quantitatively by flow cytometry(FCM) and diphenylamine (DPA)methods. Results: DNA ladders of murine thymocytes were detectable, the typical apoptosis of thymocytes could be observed morphologically by means of light and electron microscopy at 6 h after fission-neutron irradiation with doses ranging from 0.5 to 5.0 Gy, meanwhile the percentages of apoptosis increased with increasing doses. After exposure to γ-rays with doses ranging from 1.0 to 30 Gy, the experimental results were similar to those from neutron radiation. The incidence of apoptosis peaked at about 20 Gy, the percentages did not increase further when doses increased. Conclusion: Apoptosis of murine thymocytes can be induced when mice are exposed to either fission-neutron (0.5-5.0 Gy) or to γ-ray (1-30 Gy). Although the relationship between apoptosis and radiation doses is similar, the percentage of apoptosis induced by neutron irradiation is higher than that induced by γ-irradiation. The RBE values of fission-neutron for inducing apoptosis murine thymocytes are 2.09 (by FCM method) and 2.37 (by DPA method), respectively. These results also suggest that fission-neutron-induced murine immune tissue is more severe than that induced by γ-rays at several hours post-irradiation and this might be the basis for heavy damage to immune tissues induced by fission-neutron-irradiation in later period

  9. Podocyte changes after induction of acute albuminuria in mice by anti-aminopeptidase A mAb.

    Science.gov (United States)

    Dijkman, Henry B P M; Gerlofs-Nijland, Miriam E; van der Laak, Jeroen A W M; Wetzels, Jack F M; Groenen, Patricia J T A; Assmann, Karel J M

    2003-01-01

    Administration of a specific combination of anti-aminopeptidase A (APA) mAb (ASD-37/41) in mice induces an acute albuminuria which is independent of angiotensin II, a well-known substrate of APA. In the present experiments, we examined whether binding of the mAb initiated changes in the podocytic expression of cytoskeleton (-associated), adhesion and slit-diaphragm proteins in relation to the time course of albuminuria. In addition, we measured ultrastructurally the extent of foot process retraction (the number of foot processes per microm GBM) and the width of the slit pore between the podocytes by morphometric methods. An injection of the mAb combination ASD-37/41 induced a massive but transient albuminuria that started at 6 h, and peaked at 8 h, after which it declined. However, even at day 7 after injection of the mAbs some albuminuria was present. Injection of the combination ASD-3/41 or saline did not induce an albuminuria. Notably, we observed changes in the staining of CD2AP and podocin, two slit-pore-associated proteins that coincided with the start of the albuminuria. Nephrin staining was reduced and podocytic actin staining became more granular only at a time albuminuria was declining (24 h). The number of foot processes per microm GBM was already decreased at 4 h with a further reduction thereafter. The width of the slit pore was unchanged at the time of peak albuminuria and gradually decreased thereafter. At day 7, podocytic foot process effacement was even more prominent although albuminuria was only slightly abnormal. Expression of CD2AP was still granular. We observed however a change toward normal in the expression of podocin. Injection of saline or ASD-3/41 had no effect on the expression of podocytic proteins, the number of foot processes or width of the slit pore. Our data show that the onset of albuminuria in the anti-APA model is related to alterations in CD2AP and podocin, proteins that are important for maintaining slit-diaphragm structure

  10. Growth suppression of colorectal cancer by plant-derived multiple mAb CO17-1A × BR55 via inhibition of ERK1/2 phosphorylation.

    Science.gov (United States)

    Kwak, Dong Hoon; Moussavou, Ghislain; Lee, Ju Hyoung; Heo, Sung Youn; Ko, Kisung; Hwang, Kyung-A; Jekal, Seung-Joo; Choo, Young-Kug

    2014-11-14

    We have generated the transgenic Tabaco plants expressing multiple monoclonal antibody (mAb) CO7-1A × BR55 by cross-pollinating with mAb CO17-1A and mAb BR55. We have demonstrated the anti-cancer effect of plant-derived multiple mAb CO17-1A × BR55. We find that co-treatment of colorectal mAbs (anti-epithelial cellular adhesion molecule (EpCAM), plant-derived monoclonal antibody (mAb(P)) CO17-1A and mAb(P) CO17-1A × BR55) with RAW264.7 cells significantly inhibited the cell growth in SW620 cancer cells. In particular, multi mAb(P) CO17-1A × BR55 significantly and efficiently suppressed the growth of SW620 cancer cells compared to another mAbs. Apoptotic death-positive cells were significantly increased in the mAb(P) CO17-1A × BR55-treated. The mAb(P) CO17-1A × BR55 treatment significantly decreased the expression of B-Cell lymphoma-2 (BCl-2), but the expression of Bcl-2-associated X protein (Bax), and cleaved caspase-3 were markedly increased. In vivo, the mAb(P) CO17-1A × BR55 significantly and efficiently inhibited the growth of colon tumors compared to another mAbs. The apoptotic cell death and inhibition of pro-apoptotic proteins expression were highest by treatment with mAb(P) CO17-1A × BR55. In addition, the mAb(P) CO17-1A × BR55 significantly inhibited the extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in cancer cells and tumors. Therefore, this study results suggest that multiple mAb(P) CO17-1A × BR55 has a significant effect on apoptosis-mediated anticancer by suppression of ERK1/2 phosphorylation in colon cancer compared to another mAbs. In light of these results, further clinical investigation should be conducted on mAb(P) CO17-1A × BR55 to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.

  11. Controlling the Glycosylation Profile in mAbs Using Time-Dependent Media Supplementation

    Directory of Open Access Journals (Sweden)

    Devesh Radhakrishnan

    2017-12-01

    Full Text Available In order to meet desired drug product quality targets, the glycosylation profile of biotherapeutics such as monoclonal antibodies (mAbs must be maintained consistently during manufacturing. Achieving consistent glycan distribution profiles requires identifying factors that influence glycosylation, and manipulating them appropriately via well-designed control strategies. Now, the cell culture media supplement, MnCl2, is known to alter the glycosylation profile in mAbs generally, but its effect, particularly when introduced at different stages during cell growth, has yet to be investigated and quantified. In this study, we evaluate the effect of time-dependent addition of MnCl2 on the glycan profile quantitatively, using factorial design experiments. Our results show that MnCl2 addition during the lag and exponential phases affects the glycan profile significantly more than stationary phase supplementation does. Also, using a novel computational technique, we identify various combinations of glycan species that are affected by this dynamic media supplementation scheme, and quantify the effects mathematically. Our experiments demonstrate the importance of taking into consideration the time of addition of these trace supplements, not just their concentrations, and our computational analysis provides insight into what supplements to add, when, and how much, in order to induce desired changes.

  12. Monoclonal antibodies protect from Staphylococcal Enterotoxin K (SEK) induced toxic shock and sepsis by USA300 Staphylococcus aureus.

    Science.gov (United States)

    Aguilar, Jorge L; Varshney, Avanish K; Pechuan, Ximo; Dutta, Kaushik; Nosanchuk, Joshua D; Fries, Bettina C

    2017-08-18

    Staphylococcus aureus is a leading infectious cause of life-threatening disease in humans, yet there is currently no vaccine to combat this bacterium. The pathogenesis of S. aureus is mediated by a diverse array of protein toxins including a large family of secreted pyrogenic superantigens. Neutralization of superantigens, including SEB and TSST-1, has proven to be protective in several animal models of toxic shock and sepsis. We demonstrate, for the first time, that a far more prevalent staphylococcal superantigen, SEK, can also induce lethal shock in mice. Additionally, we describe monoclonal antibodies (mAbs) that inhibit SEK-induced mitogenicity as well as protect against SEK-induced lethality, and enhance survival from S. aureus septicemia in murine models. MAb-4G3 (IgG2b), mAb-5G2 (IgG1), and mAb-9H2 (IgG1), all inhibit SEK-induced proliferation and cytokine production of human immune cells. We then demonstrate that passive immunization with a combination of mAb-4G3 and mAb-5G4, 2 mAbs that do not compete for epitope(s) on SEK, significantly enhance survival in a murine model of SEK-induced toxic shock (p = 0.006). In the setting of sepsis, passive immunization with this combination of mAbs also significantly enhances survival in mice after challenge with CA-MRSA strain USA300 (p = 0.03). Furthermore, septic mice that received mAb treatment in conjunction with vancomycin exhibit less morbidity than mice treated with vancomycin alone. Taken together, these findings suggest that the contribution of SEK to S. aureus pathogenesis may be greater than previously appreciated, and that adjunctive therapy with passive immunotherapy against SEs may be beneficial.

  13. Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays

    NARCIS (Netherlands)

    Buist, M. R.; Kenemans, P.; van Kamp, G. J.; Haisma, H. J.

    1995-01-01

    The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab')2 (n = 28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18; n = 24, 3 mg).

  14. MINOR HUMAN-ANTIBODY RESPONSE TO A MOUSE AND CHIMERIC MONOCLONAL-ANTIBODY AFTER A SINGLE IV INFUSION IN OVARIAN-CARCINOMA PATIENTS - A COMPARISON OF 5 ASSAYS

    NARCIS (Netherlands)

    BUIST, MR; KENEMANS, P; VANKAMP, GJ; Haisma, Hidde

    The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab')(2) (n = 28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18; n = 24, 3

  15. 64Cu-DOTA-Anti-CTLA-4 mAb Enabled PET Visualization of CTLA-4 on the T-Cell Infiltrating Tumor Tissues

    Science.gov (United States)

    Higashikawa, Kei; Yagi, Katsuharu; Watanabe, Keiko; Kamino, Shinichiro; Ueda, Masashi; Hiromura, Makoto; Enomoto, Shuichi

    2014-01-01

    Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) targeted therapy by anti-CTLA-4 monoclonal antibody (mAb) is highly effective in cancer patients. However, it is extremely expensive and potentially produces autoimmune-related adverse effects. Therefore, the development of a method to evaluate CTLA-4 expression prior to CTLA-4-targeted therapy is expected to open doors to evidence-based and cost-efficient medical care and to avoid adverse effects brought about by ineffective therapy. In this study, we aimed to develop a molecular imaging probe for CTLA-4 visualization in tumor. First, we examined CTLA-4 expression in normal colon tissues, cultured CT26 cells, and CT26 tumor tissues from tumor-bearing BALB/c mice and BALB/c nude mice by reverse transcription polymerase chain reaction (RT-PCR) analysis and confirmed whether CTLA-4 is strongly expressed in CT26 tumor tissues. Second, we newly synthesized 64Cu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid-anti-mouse CTLA-4 mAb (64Cu-DOTA-anti-CTLA-4 mAb) and evaluated its usefulness in positron emission tomography (PET) and ex-vivo biodistribution analysis in CT26-bearing BALB/c mice. High CTLA-4 expression was confirmed in the CT26 tumor tissues of tumor-bearing BALB/c mice. However, CTLA-4 expression was extremely low in the cultured CT26 cells and the CT26 tumor tissues of tumor-bearing BALB/c nude mice. The results suggested that T cells were responsible for the high CTLA-4 expression. Furthermore, 64Cu-DOTA-anti-CTLA-4 mAb displayed significantly high accumulation in the CT26 tumor, thereby realizing non-invasive CTLA-4 visualization in the tumor. Together, the results indicate that 64Cu-DOTA-anti-CTLA-4 mAb would be useful for the evaluation of CTLA-4 expression in tumor. PMID:25365349

  16. Signaling pathways regulating murine pancreatic development

    DEFF Research Database (Denmark)

    Serup, Palle

    2012-01-01

    The recent decades have seen a huge expansion in our knowledge about pancreatic development. Numerous lineage-restricted transcription factor genes have been identified and much has been learned about their function. Similarly, numerous signaling pathways important for pancreas development have...... been identified and the specific roles have been investigated by genetic and cell biological methods. The present review presents an overview of the principal signaling pathways involved in regulating murine pancreatic growth, morphogenesis, and cell differentiation....

  17. Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV).

    Science.gov (United States)

    Satoh, Ryoichi; Kaku, Ayumi; Satomura, Megumi; Kohori, Michiyo; Noura, Kanako; Furukawa, Tomoko; Kotake, Masako; Takano, Tomomi; Hohdatsu, Tsutomu

    2011-06-01

    Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats. Copyright © 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.

  18. [International classification of various types of monoclonal antibodies].

    Science.gov (United States)

    Scheen, A J

    2009-01-01

    Significant advances in the development of monoclonal antibodies ("mabs") have been acknowledged during the last two decades. Successive developments led to the marketing of murine antibodies ("o-mab" first, followed by chimeric antibodies ("xi-mab"), humanised antibodies ("zu-mab") and, finally, human monoclonal antibodies ("u-mab"). In order to facilitate the distinction between the various monoclonal antibodies used in clinical practice, an international nomenclature has been proposed with the use of a specific suffix corresponding to the origine/source of "mabs" preceded by an infix referring to the medicine's target. The efforts in developing new types of monoclonal antibodies aimed at improving their pharmacokinetics (longer half-life), pharmacodynamics (better efficacy because of stronger affinity to human receptor), and safety profile (less antigenic and immunogenic reactions). These progresses could be obtained thanks to the remarkable development of molecular biotechnology.

  19. Smad1/5/8 are myogenic regulators of murine and human mesoangioblasts

    NARCIS (Netherlands)

    Costamagna, D. (Domiziana); Quattrocelli, M. (Mattia); F.H.J. van Tienen; L. Umans (Lieve); De Coo, I.F.M. (Irineus F.M.); A. Zwijsen (An); D. Huylebroeck (Danny); Sampaolesi, M. (Maurilio)

    2016-01-01

    textabstractMesoangioblasts (MABs) are vessel-associated stem cells that express pericyte marker genes and participate in skeletal muscle regeneration. Molecular circuits that regulate the myogenic commitment of MABs are still poorly characterized. The critical role of bone morphogenetic protein

  20. Clearance of 131I-labeled murine monoclonal antibody from patients' blood by intravenous human anti-murine immunoglobulin antibody

    International Nuclear Information System (INIS)

    Stewart, J.S.; Sivolapenko, G.B.; Hird, V.; Davies, K.A.; Walport, M.; Ritter, M.A.; Epenetos, A.A.

    1990-01-01

    Five patients treated with intraperitoneal 131I-labeled mouse monoclonal antibody for ovarian cancer also received i.v. exogenous polyclonal human anti-murine immunoglobulin antibody. The pharmacokinetics of 131I-labeled monoclonal antibody in these patients were compared with those of 28 other patients receiving i.p.-radiolabeled monoclonal antibody for the first time without exogenous human anti-murine immunoglobulin, and who had no preexisting endogenous human anti-murine immunoglobulin antibody. Patients receiving i.v. human anti-murine immunoglobulin antibody demonstrated a rapid clearance of 131I-labeled monoclonal antibody from their circulation. The (mean) maximum 131I blood content was 11.4% of the injected activity in patients receiving human anti-murine immunoglobulin antibody compared to 23.3% in patients not given human anti-murine immunoglobulin antibody. Intravenous human anti-murine immunoglobulin antibody decreased the radiation dose to bone marrow (from 131I-labeled monoclonal antibody in the vascular compartment) 4-fold. Following the injection of human anti-murine immunoglobulin antibody, 131I-monoclonal/human anti-murine immunoglobulin antibody immune complexes were rapidly transported to the liver. Antibody dehalogenation in the liver was rapid, with 87% of the injected 131I excreted in 5 days. Despite the efficient hepatic uptake of immune complexes, dehalogenation of monoclonal antibody was so rapid that the radiation dose to liver parenchyma from circulating 131I was decreased 4-fold rather than increased. All patients developed endogenous human anti-murine immunoglobulin antibody 2 to 3 weeks after treatment

  1. Rituximab (MabThera) til behandling af aktiv reumatoid artritis

    DEFF Research Database (Denmark)

    El Fassi, Daniel; Nielsen, Claus Henrik; Bendtzen, Klaus

    2006-01-01

    Rituximab (RTX) is a murine/human monoclonal antibody to CD20, a protein expressed almost exclusively on human B-lymphocytes. RTX induces rapid and marked B-cell depletion with beneficial clinical effects in 1/3 to 1/2 of rheumatoid arthritis patients. Treatment is given as two iv. infusions with...

  2. Production and characterization of a murine monoclonal IgM antibody to human C1q receptor (C1qR)

    International Nuclear Information System (INIS)

    Ghebrehiwet, B.

    1986-01-01

    A hybridoma cell line that produces a monoclonal antibody (MAb) to cell surface C1q receptor (C1qr) has been produced by fusion of the P3 x 63-Ag8.653 mouse myeloma cell line with the spleen cells of a CD-1 mouse that had been hyperimmunized with viable Raji cell suspensions (5 x 10 7 cells/inoculum). This MAb, designated II1/D1, is an IgM antibody with lambda-light chain specificity. Radiolabeled or unlabeled, highly purified II1/D1 was used to determine that: a) this antibody competes for C1q binding sites on C1qR-bearing cells; b) the molecule recognized by this MAb is the C1qR; and c) cells that are known to bind C1q also bind II1/D1 in a specific manner. Western blot analysis of solubilized Raji, or U937 cell membranes, showed that the 125 I-MAb detected a major protein band of approximately 85000 m.w. in its unreduced state, indicating that the C1qR is similar, if not identical, in both types of cells. Analyses of 125 I-II/D1 binding experiments revealed that the antibody bound to Raji cells or u937 cells in a specific manner. Uptake of the antibody was saturable, with equilibrium virtually attained within 35 min. Scatchard analysis of the binding data using the intact MAb suggests that the affinity constant K/sub D/ is 2.9 x 10 -10 M, and at apparent saturation, 24.6 ng of the antibody were bound per 2 x 10 6 cells, giving an estimated 7.8 x 10 3 antibody molecules bound per cell. That the II1/D1 antibody is specifically directed to the C1q was further evidenced by an ELISA in which the ability of C1qR-bearing cells to bind the MAb was abrogated by c-C1q in a specific dose-dependent manner

  3. Immunomodulatory role of interleukin-10 in visceral leishmaniasis: defective activation of protein kinase C-mediated signal transduction events.

    Science.gov (United States)

    Bhattacharyya, S; Ghosh, S; Jhonson, P L; Bhattacharya, S K; Majumdar, S

    2001-03-01

    Leishmania donovani, an intracellular protozoan parasite, challenges host defense mechanisms by impairing the signal transduction of macrophages. In this study we investigated whether interleukin-10 (IL-10)-mediated alteration of signaling events in a murine model of visceral leishmaniasis is associated with macrophage deactivation. Primary in vitro cultures of macrophages infected with leishmanial parasites markedly elevated the endogenous release of IL-10. Treatment with either L. donovani or recombinant IL-10 (rIL-10) inhibited both the activity and expression of the Ca2+-dependent protein kinase C (PKC) isoform. However, preincubation with neutralizing anti-IL-10 monoclonal antibody (MAb) restored the PKC activity in the parasitized macrophage. Furthermore, we observed that coincubation of macrophages with rIL-10 and L. donovani increased the intracellular parasite burden, which was abrogated by anti-IL-10 MAb. Consistent with these observations, generation of superoxide (O2-) and nitric oxide and the release of murine tumor necrosis factor-alpha were attenuated in response to L. donovani or rIL-10 treatment. On the other hand, preincubation of the infected macrophages with neutralizing anti-IL-10 MAb significantly blocked the inhibition of nitric oxide and murine tumor necrosis factor-alpha release by the infected macrophages. These findings imply that infection with L. donovani induces endogenous secretion of murine IL-10, which in turn facilitates the intracellular survival of the protozoan and orchestrates several immunomodulatory roles via selective impairment of PKC-mediated signal transduction.

  4. A fully human IgG1 anti-PD-L1 MAb in an in vitro assay enhances antigen-specific T-cell responses

    OpenAIRE

    Grenga, Italia; Donahue, Renee N; Lepone, Lauren M; Richards, Jacob; Schlom, Jeffrey

    2016-01-01

    Monoclonal antibodies (MAbs) that interfere with checkpoint molecules are being investigated for the treatment of infectious diseases and cancer, with the aim of enhancing the function of an impaired immune system. Avelumab (MSB0010718C) is a fully human IgG1 MAb targeting programmed death-ligand 1 (PD-L1), which differs from other checkpoint-blocking antibodies in its ability to mediate antibody-dependent cell-mediated cytotoxicity. These studies were conducted to define whether avelumab cou...

  5. CCR 20th anniversary commentary: a chimeric antibody, C225, inhibits EGFR activation and tumor growth.

    Science.gov (United States)

    Mendelsohn, John; Prewett, Marie; Rockwell, Patricia; Goldstein, Neil I

    2015-01-15

    Murine mAb 225 was effective against the EGFR tyrosine kinase and inhibited tumor growth in preclinical studies. A phase I trial showed safety, tumor localization, and satisfactory pharmacokinetics. Human:murine chimeric C225 retained biologic activity, which was essential for the conduct of subsequent combination therapy trials and eventual regulatory approval. ©2015 American Association for Cancer Research.

  6. Antibody-Based Therapies in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Yu-Tzu Tai

    2011-01-01

    Full Text Available The unmet need for improved multiple myeloma (MM therapy has stimulated clinical development of monoclonal antibodies (mAbs targeting either MM cells or cells of the bone marrow (BM microenvironment. In contrast to small-molecule inhibitors, therapeutic mAbs present the potential to specifically target tumor cells and directly induce an immune response to lyse tumor cells. Unique immune-effector mechanisms are only triggered by therapeutic mAbs but not by small molecule targeting agents. Although therapeutic murine mAbs or chimeric mAbs can cause immunogenicity, the advancement of genetic recombination for humanizing rodent mAbs has allowed large-scale production and designation of mAbs with better affinities, efficient selection, decreasing immunogenicity, and improved effector functions. These advancements of antibody engineering technologies have largely overcome the critical obstacle of antibody immunogenicity and enabled the development and subsequent Food and Drug Administration (FDA approval of therapeutic Abs for cancer and other diseases.

  7. Life‐cycle and cost of goods assessment of fed‐batch and perfusion‐based manufacturing processes for mAbs

    Science.gov (United States)

    Bunnak, Phumthep; Allmendinger, Richard; Ramasamy, Sri V.; Lettieri, Paola

    2016-01-01

    Life‐cycle assessment (LCA) is an environmental assessment tool that quantifies the environmental impact associated with a product or a process (e.g., water consumption, energy requirements, and solid waste generation). While LCA is a standard approach in many commercial industries, its application has not been exploited widely in the bioprocessing sector. To contribute toward the design of more cost‐efficient, robust and environmentally‐friendly manufacturing process for monoclonal antibodies (mAbs), a framework consisting of an LCA and economic analysis combined with a sensitivity analysis of manufacturing process parameters and a production scale‐up study is presented. The efficiency of the framework is demonstrated using a comparative study of the two most commonly used upstream configurations for mAb manufacture, namely fed‐batch (FB) and perfusion‐based processes. Results obtained by the framework are presented using a range of visualization tools, and indicate that a standard perfusion process (with a pooling duration of 4 days) has similar cost of goods than a FB process but a larger environmental footprint because it consumed 35% more water, demanded 17% more energy, and emitted 17% more CO2 than the FB process. Water consumption was the most important impact category, especially when scaling‐up the processes, as energy was required to produce process water and water‐for‐injection, while CO2 was emitted from energy generation. The sensitivity analysis revealed that the perfusion process can be made more environmentally‐friendly than the FB process if the pooling duration is extended to 8 days. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1324–1335, 2016 PMID:27390260

  8. Immunoreactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Epithelial Malignant Tumors from Digestive System

    OpenAIRE

    Blanco, Rancés; Rengifo, Enrique; Cedeño, Mercedes; Rengifo, Charles E.; Alonso, Daniel F.; Carr, Adriana

    2011-01-01

    The limited expression of N-Glycolyl GM3 (NeuGcGM3) ganglioside in human normal tissues, as well as its presence in melanoma and breast carcinoma using 14F7 Mab (anti-NeuGcGM3), has been previously reported. In this work we evaluated for the first time the 14F7 Mab immunorecognition in some digestive system tumors. Immunohistochemical assays were made with 14F7, followed by anti-mouse biotinylated antibody and ABC/HRP system in normal and pathological human tissues were made. No immunoreactio...

  9. Targeting Alpha Toxin and ClfA with a Multimechanistic Monoclonal-Antibody-Based Approach for Prophylaxis of Serious Staphylococcus aureus Disease

    Directory of Open Access Journals (Sweden)

    C. Tkaczyk

    2016-06-01

    Full Text Available Staphylococcus aureus produces numerous virulence factors, each contributing different mechanisms to bacterial pathogenesis in a spectrum of diseases. Alpha toxin (AT, a cytolytic pore-forming toxin, plays a key role in skin and soft tissue infections and pneumonia, and a human anti-AT monoclonal antibody (MAb, MEDI4893*, has been shown to reduce disease severity in dermonecrosis and pneumonia infection models. However, interstrain diversity and the complex pathogenesis of S. aureus bloodstream infections suggests that MEDI4893* alone may not provide adequate protection against S. aureus sepsis. Clumping factor A (ClfA, a fibrinogen binding protein, is an important virulence factor facilitating S. aureus bloodstream infections. Herein, we report on the identification of a high-affinity anti-ClfA MAb, 11H10, that inhibits ClfA binding to fibrinogen, prevents bacterial agglutination in human plasma, and promotes opsonophagocytic bacterial killing (OPK. 11H10 prophylaxis reduced disease severity in a mouse bacteremia model and was dependent on Fc effector function and OPK. Additionally, prophylaxis with 11H10 in combination with MEDI4893* provided enhanced strain coverage in this model and increased survival compared to that obtained with the individual MAbs. The MAb combination also reduced disease severity in murine dermonecrosis and pneumonia models, with activity similar to that of MEDI4893* alone. These results indicate that an MAb combination targeting multiple virulence factors provides benefit over a single MAb neutralizing one virulence mechanism by providing improved efficacy, broader strain coverage, and protection against multiple infection pathologies.

  10. Tissue Reactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Tumors of Neuroectodermal, Mesodermal, and Epithelial Origin

    Science.gov (United States)

    Blanco, Rancés; Quintana, Yisel; Blanco, Damián; Cedeño, Mercedes; Rengifo, Charles E.; Frómeta, Milagros; Ríos, Martha; Rengifo, Enrique; Carr, Adriana

    2013-01-01

    The expression of N-glycolylneuraminic acid forming the structure of gangliosides and/or other glycoconjugates (Hanganutziu-Deicher antigen) in human has been considered as a tumor-associated antigen. Specifically, some reports of 14F7 Mab (a highly specific Mab raised against N-glycolyl GM3 ganglioside) reactivity in human tumors have been recently published. Nevertheless, tumors of epithelial origin have been mostly evaluated. The goal of the present paper was to evaluate the immunohistochemical recognition of 14F7 Mab in different human tumors of neuroectodermal, mesodermal, and epithelial origins using an immunoperoxidase staining method. Samples of fetal, normal, and reactive astrocytosis of the brain were also included in the study. In general, nontumoral tissues, as well as, low-grade brain tumors showed no or a limited immunoreaction with 14F7 Mab. Nevertheless, high-grade astrocytomas (III-IV) and neuroblastomas, as well as, sarcomas and thyroid carcinomas were mostly reactive with 14F7. No reaction was evidenced in medulloblastomas and ependymoblastomas. Our data suggest that the expression of N-glycolyl GM3 ganglioside could be related to the aggressive behavior of malignant cells, without depending on the tumor origin. Our data could also support the possible use of N-glycolyl GM3 as a target for both active and passive immunotherapies of malignancies expressing this molecule. PMID:26317019

  11. Generation of a monoclonal antibody against the glycosylphosphatidylinositol-linked protein Rae-1 using genetically engineered tumor cells.

    Science.gov (United States)

    Hu, Jiemiao; Vien, Long T; Xia, Xueqing; Bover, Laura; Li, Shulin

    2014-02-04

    Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly. We used Rae-1-overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti-Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining. Our cell line-based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.

  12. Russian biosphere reserves at the youth MAB-2017 Forum in Italy

    Directory of Open Access Journals (Sweden)

    Elena A. Shuyskaya

    2018-02-01

    Full Text Available Russia was represented by 9 participants from Biosphere Reserves at the MAB Youth Forum in Italy. The main question of the Forum was «How to involve the young in the work of biosphere reserves?» The debate resulted in a Declaration, elaborated by participants from around the world (282 delegates from 85 countries. Measures to improve scientific cooperation, data exchange in the sphere of educational tourism and administrative management, development of joint projects in environmental education were formulated. The article contains a number of recommendations for the network of biosphere reserves in Russia, based on Seville strategy and Lima Action Plan.

  13. Porcine humoral immune responses to multiple injections of murine monoclonal antibodies

    DEFF Research Database (Denmark)

    Lohse, Louise; Nielsen, Jens; Kamstrup, Søren

    2005-01-01

    In humans and cattle, multiple injections of murine monoclonal antibodies (m-mAbs) induce anti-mouse antibody responses. The objectives of the present. study were to investigate whether a similar response could be seen when pigs were subjected to m-mAb therapy, and to study the kinetics of such a...

  14. Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors

    Directory of Open Access Journals (Sweden)

    Mayumi Oakland

    2013-01-01

    Full Text Available Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better transduction of GP64-pseudotyped vesicular stomatitis virus (VSV in the nasal epithelia compared with the intrapulmonary airways in mice. VSVG glycoprotein pseudotyped VSV transduced intrapulmonary epithelia with similar efficiency as nasal epithelia. Our results suggest that the differential transduction efficiency of nasal versus intrapulmonary airways by FIV vector is not a result of immunological barriers or surface area, but rather differential expression of cellular factors specific for FIV vector transduction.

  15. A literatura como resistência política: traços neorrealistas na produção literária do Movimento dos Atingidos por Barragens (MAB

    Directory of Open Access Journals (Sweden)

    Carolina Alves Pereira

    2018-04-01

    Full Text Available Este trabalho busca analisar as relações existentes entre as produções literárias do inicio do século XX, com o surgimento da corrente neo-realista, e a literatura produzida pelo Movimento dos Atingidos por Barragens (MAB, como manifestação de resistência à cultura elitista, no que tange ao espaço das criações artísticas. Para tanto, discute-se a característica do neo-realismo como expressão literária definidora dos aspectos sociais e políticos representados nas obras da época. Analisa-se, também, o conceito de resistência como determinante para a definição da literatura marginalizada do movimento campesino (MAB. Por fim, a seguinte pesquisa ratifica a importância da literatura e das artes produzida para além das universidades e dos cânones habituais. Palavras-chave: Literatura de resistência, Neorrealismo, Movimento dos Atingidos por Barragens (MAB. _________________________ This paper analyzes the relationship between the literary productions of the early twentieth century, with the rise of neo-realist current, and the literature produced by the Brazilian social group called Movimento do Atingidos por Barragens (MAB as a manifestation of resistance to elitist culture in terms the space of artistic creations. To this aim, I discuss the characteristic of neorealism as the defining literary expression of social and political represented in the works of the time aspects. In addition, I analyze the concept of resistance as a determinant for defining the literature of marginalized campesino movement (MAB. Finally, the following research confirms the importance of literature and the arts produced in excess of the usual canons and universities. Keywords: Literature of Resistance, Neorealism, Movimento dos Atingidos por Barragens (MAB.

  16. Linear pharmacokinetic parameters for monoclonal antibodies are similar within a species and across different pharmacological targets: A comparison between human, cynomolgus monkey and hFcRn Tg32 transgenic mouse using a population-modeling approach.

    Science.gov (United States)

    Betts, Alison; Keunecke, Anne; van Steeg, Tamara J; van der Graaf, Piet H; Avery, Lindsay B; Jones, Hannah; Berkhout, Jan

    2018-04-10

    The linear pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs) can be considered a class property with values that are similar to endogenous IgG. Knowledge of these parameters across species could be used to avoid unnecessary in vivo PK studies and to enable early PK predictions and pharmacokinetic/pharmacodynamic (PK/PD) simulations. In this work, population-pharmacokinetic (popPK) modeling was used to determine a single set of 'typical' popPK parameters describing the linear PK of mAbs in human, cynomolgus monkey and transgenic mice expressing the human neonatal Fc receptor (hFcRn Tg32), using a rich dataset of 27 mAbs. Non-linear PK was excluded from the datasets and a 2-compartment model was applied to describe mAb disposition. Typical human popPK estimates compared well with data from comparator mAbs with linear PK in the clinic. Outliers with higher than typical clearance were found to have non-specific interactions in an affinity-capture self-interaction nanoparticle spectroscopy assay, offering a potential tool to screen out these mAbs at an early stage. Translational strategies were investigated for prediction of human linear PK of mAbs, including use of typical human popPK parameters and allometric exponents from cynomolgus monkey and Tg32 mouse. Each method gave good prediction of human PK with parameters predicted within 2-fold. These strategies offer alternative options to the use of cynomolgus monkeys for human PK predictions of linear mAbs, based on in silico methods (typical human popPK parameters) or using a rodent species (Tg32 mouse), and call into question the value of completing extensive in vivo preclinical PK to inform linear mAb PK.

  17. Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

    Science.gov (United States)

    Giuntini, Serena; Reason, Donald C; Granoff, Dan M

    2011-09-01

    Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

  18. Monoclonal antibodies (MAb) made against insect-derived metacyclic trypomastigotes (IMT) of Trypanosoma cruzi (TC) cross-react with other parasite forms

    International Nuclear Information System (INIS)

    Kirchhoff, L.V.; Gilliam, F.C.

    1986-01-01

    Considerable information has been generated in recent years about stage-specific surface membrane antigens of a number of protozoa, and this phenomenon has been observed among several stages of TC as well. However, little is known about the surface antigens of IMT, the true infective stage of TC, because of the difficulty of obtaining sufficient numbers of these organisms for analysis. The Tulahuen strain of TC was maintained in the reduviid vector Dipetalogaster maximus by repeated feeding on mice with high parasitemias. IMT collected with insect urine were irradiated (150 krad) and used to immunize a BALB/c mouse for hybridoma production. Supernatants were screened by immunofluorescence assay for the presence of IgG MAb that react with methanol-fixed IMT, epimastogotes (EPI) and culture-derived metacyclic trypomastigoes (CMT). Of 41 MAb obtained, 40 reacted with IMT, 37 with EPI and 38 with CMT. Four MAb immunoprecipitated radioiodinated proteins or protein conjugates of M/sub r/ 80, 72, 45 and 45 from lysates of 125 I surface-labeled EPI. These results indicate that, at least at the epitopic level, there is considerable overlap among IMT, EPI and CMT surface antigens. This finding suggests that analysis of surface proteins of the latter 2 parasite forms may lead to identification of molecules useful for vaccine development

  19. IL-2/anti-IL-2 mAb immunocomplexes: A renascence of IL-2 in cancer immunotherapy?

    Czech Academy of Sciences Publication Activity Database

    Tomala, Jakub; Kovář, Marek

    2016-01-01

    Roč. 5, č. 3 (2016), e1102829 ISSN 2162-402X R&D Projects: GA ČR GA13-12885S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Anti-IL-2 mAb * cancer immunotherapy * IL-2 Subject RIV: EE - Microbiology, Virology Impact factor: 7.719, year: 2016

  20. Discriminating between Interstitial and Circulating Leukocytes in Tissues of the Murine Oral Mucosa Avoiding Nasal-Associated Lymphoid Tissue Contamination.

    Science.gov (United States)

    Bittner-Eddy, Peter D; Fischer, Lori A; Tu, Andy A; Allman, Daniel A; Costalonga, Massimo

    2017-01-01

    Periodontitis is a chronic inflammatory response to a microbial biofilm that destroys bone and soft tissues supporting the teeth. Murine models of periodontitis based on Porphyromonas gingivalis ( Pg ) colonization have shown that extravasation of leukocytes into oral tissue is critical to driving alveolar bone destruction. Identifying interstitial leukocytes is key to understanding the immunopathogenesis of periodontitis. Here, we describe a robust flow cytometry assay based on intravenous FITC-conjugated anti-mouse CD45 mAb that distinguishes interstitial leukocytes in the oral mucosa of mice from those circulating within the vasculature or in post-dissection contaminating blood. Unaccounted circulating leukocytes skewed the relative frequency of B cells and granulocytes and inflated the numbers of all leukocyte cell types. We also describe a dissection technique that avoids contamination of oral mucosal tissues with nasal-associated lymphoid tissues (NALT), a B cell rich organ that can inflate leukocyte numbers at least 10-fold and skew the assessment of interstitial CD4 T cell phenotypes. Unlike circulating CD4 T cells, interstitial CD4 T cells were almost exclusively antigen-experienced cells (CD44 hi ). We report for the first time the presence of antigen-experienced Pg -specific CD4 T cells in NALT following oral feeding of mice with Pg . This new combined flow cytometry and dissection approach allows identification of leukocytes infiltrating the connective tissues of the murine oral mucosa and avoids confounding analyses of leukocytes not recruited to inflamed oral mucosal tissues in disease conditions like periodontitis, candidiasis, or sialadenitis.

  1. Differential expression of Meis2, Mab21l2 and Tbx3 during limb development associated with diversification of limb morphology in mammals.

    Science.gov (United States)

    Dai, Mengyao; Wang, Yao; Fang, Lu; Irwin, David M; Zhu, Tengteng; Zhang, Junpeng; Zhang, Shuyi; Wang, Zhe

    2014-01-01

    Bats are the only mammals capable of self-powered flight using wings. Differing from mouse or human limbs, four elongated digits within a broad wing membrane support the bat wing, and the foot of the bat has evolved a long calcar that spread the interfemoral membrane. Our recent mRNA sequencing (mRNA-Seq) study found unique expression patterns for genes at the 5' end of the Hoxd gene cluster and for Tbx3 that are associated with digit elongation and wing membrane growth in bats. In this study, we focused on two additional genes, Meis2 and Mab21l2, identified from the mRNA-Seq data. Using whole-mount in situ hybridization (WISH) we validated the mRNA-Seq results for differences in the expression patterns of Meis2 and Mab21l2 between bat and mouse limbs, and further characterize the timing and location of the expression of these two genes. These analyses suggest that Meis2 may function in wing membrane growth and Mab21l2 may have a role in AP and DV axial patterning. In addition, we found that Tbx3 is uniquely expressed in the unique calcar structure found in the bat hindlimb, suggesting a role for this gene in calcar growth and elongation. Moreover, analysis of the coding sequences for Meis2, Mab21l2 and Tbx3 showed that Meis2 and Mab21l2 have high sequence identity, consistent with the functions of genes being conserved, but that Tbx3 showed accelerated evolution in bats. However, evidence for positive selection in Tbx3 was not found, which would suggest that the function of this gene has not been changed. Together, our findings support the hypothesis that the modulation of the spatiotemporal expression patterns of multiple functional conserved genes control limb morphology and drive morphological change in the diversification of mammalian limbs.

  2. At least two Fc Neu5Gc residues of monoclonal antibodies are required for binding to anti-Neu5Gc antibody.

    Science.gov (United States)

    Yu, Chuanfei; Gao, Kai; Zhu, Lei; Wang, Wenbo; Wang, Lan; Zhang, Feng; Liu, Chunyu; Li, Meng; Wormald, Mark R; Rudd, Pauline M; Wang, Junzhi

    2016-01-29

    Two non-human glycan epitopes, galactose-α-1,3-galactose (α-gal) and Neu5Gc-α-2-6-galactose (Neu5Gc) have been shown to be antigenic when attached to Fab oligosaccharides of monoclonal antibodies (mAbs) , while α-gal attached to Fc glycans was not. However, the antigenicity of Neu5Gc on the Fc glycans remains unclear in the context that most mAbs carry only Fc glycans. After studying two clinical mAbs carrying significant amounts of Fc Neu5Gc, we show that their binding activity with anti-Neu5Gc antibody resided in a small subset of mAbs carrying two or more Fc Neu5Gc, while mAbs harboring only one Neu5Gc showed no reactivity. Since most Neu5Gc epitopes were distributed singly on the Fc of mAbs, our results suggest that the potential antigenicity of Fc Neu5Gc is low. Our study could be referenced in the process design and optimization of mAb production in murine myeloma cells and in the quality control of mAbs for industries and regulatory authorities.

  3. mEBT: multiple-matching Evidence-based Translator of Murine Genomic Responses for Human Immunity Studies.

    Science.gov (United States)

    Tae, Donghyun; Seok, Junhee

    2018-05-29

    In this paper, we introduce multiple-matching Evidence-based Translator (mEBT) to discover genomic responses from murine expression data for human immune studies, which are significant in the given condition of mice and likely have similar responses in the corresponding condition of human. mEBT is evaluated over multiple data sets and shows improved inter-species agreement. mEBT is expected to be useful for research groups who use murine models to study human immunity. http://cdal.korea.ac.kr/mebt/. jseok14@korea.ac.kr. Supplementary data are available at Bioinformatics online.

  4. Combined therapy of interleukin-12 and interleukin-18 against cryptococcus neoformans infection in a murine model

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective To explore adverse effects of combined treatment of interleukin-12 (IL-12) and interleukin-18 (IL-18) against cryptococcosis in a murine model.Methods Infected mice were treated with a combination of IL-12 and IL-18. Their body weight and intake of water and food were observed and recorded. Serum levels of leptin were detected with an enzyme-linked immuno sorbent assay (ELISA).Results In the combined treatment group, the intake volume of water and food were reduced, leading to weight loss and undetectable levels of leptin in the serum. These adverse effects were more profound in mice that had received higher doses of cytokines, which sometimes led to a fatal outcome. There was a significant difference compared with the control group. Neutralization of endogenous tumor necrosis factor-α (TNF-α) by its specific mAb did not alter the wasting effect of this treatment.Conclusions The combined IL-12/IL-18 treatment may cause a number of adverse effects independent of TNF-α and leptin synthesis. Further investigations for resolving these adverse effects are required before clinical application of these cytokines.

  5. Comparison between anti-CEA and anti-HER2 212Pb-labeled mAbs during α-RIT of small volume peritoneal carcinomatosis - Role of activity distribution on therapeutic efficacy and toxicity?

    International Nuclear Information System (INIS)

    Elgqvist, J.; Boudousq, V.; Bobyk, L.; Busson, M.; Lozza, C.; Navarro-Teulon, I.; Pouget, J.P.; Maquaire, P.; Torgue, J.

    2015-01-01

    Full text of publication follows. Objectives: we investigated the role of internalizing/non-internalizing monoclonal antibodies (mAbs) on the final outcome (efficacy/toxicity) of mice treated with alpha radioimmunotherapy (α- RIT) using 212 Pb-labeled mAbs. The relationship between distribution of radioactivity at the tissue level and biological parameters was also assessed. Methods: nude mice bearing 2-3 mm peritoneal nodules obtained by xenograft of A-431 tumor cells, expressing low and high level of HER2 and CEA receptors, respectively, were intraperitoneally (i.p.) injected with increasing activities (370-1480 kBq; 37 MBq/mg) of either 35A7 (non-internalising anti-CEA), Trastuzumab (internalizing anti-HER2) or PX (non-specific) 212 Pb-labeled mAbs. Control groups were injected with corresponding amount of unlabeled mAbs or with NaCl. Tumor growth was followed by bioluminescence and median survival (MS) of control and treated mice was determined. 212 Pb-35A7 and 212 Pb-Trastuzumab biodistribution was used to determine the cumulative uptake of radioactivity (UOR) in organs and tumors. Mean absorbed doses were calculated using the MIRD formalism. Haematological, liver and kidney toxicities were also assessed. Distribution of radioactivity at the tissue level was determined by digital micro-autoradiography and the relationship with biological markers of tissue damage was investigated using immunohistochemistry. Results: a mild and transient haematological toxicity in groups treated with the highest amount of activity was observed. MS of the groups treated either with internalizing or non-internalizing 212 Pb-labeled mAbs was significantly improved compared to those treated with non-specific 212 Pb-PX or those only given unlabeled mAbs or just NaCl. MS ranged from 42 days to 94 days using various activity levels of anti-CEA 212 Pb-35A7 while MS was not reached over the follow-up period of 130 days for mice treated with anti-HER2 212 Pb-Trastuzumab. However, UOR and

  6. Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K

    2015-10-21

    A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples.

  7. Development of a Monoclonal Antibody-Based Sandwich ELISA for Peanut Allergen Ara h 1 in Food

    Directory of Open Access Journals (Sweden)

    Chuanlai Xu

    2013-07-01

    Full Text Available We have established a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA based on two monoclonal antibodies (mAb to measure the content of the major peanut allergen Ara h 1 in foods. Two mAbs were selected out of 12 murine hybridoma cells secreting Ara h 1-specific antibody. Using mAb 6 as the capture antibody and HRP-labelled mAb 4 as the detection antibody, the limit of detection (LOD the assay was 0.34 ng/mL. Cross-reaction analysis showed that this method was strongly specific and had no cross-reactions with Ara h 2, pea protein or soy protein. Sample analysis showed that this ELISA was a useful tool to monitor peanut allergens in food products by measuring Ara h 1 content.

  8. Characterization of a Novel Murine Model to Study Zika Virus.

    Science.gov (United States)

    Rossi, Shannan L; Tesh, Robert B; Azar, Sasha R; Muruato, Antonio E; Hanley, Kathryn A; Auguste, Albert J; Langsjoen, Rose M; Paessler, Slobodan; Vasilakis, Nikos; Weaver, Scott C

    2016-06-01

    The mosquito-borne Zika virus (ZIKV) is responsible for an explosive ongoing outbreak of febrile illness across the Americas. ZIKV was previously thought to cause only a mild, flu-like illness, but during the current outbreak, an association with Guillain-Barré syndrome and microcephaly in neonates has been detected. A previous study showed that ZIKV requires murine adaptation to generate reproducible murine disease. In our study, a low-passage Cambodian isolate caused disease and mortality in mice lacking the interferon (IFN) alpha receptor (A129 mice) in an age-dependent manner, but not in similarly aged immunocompetent mice. In A129 mice, viremia peaked at ∼10(7) plaque-forming units/mL by day 2 postinfection (PI) and reached high titers in the spleen by day 1. ZIKV was detected in the brain on day 3 PI and caused signs of neurologic disease, including tremors, by day 6. Robust replication was also noted in the testis. In this model, all mice infected at the youngest age (3 weeks) succumbed to illness by day 7 PI. Older mice (11 weeks) showed signs of illness, viremia, and weight loss but recovered starting on day 8. In addition, AG129 mice, which lack both type I and II IFN responses, supported similar infection kinetics to A129 mice, but with exaggerated disease signs. This characterization of an Asian lineage ZIKV strain in a murine model, and one of the few studies reporting a model of Zika disease and demonstrating age-dependent morbidity and mortality, could provide a platform for testing the efficacy of antivirals and vaccines. © The American Society of Tropical Medicine and Hygiene.

  9. Solid-state mAbs and ADCs subjected to heat-stress stability conditions can be covalently modified with buffer and excipient molecules.

    Science.gov (United States)

    Valliere-Douglass, John F; Lewis, Patsy; Salas-Solano, Oscar; Jiang, Shan

    2015-02-01

    We report that a unique type of chemical modification occurs on lyophilized proteins. Freeze-dried mAbs and antibody-drug conjugates (ADCs) can be covalently modified with buffer and excipient molecules on the side chains of Glu, Asp, Thr, and Ser amino acids when subjected to temperature stress. The reaction occurs primarily via condensation of common buffers and excipients such as histidine, tris, trehalose and sucrose, with Glu and Asp carboxylates in the primary sequence of proteins. The reaction was also found to proceed through condensation of carboxylate containing buffers such as citrate, with Thr and Ser hydroxyls in the primary sequence of proteins. Based on the mass of the covalent adducts observed on mAbs and ADCs, it is apparent that the reaction produces water as a product and is thus favored in a low moisture environments such as a lyophilized protein cake. Herein, we present the evidence for the covalent modification of proteins drawn from case studies of in-depth characterization of heat-stressed mAbs and ADCs in the solid state. We also demonstrate how common charge variant assays such as imaged capillary isoelectric focusing and mass spectrometry can be used to monitor this specific class of protein modification. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  10. Interleukin-2 production by human leukemia cell lines of pre-B cell origin

    International Nuclear Information System (INIS)

    Holan, V.; Minowada, J.

    1993-01-01

    Cells of 7 tested human leukemia cell lines of pre-B cell origin (as characterized by immunophenotyping and by the expression of cytoplasmic micro chains, but not by surface immunoglobulins) produced after stimulation with bacterial lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) a lymphokine activity which supported the growth of the interleukin-2 (IL-2)-dependent CTLL-2 cell line. Three pieces of evidence indicate that the secreted lymphokine was functionally and antigenically very similar, if not identical, to human IL-2: (1) The lymphokine supported the growth of murine IL-2-dependent CTLL-2 cells, which did not respond to human lymphokines other than IL-2, but it did not stimulate the growth of murine IL-3-dependent FDC-P2 cells, (2) the biological activity of the lymphokine was was inhibited by monoclonal antibody (mAb) anti-human-IL-2, and (3) the proliferation of IL-2-dependent cells in the presence of the active materials was completely inhibited by the inclusion of the anti-mouse-IL-2 receptor (IL-2R) mAb. Since leukemia cells of immature B-cell origin also synthesize IL-2R, the human pre-B cell leukemias could represent another type of hematological malignancy where the autocrine processes of IL-2 production and utilization are involved in the expansion of the disease. (author)

  11. Correction of murine mucopolysaccharidosis VII by a human. beta. -glucuronidase transgene

    Energy Technology Data Exchange (ETDEWEB)

    Kyle, J.W.; Vogler, C.; Hoffmann, J.W.; Sly, W.S. (St. Louis Univ. School of Medicine, MO (USA)); Birkenmeier, E.H.; Gwynn, B. (Jackson Laboratory, Bar Harbor, ME (USA))

    1990-05-01

    The authors recently described a murine model for mucopolysaccharidosis VII in mice that have an inherited deficiency of {beta}-glucuronidase. Affected mice, of genotype gus{sup mps}/gus{sup mps}, present clinical manifestations similar to those of humans with mucopolysaccharidosis VII (Sly syndrome) and are shown here to have secondary elevations of other lysosomal enzymes. The mucopolysaccharidosis VII phenotype in both species includes dwarfism, skeletal deformities, and premature death. Lysosome storage is visualized within enlarged vesicles and correlates biochemically with accumulation of undegraded and partially degraded glycosaminoglycans. In this report they describe the consequences of introducing the human {beta}-glucuronidase gene, GUSB, into gus{sup mps}/gus{sup mps} mice that produce virtually no murine {beta}-glucuronidase. Transgenic mice homozygous for the mucopolysaccharidosis VII mutation expressed high levels of human {beta}-glucuronidase activity in all tissues examined and were phenotypically normal. Biochemically, both the intralysosomal storage of glycosaminoglycans and the secondary elevation of other acid hydrolases were corrected. These findings demonstrate that the GUSB transgene is expressed in gus{sup mps}/gus{sup mps} mice and that human {beta}-glucuronidase corrects the murine mucopolysaccharidosis storage disease.

  12. The Murine Factor H-Related Protein FHR-B Promotes Complement Activation

    Directory of Open Access Journals (Sweden)

    Marcell Cserhalmi

    2017-09-01

    Full Text Available Factor H-related (FHR proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH. While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3, and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

  13. Radioimmunoscintigraphy with monoclonal antibody Technetium-99m-Anti-EGF-Receptor (R3-MAB) for the detection of head and neck tumours, metastasis and recurrence. Final report for the period 15 April 1995 - 15 April 1997

    International Nuclear Information System (INIS)

    Oliva Gonzalez, J.P.

    1998-03-01

    A clinical study was carried out to determine the sensitivity of radioimmunoscintigraphy (RIS) using indigenously produced mouse monoclonal antibody (MAB) against epidermal growth factor receptor in the detection of primary, recurrent and metastatic malignant epithelial tumours of the head and neck region in 13 patients. The MAB was labelled with 99m Tc and imaging was carried out using gamma camera and SPECT. The results were correlated with histopathological findings. RIS gave a sensitivity of 76.9%. This study showed that the indigenously produced MAB can be used for the detection of malignant epithelial tumours in the head and neck region but the MAB will be further characterized to improve its sensitivity in the detection of the neoplasia. (author)

  14. Radioimmunodetection of human tumor xenografts by monoclonal antibody F(ab')/sub 2/ fragments

    Energy Technology Data Exchange (ETDEWEB)

    Herlyn, D.; Munz, D.L.; Herlyn, M.; Koprowski, H.; Powe, J.; Alavi, A.; Meinken, G.E.; Srivastava, S.C.

    1986-01-01

    Procedures are described for the radiolocalization of human tumors by murine monoclonal antibodies (MAb) in animal model systems. Visualization of tumor xenografts was clearer in nude mice compared to experimentally immunosuppressed mice due to the higher tumor viability. MAb localization in tumor tissue was greatly enhanced when F(ab')/sub 2/ fragments rather than intact antibody molecules were used. Although tumors could be visualized with /sup 131/I-, /sup 123/I-or /sup 111/In-labeled MAb fragments without background subtraction, tumor-to-background ratios of radioactivity were highest for /sup 131/I-labeled fragments. /sup 131/I-labeled F(ab')/sub 2/ fragments of eight MAb against human colorectal carcinoma, melanoma or lung carcinoma localized specifically only in those tumors that bound the MAb in vitro and not in unrelated tumors. Radiolabeled fragments of MAb with other specificities (anti-hepatitis virus MAb) did not localize in tumors. All MAb that inhibited tumor growth in nude mice effectively localized these tumors by ..gamma..-scintigraphy. Some MAb were effective in localizing tumors but ineffective in inhibiting their growth. The ability of the specific radiolabeled F(ab')/sub 2/ fragments to localize in tumor grafts correlated significantly with MAb binding affinity and density of antigenic sites on tumor cells together, but not with either in vitro binding parameter alone.

  15. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    International Nuclear Information System (INIS)

    Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.

    2009-01-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL

  16. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, Kausik [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Ramagopal, Udupi A. [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Nathenson, Stanley G., E-mail: nathenso@aecom.yu.edu [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Almo, Steven C., E-mail: nathenso@aecom.yu.edu [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States)

    2009-05-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  17. Multiple epitopes in a dodecapeptide of myelin basic protein determined bymonoclonal antibodies

    International Nuclear Information System (INIS)

    Price, J.O.; Whitaker, J.N.; Vasu, R.I.; Metzger, D.W.

    1986-01-01

    Three custom synthesized myelin basic protein (MBP) peptides, bovine peptide 79-88, human peptide 80-89, and human peptide 82-91, were used to produce four murine monoclonal antibodies (MAb) that were selected on the basis of reaction in a solid phase radioimmunoassay (SRIA) with human MBP. The MAb were compared with respect to antigen specificity against intact MBP and 10 overlapping MBP peptides. One MAb recognized an epitope near the amino-terminus of bovine MBP peptide 79-88. A second MAb was directed towards an epitope that is more reactive in human MBP peptide 45-89 than in intact MBP, but is not recognized in any of the small MBP peptides examined. The third MAb detected an epitope near the middle of human MBP peptide 80-89, whereas the fourth MAb reacted with the carboxyl-terminal portion of human MBP peptide 82-91. Epitopes recognized in SRIA were sometimes not detected by the same MAb in a fluid phase double antibody radioimmunoassay. These results demonstrate the multiplicity of potential epitopes in a dodecapeptide of MBP and do not support the concept of a single, dominant epitope in the region of MBP peptide 80-89

  18. Chemical changes demonstrated in cartilage by synchrotron infrared microspectroscopy in an antibody-induced murine model of rheumatoid arthritis

    Science.gov (United States)

    Croxford, Allyson M.; Selva Nandakumar, Kutty; Holmdahl, Rikard; Tobin, Mark J.; McNaughton, Don; Rowley, Merrill J.

    2011-06-01

    Collagen antibody-induced arthritis develops in mice following passive transfer of monoclonal antibodies (mAbs) to type II collagen (CII) and is attributed to effects of proinflammatory immune complexes, but transferred mAbs may react directly and damagingly with CII. To determine whether such mAbs cause cartilage damage in vivo in the absence of inflammation, mice lacking complement factor 5 that do not develop joint inflammation were injected intravenously with two arthritogenic mAbs to CII, M2139 and CIIC1. Paws were collected at day 3, decalcified, paraffin embedded, and 5-μm sections were examined using standard histology and synchrotron Fourier-transform infrared microspectroscopy (FTIRM). None of the mice injected with mAb showed visual or histological evidence of inflammation but there were histological changes in the articular cartilage including loss of proteoglycan and altered chondrocyte morphology. Findings using FTIRM at high lateral resolution revealed loss of collagen and the appearance of a new peak at 1635 cm-1 at the surface of the cartilage interpreted as cellular activation. Thus, we demonstrate the utility of synchrotron FTIRM for examining chemical changes in diseased cartilage at the microscopic level and establish that arthritogenic mAbs to CII do cause cartilage damage in vivo in the absence of inflammation.

  19. 4-1BB Aptamer-Based Immunomodulation Enhances the Therapeutic Index of Radiation Therapy in Murine Tumor Models

    Energy Technology Data Exchange (ETDEWEB)

    Benaduce, Ana Paula; Brenneman, Randall; Schrand, Brett; Pollack, Alan; Gilboa, Eli; Ishkanian, Adrian, E-mail: aishkanian@med.miami.edu

    2016-10-01

    Purpose: To report a novel strategy using oligonucleotide aptamers to 4-1BB as an alternate method for costimulation, and show that combinatorial therapy with radiation improves the therapeutic ratio over equivalent monoclonal antibodies. Methods and Materials: Subcutaneous 4T1 (mouse mammary carcinoma) tumors were established (approximately 100 mm{sup 3}), and a radiation therapy (RT) dose/fractionation schedule that optimally synergizes with 4-1BB monoclonal antibody (mAb) was identified. Comparable tumor control and animal survival was observed when either 4-1BB antibody or aptamer were combined with RT using models of breast cancer and melanoma (4T1 and B16-F10). Off-target CD8{sup +} T-cell toxicity was evaluated by quantification of CD8{sup +} T cells in livers and spleens of treated animals. Results: When combined with 4-1BB mAb, significant differences in tumor control were observed by varying RT dose and fractionation schedules. Optimal synergy between RT and 4-1BB mAb was observed at 5 Gy × 6. Testing 4-1BB mAb and aptamer independently using the optimal RT (5 Gy × 6 for 4T1/Balb/c and 12 Gy × 1 for B16/C57BL6J mouse models) revealed equivalent tumor control using 4-1BB aptamer and 4-1BB mAb. 4-1BB mAb, but not 4-1BB aptamer-treated animals, exhibited increased lymphocytic liver infiltrates and increased splenic and liver CD8{sup +} T cells. Conclusions: Radiation therapy synergizes with 4-1BB mAb, and this effect is dependent on RT dose and fractionation. Tumor control by 4-1BB aptamer is equivalent to 4-1BB mAb when combined with optimal RT dose, without eliciting off-target liver and spleen CD8{sup +} expansion. 4-1BB aptamer-based costimulation affords a comparable and less toxic strategy to augment RT-mediated tumor control.

  20. Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition.

    Science.gov (United States)

    Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

    2016-01-01

    We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro . Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8 + and CD4 + ). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs.

  1. Imported rickettsioses : think of murine typhus

    NARCIS (Netherlands)

    van der Kleij, FGH; Gansevoort, RT; Kreeftenberg, HG

    Murine typhus is a disease still prevalent in many parts of the world. Because the incidence in the US and Europe has declined rapidly, physicians in these continents have become unfamiliar with the clinical picture. Murine typhus is associated with significant morbidity and fatalities do occur,

  2. Generation and characterisation of murine monoclonal antibodies specific for cervine immunoglobulin light chain, IgM and IgG

    International Nuclear Information System (INIS)

    Hibma, M.; Griffin, J.F.T.

    1992-01-01

    Monoclonal antibodies (mAb) which react with cervine immunoglobulin (Ig) light chain, IgM and IgG were produced using conventional cell fusion technology. Hybridoma supernatants were initially screened for specificity against cervine Ig using an enzyme-linked immunosorbent assay (ELISA). The specificity of supernatants against size-fractionated cervine Ig was further determined. Supernatants were characterised using western blotting and autoradiographic techniques. The mAb OU1G, OU2G and OU3G were specific for cervine gamma-chain of IgG, whereas OU1L was specific for light chain of Ig. A further mAb (OU1M) bound IgM and not IgG. These mAb were found to have varying cross-reactivity against Ig from other species

  3. The development of a murine model for Forcipomyia taiwana (biting midge) allergy.

    Science.gov (United States)

    Lee, Mey-Fann; Yang, Kai-Jei; Wang, Nancy M; Chiu, Yung-Tsung; Chen, Pei-Chih; Chen, Yi-Hsing

    2014-01-01

    Forcipomyia taiwana (biting midge) allergy is the most prevalent biting insect allergy in Taiwan. An animal model corresponding to the human immuno-pathologic features of midge allergy is needed for investigating the mechanisms and therapies. This study successfully developed a murine model of Forcipomyia taiwana allergy. BALB/c mice were sensitized intra-peritoneally with midge extract on days 0, 7, 14, 21 then intra-dermally on days 28, 31 and 35. Serum midge-specific IgE, IgG1, and IgG2a were measured every 14 days by indirect ELISA. The mice were challenged intradermally with midge extract at day 40 and then sacrificed. Proliferation and cytokine production of splenocytes after stimulation with midge extract were determined by MTT assay and ELISA, respectively. The cytokine mRNA expression in response to midge stimulation was analyzed by RT-PCR. Serum IgE, total IgG, and IgG1 antibody levels against midge extract were significantly higher in the midge-sensitized mice than in the control mice. After the two-step sensitization, all mice in the midge-sensitized group displayed immediate itch and plasma extravasation reactions in response to challenge with midge extract. Skin histology from midge-sensitized mice showed marked eosinophil and lymphocyte infiltrations similar to that observed in humans. Stimulation of murine splenocytes with midge extract elicited significant proliferation, IL-4, IL-10, IL-13 and IFN-γ protein production, and up-regulation of mRNA in a dose-dependent manner in the midge-sensitized group, but not in the control group. A murine model of midge bite allergy has been successfully developed using a two-step sensitization protocol. The sensitized mice have very similar clinical and immunologic reactions to challenge with midge proteins as the reactions of human to midge bites. This murine model may be a useful platform for future research and the development of treatment strategies for insect bite allergy.

  4. Characterization, expression and complex formation of the murine Fanconi anaemia gene product Fancg.

    Science.gov (United States)

    van de Vrugt, Henri J; Koomen, Mireille; Berns, Mariska A D; de Vries, Yne; Rooimans, Martin A; van der Weel, Laura; Blom, Eric; de Groot, Jan; Schepers, Rik J; Stone, Stacie; Hoatlin, Maureen E; Cheng, Ngan Ching; Joenje, Hans; Arwert, Fré

    2002-03-01

    Fanconi anaemia (FA) is an autosomal recessive chromosomal instability disorder. Six distinct FA disease genes have been identified, the products of which function in an integrated pathway that is thought to support a nuclear caretaker function. Comparison of FA gene characteristics in different species may help to unravel the molecular function of the FA pathway. We have cloned the murine homologue of the Fanconi anaemia complementation group G gene, FANCG/XRCC9. The murine Fancg protein shows an 83% similarity to the human protein sequence, and has a predicted molecular weight of 68.5 kDa. Expression of mouse Fancg in human FA-G lymphoblasts fully corrects their cross-linker hypersensitivity. At mRNA and protein levels we detected the co-expression of Fancg and Fanca in murine tissues. In addition, mouse Fancg and Fanca proteins co-purify by immunoprecipitation. Upon transfection into Fanca-deficient mouse embryonic fibroblasts EGFP-Fancg chimeric protein was detectable in the nucleus. We identified a murine cDNA, Fancg, which cross-complements the cellular defect of human FA-G cells and thus represents a true homologue of human FANCG. Spleen, thymus and testis showed the highest Fancg expression levels. Although Fancg and Fanca are able to form a complex, this interaction is not required for Fancg to accumulate in the nuclear compartment.

  5. Coagulation and inflammation in scrub typhus and murine typhusu-a prospective comparative study from Laos

    NARCIS (Netherlands)

    Paris, D. H.; Chansamouth, V.; Nawtaisong, P.; Löwenberg, E. C.; Phetsouvanh, R.; Blacksell, S. D.; Lee, S. J.; Dondorp, A. M.; van der Poll, T.; Newton, P. N.; Levi, M. [=Marcel M.; Day, N. P. J.

    2012-01-01

    Scrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited.

  6. Myeloablative radioimmunotherapy with {sup 188}Re-CD66mAb before stem cell transplantation. No increase of proinflammatory cytokine levels of TNF-{alpha}; Myeloablative Radioimmuntherapie mit {sup 188}Re-CD66mAb vor Stammzelltransplantation. Kein Anstieg proinflammatorischer Zytokinspiegel von TNF-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Mutschler, J.; Reske, S.N. [Universitaetsklinik Ulm (Germany). Klinik fuer Nuklearmedizin; Steinbach, G. [Universitaetsklinik Ulm (Germany). Abt. Klinische Chemie; Bunjes, D. [Universitaetsklinik Ulm (Germany). Medizinische Klinik III; Buchmann, I. [Universitaetsklinik Heidelberg (Germany). Abt. fuer Nuklearmedizin

    2009-07-01

    Tumour necrosis factor-{alpha} (TNF-{alpha}) serum levels may increase due to intensive conditioning regimes with high-dose chemotherapy and total body irradiation (TBI) before stem cell transplantation. This increases the risk for developing acute graft versus host disease (aGvHD) after stem cell transplantation. In this prospective study we investigated the influence of radioimmunotherapy with {sup 188}Re-CD-66-mAb on changes on TNF-{alpha} serum levels. Patients, methods: In 18 patients we measured TNF-{alpha} before and up to 96 hours after radioimmunotherapy, in 2 patients in addition following TBI, in 9 patients also following chemotherapy. For measuring TNF-{alpha} we used an automated immunochemiluminescence assay (Immulite 1000 DPC Biermann, Bad Nauheim). The mean follow up period to record incidence of aGVHD was 100 days after stem cell transplantation. Compared to the basal levels before, the levels of TNF-{alpha} after conditioning with {sup 188}Re-CD-66-mAb did not increase significantly and remained in the physiological range. In contrast, these initial physiological cytokine levels increased and became pathological following 48 h after total body irradiation (13.2 {+-} 6.6 pg/ml) and chemotherapy (10.8 {+-} 15.7 pg/ml). In our study we found a low incidence of aGvHD (22.2%, n = 4/18). Conclusion: These results demonstrate that additional conditioning therapy with {sup 188}Re-CD-66-mAb does not increase proinflammatory cytokine levels of TNF-{alpha}. This finding may indicate that additive radioimmunotherapy may not be a significant factor for increasing the rate of conditioning- associated aGvHD. (orig.)

  7. Effects of anti-CD40 mAb on inducing malignant B cells proliferation arrest and apoptosis and its mechanism

    International Nuclear Information System (INIS)

    Tang Lin; Zhuang Yumei; Zhou Zhaohua; Yu Gehua; Pan Jianzhong; Zhang Xueguang

    2002-01-01

    Objective: To study the expression of CD 40 molecule and the biological effects mediated by CD 40 molecules on malignant B cells. Methods: Agonistic anti-human CD 40 monoclonal antibody (clone 5C11) was added to cell culture system. Cell counting, PI staining, Annexin-V staining and flow cytometric analysis were used to study the behavior of malignant B cell lines after treatment with mAb clone 5C11. Results: 5C11 induced homotypic aggregation and proliferation arrest and mediated apoptosis in multiple myeloma cell line XG2 that expressed CD 40 strongly; 5C11 induced B lymphoma cell line Daudi homotypic aggregation and proliferation arrest and apoptosis, the apoptosis of XG2 and Daudi by CD40 activation was not mediated by TNF. Conclusion: Agonistic anti-CD 40 mAb 5C11 can inhibit the proliferation of malignant B cells by inducing them to die apoplectically

  8. Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

    Science.gov (United States)

    Zhou, Shiyue; Tello, Nadia; Harvey, Alex; Boyes, Barry; Orlando, Ron; Mechref, Yehia

    2016-06-01

    Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. 47-mG2a: A Mouse IgG2a-Type of PcMab-47 Useful for Detecting Podocalyxin in Esophageal Cancers by Immunohistochemistry.

    Science.gov (United States)

    Kaneko, Mika K; Itai, Shunsuke; Yamada, Shinji; Kato, Yukinari

    2018-04-09

    Esophageal cancer is one of the highly malignant cancers. It comprises two of the most common histological tumor types: squamous cell carcinoma (SCC) and adenocarcinoma. SCC accounts for about 90% of esophageal cancers. Despite developments in treatment strategies, the prognosis and survival rate remain poor. Podocalyxin (PODXL) is a highly glycosylated type-I transmembrane protein. It is expressed in normal tissues such as kidney, heart, breast, and pancreas. Upregulation of PODXL correlates with tumor progression, invasion, and metastasis. Therefore, this glycoprotein could be a potential biomarker for predicting the prognosis of some cancers, for instance, brain, colorectal, oral, lung, bladder, prostate, and ovarian cancers. We previously developed a specific and sensitive anti-PODXL monoclonal antibody (mAb), PcMab-47 (mouse IgG 1 , kappa) and its mouse IgG 2a -type (47-mG 2a ). We showed their utility in immunohistochemical analysis of oral cancers. Herein, we demonstrate that PcMab-47 and 47-mG 2a can also be used to detect esophageal squamous cell carcinoma (ESCC) with this technique. These two antibodies, respectively, stained 123/130 (94.6%) and 127/130 (97.7%) ESCC cases, indicating that they can detect PODXL with high sensitivity in this carcinoma. Of more than 3+ cases, 47-mG 2a was more effective than PcMab-47, respectively, staining 56/127 (44.1%) and 41/123 (33.3%). Therefore, 47-mG 2a can be used for the detection of PODXL in ESCC using immunohistochemical analysis.

  10. Aeroallergen analyses and their clinical relevance. I. Immunochemical quantification of allergens by RAST-inhibition, Mab-ELISA, basophil histamine release, and counter current immuno electrophoresis

    DEFF Research Database (Denmark)

    Johnsen, C R; Abrahamsen, L; Stahl Skov, P

    1991-01-01

    The aim was to compare IgE and IgG4 RAST-inhibition assay (RI), monoclonal antibody ELISA (Mab-ELISA), counter current immuno electrophoresis (CCIE) and histamine release from basophil leukocytes (HR) for allergen quantification with special reference to aeroallergen detection. As components......-U/ml) than IgE-RI (2*10(3) SQ-U/ml). The ranges of allergen detection limits for the Mab-ELISA were equal for cat and Derm. pter. (10-10(2) SQ-U/ml). The range of allergen detection limits for CCIE, assaying dog were 10(4)-10(5) SQ-U/ml. The ranges of allergen detection limits for HR were equal for cat...

  11. The expression analysis of ICOS-L on activated T cells and immature dendritic cells as well as malignant B cells and Grave's-disease-derived thyroid tissues by two novel mAbs against human ICOS-L.

    Science.gov (United States)

    Wang, F; Zhu, W; Liu, T; Sun, Z; Ju, S; Ju, S; Yu, G; Xie, W; Deng, Z; Lu, B; Zhang, X

    2007-01-01

    ICOS-L, a newly identified member of B7 superfamily, plays an important role in immune responses. In this article, we report on two novel mouse anti-human ICOS-L monoclonal antibodies (mAbs) named as 11C4 and 12B11, whose specificities were verified by methods of flow cytometry, western blotting, and epitope competition assay. The two mAbs bound to distinct ICOS-L epitopes on B cells. Interestingly, mAb 11C4 could well recognize ICOS-L molecule on activated T cells and Jurkat cell lines, which is different from commercial anti-ICOS-L mAb (clone number MIH12) and the other mAb 12B11. In addition, we found that the expression of ICOS-L molecule was only detected on the surface of immature monocyte-derived dendritic cells (Mo-DCs) and was sharply decreased after induction of mature Mo-DCs activated by tumor necrosis factor-alpha or CD40. Furthermore, we showed that 11C4 could effectively suppress the maturation of Mo-DCs in vitro as evidenced by the low expression of CD80, CD86, CD83, and human leukocyte antigen-DR, which suggested that ICOS-L may be involved in the maturation of Mo-DCs. Using immunohistochemistry staining with mAb 11C4, the expression of ICOS-L was found in B lymphoma tissues and thyroid tissues from the Grave's disease but not in thyroid adenoma and normal thyroid tissues.

  12. Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes Internalin A for enhanced infectivity in the murine oral infection model

    LENUS (Irish Health Repository)

    Monk, Ian R

    2010-12-13

    Abstract Background Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. Results We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26), multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlA m*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlA m* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain. Conclusions We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human

  13. Directed evolution and targeted mutagenesis to murinize listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model

    Directory of Open Access Journals (Sweden)

    Hill Colin

    2010-12-01

    Full Text Available Abstract Background Internalin A (InlA is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. Results We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26, multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlAm*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlAm* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain. Conclusions We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced

  14. MRI of Mouse Models for Gliomas Shows Similarities to Humans and Can Be Used to Identify Mice for Preclinical Trials

    Directory of Open Access Journals (Sweden)

    Jason A. Koutcher

    2002-01-01

    Full Text Available Magnetic resonance imaging (MRI has been utilized for screening and detecting brain tumors in mice based upon their imaging characteristics appearance and their pattern of enhancement. Imaging of these tumors reveals many similarities to those observed in humans with identical pathology. Specifically, high-grade murine gliomas have histologic characteristics of glioblastoma multiforme (GBM with contrast enhancement after intravenous administration of gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA, implying disruption of the blood-brain barrier in these tumors. In contrast, low-grade murine oligodendrogliomas do not reveal contrast enhancement, similar to human tumors. MRI can be used to identify mice with brain neoplasms as inclusion criteria in preclinical trials.

  15. Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

    Science.gov (United States)

    Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

    2015-08-01

    We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  16. Anti-respiratory syncytial virus (RSV) G monoclonal antibodies reduce lung inflammation and viral lung titers when delivered therapeutically in a BALB/c mouse model.

    Science.gov (United States)

    Caidi, Hayat; Miao, Congrong; Thornburg, Natalie J; Tripp, Ralph A; Anderson, Larry J; Haynes, Lia M

    2018-06-01

    RSV continues to be a high priority for vaccine and antiviral drug development. Unfortunately, no safe and effective RSV vaccine is available and treatment options are limited. Over the past decade, several studies have focused on the role of RSV G protein on viral entry, viral neutralization, and RSV-mediated pathology. Anti-G murine monoclonal antibody (mAb) 131-2G treatment has been previously shown to reduce weight loss, bronchoalveolar lavage (BAL) cell number, airway reactivity, and Th2-type cytokine production in RSV-infected mice more rapidly than a commercial humanized monoclonal antibody (mAb) against RSV F protein (Palivizumab). In this study, we have tested two human anti-RSV G mAbs, 2B11 and 3D3, by both prophylactic and therapeutic treatment for RSV in the BALB/c mouse model. Both anti-G mAbs reduced viral load, leukocyte infiltration and IFN-γ and IL-4 expression in cell-free BAL supernatants emphasizing the potential of anti-G mAbs as anti-inflammatory and antiviral strategies. Published by Elsevier B.V.

  17. Improved radioimmunotherapy of hematologic malignancies

    International Nuclear Information System (INIS)

    Press, O.W.

    1989-01-01

    In the seven months which have elapsed since initial funding of this project, considerable progress has been made towards achieving the objectives of this research. These objectives include: to study the relative rates of metabolic degradation of radiolabeled monoclonal antibodies (mAbs) targeting tumor associated antigens; to examine the effects of lysomotropic amines (ammonium chloride, chloroquine, amantadine), carboxylic ionophores (monensin), and thionamides (propylthiouracil), on the retention of radiolabeled mAbs by tumor cells; to identify the subcellular site of radioimmunoconjugate degradation, and to quantify the sizes of the fragments generated by intracellular metabolism of radiolabeled mAbs; to examine the effects of lysomotropic agents on the quality of external gamma camera imaging and radiation dosimetry generated in tumor-bearing mice injected with radiolabeled mAbs; to examine the effects of lysomotropic agents on the radiotherapeutic efficacy of radiolabeled mAbs in murine tumors and human tumors xenografted to nude mice; and to compare the effects of lysomotropic agents on the degradation of radioimmunoconjugates made with different radionuclides and different conjugation methods

  18. Mapping of epitopes on Poa p I and Lol p I allergens with monoclonal antibodies.

    Science.gov (United States)

    Lin, Z W; Ekramoddoullah, A K; Jaggi, K S; Dzuba-Fischer, J; Rector, E; Kisil, F T

    1990-01-01

    Allergen Poa p I isolated from the dialysed aqueous extract of Kentucky blue grass pollen by affinity chromatography with an anti-Lol p I murine monoclonal antibody (MAb) 290A-167 was previously shown to consist of a 35.8-kilodalton (kD) component with a pI of 6.4, designated as Poa p Ia, and a 33-kD component with a pI of 9.1, designated as Poa p Ib. The present study reports on the comparative antigenic analyses of these two components, using MAbs produced separately against Poa p I and Lol p I. Thus, anti-Poa p I MAbs 60 and 61 and anti-Lol p I MAb 290A-167 recognized Poa p Ia and Poa p Ib whereas anti-Poa p I MAbs 62, 63 and 64 and anti-Lol p I MAb 348A-6 recognized only Poa p Ia. The specificities of the MAbs were further resolved by comparing their respective abilities to inhibit the binding of 125I-Poa p I or 125I-Lol p I to the different MAbs prepared in the form of solid phase. These studies revealed that at least 4 distinct epitopes (designated as E1, E2, E3 and E4) were shared by both Poa p I and Lol p I. All 4 epitopes were present on Poa p Ia whereas only E1 and E3 were detected on Poa p Ib. E1 was recognized by MAbs 60 and 61, E2 by MAbs 62, 63 and 64, E3 by MAb 290A-167 and E4 by MAb 348A-6.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Effects of radiolabelled monoclonal antibody infusion on blood leukocytes in cancer patients

    International Nuclear Information System (INIS)

    Gridley, D.S.; Slater, J.M.; Stickney, D.R.

    1990-01-01

    This study was undertaken to investigate the effects of a single infusion of radiolabelled murine monoclonal antibody (MAb) on peripheral blood leukocytes in cancer patients. Eleven patients with disseminated colon cancer, malignant melanoma, or lung adenocarcinoma were infused with 111In-labelled anti-ZCE 025, anti-p97 type 96.5c, or LA 20207 MAb, respectively. Blood samples were obtained before infusion, immediately after infusion (1 hr), and at 4 and 7 days postinfusion. Flow cytometry analysis of CD3+, CD4+, CD8+, CD16+, and CD19+ lymphocytes showed increasing CD4:CD8 ratios in seven patients after infusion. This phenomenon was not restricted to antibody subclass or to type of cancer. Two of the remaining patients exhibited a marked post-infusion increase in CD8+ cells. In all three patients with malignant melanoma, decreasing levels of CD16+ lymphocytes were noted after infusion and natural killer cell cytotoxicity showed fluctuations which paralleled the changes in the CD16+ subpopulation. Oxygen radical production by phagocytic cells was markedly affected in three subjects. These results suggest that a single infusion of radiolabelled murine MAb may alter the balance of critical lymphocyte subpopulations and modulate other leukocyte responses in cancer patients

  20. Biodistribution, pharmacokinetic, and imaging studies with 186Re-labeled NR-LU-10 whole antibody in LS174T colonic tumor-bearing mice

    International Nuclear Information System (INIS)

    Goldrosen, M.H.; Biddle, W.C.; Pancook, J.; Bakshi, S.; Vanderheyden, J.L.; Fritzberg, A.R.; Morgan, A.C. Jr.; Foon, K.A.

    1990-01-01

    Biodistribution, pharmacokinetic, and radioimaging studies were performed with 186Re-labeled NR-LU-10 whole antibody in athymic nude mice bearing the LS174T tumor growing either s.c. or in an experimental hepatic metastasis model. NR-LU-10 is an IgG2b murine monoclonal antibody (MAb) that reacts with virtually all human tumors of epithelial origin. NR-BC-1, a IgG2b murine MAb that reacts with normal human B-cell and B malignancies, was used as an isotype-matched control. These MAbs were radiolabeled with 186Re by a preformed chelate approach by using the triamide thiolate ligand system. 186Re-labeled NR-LU-10 (50 microCi) was injected into nude mice bearing LS174T tumors growing s.c. Biodistribution studies revealed that the LS174T tumor retained the highest concentration of 186Re-labeled NR-LU-10 at day 6. The tumor:blood ratio ranged from 0.1:1 to 10.8:1 by day 6, the last day of analysis. In contrast the tumor:blood ratio of 186Re-labeled NR-BC-1, the isotype-matched MAb control, was 1:1 on day 6. Pharmacokinetic analysis indicated that the t1/2 beta of NR-LU-10 for blood and other tissues ranged from 21 to 25 h, while the t1/2 beta for the LS174T tumor averaged 52 h. The area under the curve for tumor compared to blood was 2.8- to 5.7-fold higher than the area under the curve for all other tissues and organs. The mean residence time for NR-LU-10 in blood and all other organs ranged from 23 to 26 h, while the mean residence time for NR-LU-10 in the LS174T tumor was 72 h. Scintigraphic images revealed selective uptake of the 186Re-labeled NR-LU-10, but not of the 186Re-labeled NR-BC-1, at the LS174T tumor site. Studies in an experimental model of hepatic metastasis revealed a similar selective pattern of 186Re-labeled NR-LU-10 accumulation. Scintigraphic images of the LS174T tumor growing within the athymic nude mouse liver were obtained

  1. Investigation of the Influence of Protein-Losing Enteropathy on Monoclonal Antibody Pharmacokinetics in Mice.

    Science.gov (United States)

    Yang, Yujie; Li, Tommy R; Balthasar, Joseph P

    2017-11-01

    Protein losing enteropathy (PLE), which is characterized by substantial loss of plasma proteins into the gastrointestinal (GI) tract, is a complication of a variety of GI diseases, including inflammatory bowel disease. Clinical studies have found that the clearance of monoclonal antibodies (mAb) is often increased in subjects with diseases known to cause PLE; however, direct relationships between PLE and mAb pharmacokinetics have not been demonstrated. This study employed a murine model of colitis to examine the influence of PLE on mAb pharmacokinetics. Mice were given dextran sodium sulfate (DSS, 2% w/v) supplemented tap water as drinking source for 6 days to induce colitis and PLE. Mice were then intravenously injected with 8C2, a murine IgG1 mAb. 8C2 plasma concentrations were measured up to 14 days post injection. Fecal alpha-1-antitrypsin (A1AT) clearance was measured as biomarker for PLE. DSS-treated mice developed PLE of clinically relevant severity. They also showed a transient increase in 8C2 plasma clearance and a decrease in 8C2 plasma exposure. The area under the 8C2 plasma concentration-time curve for the length of the study (AUC 0-14d ) reduced from 1368 ± 255 to 594 ± 224 day μg/ml following DSS treatment (p = 0.001). A quantitative relationship between A1AT clearance and 8C2 clearance was obtained via population pharmacokinetic modeling. DSS treatment substantially increased 8C2 clearance and reduced 8C2 exposure. Increased mAb plasma clearance was highly correlated with A1AT fecal clearance, suggesting the possible utility of A1AT fecal clearance as a mechanistic biomarker to predict the pharmacokinetics of therapeutic antibodies.

  2. Biological properties in vitro of a combination of recombinant murine interleukin-3 and granulocyte-macrophage colony-stimulating factor.

    Science.gov (United States)

    Riklis, I; Kletter, Y; Bleiberg, I; Fabian, I

    1989-04-01

    The effect of recombinant murine interleukin-3 (rIL-3) and recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) on in vitro murine myeloid progenitor cell (CFU-C) growth and on the function of murine resident peritoneal macrophages was investigated. Both rIL-3 and rGM-CSF are known to support the growth of CFU-C and, when combined, were found to act synergistically to induce the development of an increased number of CFU-C. The distribution pattern of myeloid colonies in the presence of these two growth factors was in general similar to that in the presence of rGM-CSF alone. Both rGM-CSF and rIL-3 enhanced the phagocytosis of Candida albicans (CA) by mature macrophages producing an increase in the percentage of phagocytosing cells as well as an increase in the number of yeast particles ingested per cell. No additive effect on the phagocytosis was observed when the two growth factors were added concurrently. rGM-CSF, but not rIL-3, enhanced the killing of CA by macrophages. This killing was inhibited by scavengers of oxygen radicals.

  3. Enhancement of CD147 on M1 macrophages induces differentiation of Th17 cells in the lung interstitial fibrosis.

    Science.gov (United States)

    Geng, Jie-jie; Zhang, Kui; Chen, Li-na; Miao, Jin-lin; Yao, Meng; Ren, Ying; Fu, Zhi-guang; Chen, Zhi-nan; Zhu, Ping

    2014-09-01

    Lung interstitial fibrosis is a chronic lung disease, and few effective therapies are available to halt or reverse the progression of the disease. In murine and human lung fibrosis, the expression of CD147 is increased. However, the role of CD147 in lung fibrosis has not been identified, and it remains to be determined whether lung fibrosis would be improved by decreasing the expression of CD147. A murine bleomycin-induced lung interstitial fibrosis model was used in the experiments, and HAb18 mAbs and CsA were administered during the induction of lung fibrosis. In our study, we found that the HAb18 mAbs markedly reduced the collagen score and down-regulated M1 macrophages and Th17 cells. In vitro, flow cytometry analysis showed that M1 macrophages induced higher Th17 differentiation than M2 macrophages. After treatment with HAb18 mAbs or after reducing the expression of CD147 by lentivirus interference in M1 macrophages, the level of Th17 cells were significantly inhibited. In conclusion, HAb18 mAbs or CsA treatment ameliorates lung interstitial fibrosis. CD147 promoted M1 macrophage and induced the differentiation of Th17 cells in lung interstitial fibrosis, perhaps by regulating some cytokines such as IL-6, IL-1β, IL-12 and IL-23. These results indicated that CD147 may play an important role in the development of lung interstitial fibrosis. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Circulating chromatin-anti-chromatin antibody complexes bind with high affinity to dermo-epidermal structures in murine and human lupus nephritis

    DEFF Research Database (Denmark)

    Fismen, S; Hedberg, A; Fenton, K A

    2009-01-01

    Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo-epidermal i......Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo......-epidermal immune complex deposits have similar molecular composition as glomerular deposits, (ii) whether chromatin fragments bind dermo-epidermal structures, and (iii) whether deposits in nephritic glomeruli predispose for accumulation of similar deposits in skin. Paired skin and kidney biopsies from nephritic...... (NZBxNZW)F1 and MRL-lpr/lpr mice and from five patients with lupus nephritis were analysed by immunofluorescence, immune electron microscopy (IEM) and co-localization TUNEL IEM. Affinity of chromatin fragments for membrane structures was determined by surface plasmon resonance. Results demonstrated (i...

  5. Therapeutic regulatory T-cell adoptive transfer ameliorates established murine chronic GVHD in a CXCR5-dependent manner

    Science.gov (United States)

    McDonald-Hyman, Cameron; Flynn, Ryan; Panoskaltsis-Mortari, Angela; Peterson, Nicholas; MacDonald, Kelli P. A.; Hill, Geoffrey R.; Luznik, Leo; Serody, Jonathan S.; Murphy, William J.; Maillard, Ivan; Munn, David H.; Turka, Laurence A.; Koreth, John; Cutler, Corey S.; Soiffer, Robert J.; Antin, Joseph H.; Ritz, Jerome

    2016-01-01

    Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation. In cGVHD, alloreactive T cells and germinal center (GC) B cells often participate in GC reactions to produce pathogenic antibodies. Although regulatory T cells (Tregs) can inhibit GC reactions, Treg numbers are reduced in cGVHD, contributing to cGVHD pathogenesis. Here, we explored 2 means to increase Tregs in cGVHD: interleukin-2/monoclonal antibody (IL-2/mAb) complexes and donor Treg infusions. IL-2/mAb complexes given over 1 month were efficacious in expanding Tregs and treating established cGVHD in a multi-organ-system disease mouse model characterized by GC reactions, antibody deposition, and lung dysfunction. In an acute GVHD (aGVHD) model, IL-2/mAb complexes given for only 4 days resulted in rapid mortality, indicating IL-2/mAb complexes can drive conventional T-cell (Tcon)-mediated injury. In contrast, Treg infusions, which uniformly suppress aGVHD, increased Treg frequency and were effective in preventing the onset of, and treating, established cGVHD. Efficacy was dependent upon CXCR5-sufficient Tregs homing to, and inhibiting, GC reactions. These studies indicate that the infusion of Tregs, especially ones enriched for GC homing, may be desirable for cGVHD therapy. Although IL-2/mAb complexes can be efficacious in cGVHD, a cautious approach needs to be taken in settings in which aGVHD elements, and associated Tcon, are present. PMID:27385791

  6. Role of cytochrome P450 IA2 in acetanilide 4-hydroxylation as determined with cDNA expression and monoclonal antibodies.

    Science.gov (United States)

    Liu, G; Gelboin, H V; Myers, M J

    1991-02-01

    The role of P450 IA2 in the hydroxylation of acetanilide was examined using an inhibitory monoclonal antibody (MAb) 1-7-1 and vaccinia cDNA expression producing murine P450 IA1 (mIA1), murine P450 IA2 (mIA2), or human P450 IA2 (hIA2). Acetanilide hydroxylase (AcOH) activity was measured using an HPLC method with more than 500-fold greater sensitivity than previously described procedures. This method, which does not require the use of radioactive acetanilide, was achieved by optimizing both the gradient system and the amount of enzyme needed to achieve detection by uv light. MAb 1-7-1 inhibits up to 80% of the AcOH activity in both rat liver microsomes and cDNA expressed mouse and human P450 IA2. MAb 1-7-1, which recognizes both P450 IA1 and P450 IA2, completely inhibits the aryl hydrocarbon hydroxylase (AHH) activity of cDNA expressed in IA1. The inhibition of only 80% of the AHH activity present in MC liver microsomes by MAb 1-7-1 suggests that additional P450 forms are contributing to the overall AHH activity present in methylcholanthrene (MC)-liver microsomes as MAb 1-7-1 almost completely inhibits the AHH activity of expressed mIA1. Maximal inhibition of IA2 by 1-7-1 results in an 80% decrease in acetanilide hydroxylase activity in both liver microsomes and expressed mouse and human IA2. The capacity of MAb 1-7-1 to produce identical levels of inhibition of acetanilide hydroxylase activity in rat MC microsomes (80%) and in expressed mouse (81%) and human P450 IA2 (80%) strongly suggests that P450 IA2 is the major and perhaps the only enzyme responsible for the metabolism of acetanilide. These results demonstrate the complementary utility of monoclonal antibodies and cDNA expression for defining the contribution of specific P450 enzymes to the metabolism of a given substrate. This complementary approach allows for a more precise determination of the inhibitory capacity of MAb with respect to the metabolic capacity of the target P450.

  7. Characterization of the mechanism of protection mediated by CS-D7, a monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB

    Directory of Open Access Journals (Sweden)

    Gregory ePancari

    2012-03-01

    Full Text Available We previously reported the development of a human monoclonal antibody (CS-D7, IgG1 with specificity and affinity for the iron regulated surface determinant B (IsdB of Staphylococcus aureus. CS-D7 mediates opsonophagocytic killing in vitro and protection in a murine sepsis model. In light of recent data indicating that IsdB specific T cells (CD4+, Th17, not Ab, mediate protection after vaccination with IsdB, it is important to investigate the mechanism of protection mediated by CS-D7. The mAb was examined to determine if it blocked heme binding to IsdB in vitro. The mAb was not found to have heme blocking activity, nor did it prevent bacterial growth under in vivo conditions, in an implanted growth chamber. To assess the role of the mAb Fc a point mutation was introduced at aa 297 (CS-D7●N297A. This point mutation removes Fc effector functions. In vitro analysis of the mutein confirmed that it lacked measurable binding to FcγR, and that it did not fix complement. The mutein had dramatically reduced in vitro opsonic OP activity compared to CS-D7. Nonetheless, the mutein conferred protection equivalent to the wild type mAb in the murine sepsis model. Both wild type and mutein mAbs were efficacious in FcγR deletion mice (including both FcγRII-/- mice and FcγRIII-/- mice, indicating that these receptors were not essential for mAb mediated protection in vivo. Protection mediated by CS-D7 was lost in Balb/c mice depleted of C3 with cobra venom factor (CFV, was lost in mice depleted of superoxide dismutase (SOD in P47phox deletion mice, and was absent in SCID mice. Enhanced clearance of S. aureus in the liver of CS-D7 treated mice and enhanced production of INF-γ, but not of IL17, may play a role in the mechanism of protection mediated by the mAb. CS-D7 apparently mediates survival in challenged mice through a mechanism involving complement, phagocytes, and lymphocytes, but which does not depend on interaction with FcγR, or on blocking heme

  8. Myeloablative radioimmunotherapy with 188Re-CD66mAb before stem cell transplantation. No increase of proinflammatory cytokine levels of TNF-α

    International Nuclear Information System (INIS)

    Mutschler, J.; Reske, S.N.; Steinbach, G.; Bunjes, D.; Buchmann, I.

    2009-01-01

    Tumour necrosis factor-α (TNF-α) serum levels may increase due to intensive conditioning regimes with high-dose chemotherapy and total body irradiation (TBI) before stem cell transplantation. This increases the risk for developing acute graft versus host disease (aGvHD) after stem cell transplantation. In this prospective study we investigated the influence of radioimmunotherapy with 188 Re-CD-66-mAb on changes on TNF-α serum levels. Patients, methods: In 18 patients we measured TNF-α before and up to 96 hours after radioimmunotherapy, in 2 patients in addition following TBI, in 9 patients also following chemotherapy. For measuring TNF-α we used an automated immunochemiluminescence assay (Immulite 1000 DPC Biermann, Bad Nauheim). The mean follow up period to record incidence of aGVHD was 100 days after stem cell transplantation. Compared to the basal levels before, the levels of TNF-α after conditioning with 188 Re-CD-66-mAb did not increase significantly and remained in the physiological range. In contrast, these initial physiological cytokine levels increased and became pathological following 48 h after total body irradiation (13.2 ± 6.6 pg/ml) and chemotherapy (10.8 ± 15.7 pg/ml). In our study we found a low incidence of aGvHD (22.2%, n = 4/18). Conclusion: These results demonstrate that additional conditioning therapy with 188 Re-CD-66-mAb does not increase proinflammatory cytokine levels of TNF-α. This finding may indicate that additive radioimmunotherapy may not be a significant factor for increasing the rate of conditioning- associated aGvHD. (orig.)

  9. Somatosensory BOLD fMRI reveals close link between salient blood pressure changes and the murine neuromatrix.

    Science.gov (United States)

    Reimann, Henning Matthias; Todiras, Mihail; Hodge, Russ; Huelnhagen, Till; Millward, Jason Michael; Turner, Robert; Seeliger, Erdmann; Bader, Michael; Pohlmann, Andreas; Niendorf, Thoralf

    2018-05-15

    The neuromatrix, or "pain matrix", is a network of cortical brain areas which is activated by noxious as well as salient somatosensory stimulation. This has been studied in mice and humans using blood oxygenation level-dependent (BOLD) fMRI. Here we demonstrate that BOLD effects observed in the murine neuromatrix in response to salient somatosensory stimuli are prone to reflect mean arterial blood pressure (MABP) changes, rather than neural activity. We show that a standard electrostimulus typically used in murine somatosensory fMRI can induce substantial elevations in MABP. Equivalent drug-induced MABP changes - without somatosensory stimulation - evoked BOLD patterns in the neuromatrix strikingly similar to those evoked by electrostimulation. This constitutes a serious caveat for murine fMRI. The regional specificity of these BOLD patterns can be attributed to the co-localization of the neuromatrix with large draining veins. Based on these findings we propose a cardiovascular support mechanism whereby abrupt elevations in MABP provide additional energy supply to the neuromatrix and other essential brain areas in fight-or-flight situations. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Effectiveness of the immunomodulatory extract of Kalanchoe pinnata against murine visceral leishmaniasis.

    Science.gov (United States)

    Gomes, D C O; Muzitano, M F; Costa, S S; Rossi-Bergmann, B

    2010-04-01

    Previously, we described the protective action of the immunomodulatory extract of Kalanchoe pinnata (Kp) in murine and human cutaneous leishmaniasis. In the present study, we investigated the effectiveness of Kp against visceral leishmaniasis, using the BALB/c mouse model of infection with Leishmania chagasi. Mice receiving oral daily doses of Kp (400 mg/kg) for 30 days displayed significantly reduced hepatic and splenic parasite burden, when compared with untreated animals. Protectiveness was accompanied by a reduction in parasite-specific IgG serum levels, and impaired capacity of spleen cells to produce IL-4, but not IFN-gamma and nitric oxide upon antigen recall in vitro. The reference drug Pentostam (72 mg/kg) given by the intra-peritoneal route on alternate days produced an anti-leishmanial effect similar to oral Kp. Our findings show that the oral efficacy of Kp, seen previously in murine cutaneous leishmaniasis, extends also to visceral leishmaniasis caused by L. chagasi, a difficult to treat and lethal disease of man.

  11. Murine nephrotoxic nephritis as a model of chronic kidney disease

    DEFF Research Database (Denmark)

    Ougaard, M. K.E.; Kvist, P. H.; Jensen, H. E.

    2018-01-01

    Using the nonaccelerated murine nephrotoxic nephritis (NTN) as a model of chronic kidney disease (CKD) could provide an easily inducible model that enables a rapid test of treatments. Originally, the NTN model was developed as an acute model of glomerulonephritis, but in this study we evaluate...... progressive mesangial expansion and significant renal fibrosis within three weeks suggesting CKD development. CD1 and C57BL/6 females showed a similar disease progression, but female mice seemed more susceptible to NTS compared to male mice. The presence of albuminuria, GFR decline, mesangial expansion...

  12. Production of antibodies which recognize opiate receptors on murine leukocytes

    Energy Technology Data Exchange (ETDEWEB)

    Carr, D.J.J.; Bost, K.L.; Blalock, J.E.

    1988-01-01

    An antibody has been developed which recognizes opiate receptors on cells of the immune system. This antibody blocks specific binding of the radiolabeled opiate receptor ligand, /sup 3/H-dihydromorphine, to receptors on murine splenocytes. Additionally, the anti-receptor antibody competes with ..beta..-endorphin, meta-enkephalin, and naloxone for the same binding site on the leukocytes. Moreover, the anti-receptor antibody possesses agonist activity similar to ..beta..-endorphin in suppressing cAMP production by lymphocytes. These results suggest the development of an antibody which recognizes classical opiate receptors on cells of the immune system.

  13. A Murine Herpesvirus Closely Related to Ubiquitous Human Herpesviruses Causes T-Cell Depletion.

    Science.gov (United States)

    Patel, Swapneel J; Zhao, Guoyan; Penna, Vinay R; Park, Eugene; Lauron, Elvin J; Harvey, Ian B; Beatty, Wandy L; Plougastel-Douglas, Beatrice; Poursine-Laurent, Jennifer; Fremont, Daved H; Wang, David; Yokoyama, Wayne M

    2017-05-01

    The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with roseoloviruses as direct drivers of pathology, because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4 + T cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By next-generation sequencing of infected tissue homogenates, we assembled a contiguous 174-kb genome sequence containing 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV-6A, HHV-6B, and HHV-7, than to another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV-6A, HHV-6B, and HHV-7. IMPORTANCE Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesviruses 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and

  14. Identification of two distinct allergenic sites on ryegrass-pollen allergen, Lol p IV.

    Science.gov (United States)

    Jaggi, K S; Ekramoddoullah, A K; Kisil, F T; Dzuba-Fischer, J M; Rector, E S; Sehon, A H

    1989-04-01

    Lol p IV is an important allergen of ryegrass pollen. For the immunochemical identification of antigenic and/or allergenic site(s), murine monoclonal antibodies (MAbs) were prepared against Lol p IV. The hybridoma cell-culture supernatants were screened for anti-Lol p IV antibodies by a combination of ELISA and Western immunoblot analyses. The MAbs were finally purified from ascites on a Mono Q ion-exchange column. In a competitive radioimmunoassay with Lol p IV as the solid phase and 125I-labeled MAbs, it was established that MAbs 90, 91, 92, 93, and 94, although they differed in their relative affinities, recognized in common with one another an epitope designated as antigenic site A, whereas MAb 12 recognized a different epitope referred to as site B. Sites A and B were also demonstrated to constitute allergenic determinants of Lol p IV. Differences in the repertoire of specificities of the human IgE antibodies directed to Lol p IV were also demonstrated. Interestingly, it was found that sera from both allergic as well as from nonatopic individuals had IgG antibodies to sites A and/or B.

  15. Mapping of linear antibody epitopes of the glycoprotein of VHSV, a salmonid rhabdovirus

    DEFF Research Database (Denmark)

    Fernandez-Alonso, M.; Lorenzo, G.; Perez, L.

    1998-01-01

    antibodies (MAbs), only 2 non-neutralizing MAbs, I10 (aa 139-153) and IP1H3 (aa 399-413), could be mapped to specific peptides in the pepscan of the gpG. Mapping of these MAbs was confirmed by immunoblotting with recombinant proteins and/or other synthetic peptides covering those sequences. None......Antibody Linear epitopes of the glycoprotein G (gpG) of the viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus of salmonids, were mapped by pepscan using overlapping 15-mer peptides covering the entire gpG sequence and ELISA with polyclonal and monoclonal murine and polyclonal trout...... antibodies. Among the regions recognized in the pepscan by the polyclonal antibodies (PAbs) were the previously identified phosphatidylserine binding heptad-repeats (Estepa & Coll 1996; Virology 216:60-70) and leucocyte stimulating peptides (Lorenzo et al. 1995; Virology 212:348-355). Among 17 monoclonal...

  16. Rapid desensitization of mice with anti-FcγRIIb/FcγRIII mAb safely prevents IgG-mediated anaphylaxis.

    Science.gov (United States)

    Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Clay, Corey D; Morris, Suzanne C; Finkelman, Fred D

    2013-12-01

    Stimulatory IgG receptors (FcγRs) on bone marrow-derived cells contribute to the pathogenesis of several autoimmune and inflammatory disorders. Monoclonal antibodies that block FcγRs might suppress these diseases, but they can induce anaphylaxis. We wanted to determine whether a rapid desensitization approach can safely suppress IgG/FcγR-mediated anaphylaxis. Mice were injected with serially increasing doses of 2.4G2, a rat mAb that blocks the inhibitory FcγR, FcγRIIb, and the stimulatory receptor, FcγRIII. Rectal temperature was used to detect the development of anaphylaxis. Passive and active IgG-mediated anaphylaxis were evaluated in mice that had been rapidly desensitized with 2.4G2 or mock-desensitized in mice in which monocyte/macrophages, basophils, or neutrophils had been depleted or desensitized and in mice in which FcγRI, FcγRIII, and/or FcγRIV had been deleted or blocked. Rapid desensitization with 2.4G2 prevented 2.4G2-induced shock and completely suppressed IgG-mediated anaphylaxis. Rapid desensitization of ovalbumin-sensitized mice with 2.4G2 was safer and more effective than rapid desensitization with ovalbumin. 2.4G2 treatment completely blocked FcγRIII and removed most FcγRI and FcγRIV from nucleated peripheral blood cells. Because IgG(2a)-mediated anaphylaxis was partially FcγRI and FcγRIV dependent, the effects of 2.4G2 on FcγRI and FcγRIV were probably crucial for its complete inhibition of IgG(2a)-mediated anaphylaxis. IgG(2a)-mediated anaphylaxis was partially inhibited by depletion or desensitization of monocyte/macrophages, basophils, or neutrophils. IgG-mediated anaphylaxis can be induced by ligation of FcγRI, FcγRIII, or FcγRIV on monocycte/macrophages, basophils, or neutrophils and can be safely suppressed by rapid desensitization with anti-FcγRII/RIII mAb. A similar approach may safely suppress other FcγR-dependent immunopathology. Published by Mosby, Inc.

  17. A comparison of targetting of neuroblastoma with MIBG and anti L1-CAM antibody mAb chCE7: therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients

    International Nuclear Information System (INIS)

    Hoefnagel, C.A.; Rutgers, M.; Buitenhuis, C.K.M.; Smets, L.A.; Kraker, J. de; Meli, M.; Carrel, F.; Schubiger, P.A.; Novak-Hofer, I.; Amstutz, H.

    2001-01-01

    Modine-131 labelled anti L1-CAM antibody mAb chCE7 was compared with the effective neuroblastoma-seeking agent 131 I-labelled metaiodobenzylguanidine (MIBG) with regard to (a) its therapeutic efficacy in treating nude mice with neuroblastoma xenografts and (b) its tumour targetting ability in neuroblastoma patients. The SK-N-SH tumour cells used in the mouse experiments show good MIBG uptake and provide a relatively low number of 6,300 binding sites/cell for mAb chCE7. Tumours were treated with single injections of 131 I-MIBG (110 MBq) and with 131 I-labelled mAb chCE7 (17 MBq) and both agents showed antitumour activity. After therapy with 131 I-chCE7, the subcutaneous tumours nearly disappeared; treatment with 131 I-MIBG was somewhat less effective, resulting in a 70% reduction in tumour volume. A calculated tumour regrowth delay of 9 days occurred with a radioactivity dose of 17 MBq of an irrelevant control antibody mAb 35, which does not bind to SK-N-SH cells, compared with a regrowth delay of 34 days with 131 I-mAb chCE7 and of 24 days with 131 I-MIBG. General toxicity appeared to be mild, as assessed by a transient, approximate 10% maximum decrease in body weight during the treatments. The superior growth inhibition achieved by 131 I-chCE7 compared with 131 I-MIBG can be explained by its prolonged retention in the tumours, due to slower normal tissue and plasma clearance. Cross-reaction of mAb chCE7 with L1-CAM present in normal human tissues was investigated by direct binding of radioiodinated mAb to frozen tissue sections. Results showed a strong reaction with normal human brain tissue and weak but detectable binding to normal adult kidney sections. Seven patients with recurrent neuroblastoma were sequentially imaged with 131 I-MIBG and 131 I-chCE7. The results underlined the heterogeneity of neuroblastoma and showed the two imaging modalities to be complementary. 131 I-chCE7 scintigraphy may have clinical utility in detecting metastases which do not

  18. Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

    International Nuclear Information System (INIS)

    Fitzpatrick, D.R.; Zamb, T.; Parker, M.D.; van Drunen Littel-van den Hurk, S.; Babiuk, L.A.; Lawman, M.J.P.

    1988-01-01

    Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK - cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1 infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1 infected bovine cells. For gI, a deficiency in N-linked glycosylated of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected of BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes

  19. Monoclonal antibodies against peptidorhamnomannans of Scedosporium apiospermum enhance the pathogenicity of the fungus.

    Directory of Open Access Journals (Sweden)

    Livia C L Lopes

    2010-10-01

    Full Text Available Scedosporium apiospermum is part of the Pseudallescheria-Scedosporium complex. Peptidorhamnomannans (PRMs are cell wall glycopeptides present in some fungi, and their structures have been characterized in S. apiospermum, S. prolificans and Sporothrix schenckii. Prior work shows that PRMs can interact with host cells and that the glycopeptides are antigenic. In the present study, three monoclonal antibodies (mAbs, IgG1 to S. apiospermum derived PRM were generated and their effects on S. apiospermum were examined in vitro and in vivo. The mAbs recognized a carbohydrate epitope on PRM. In culture, addition of the PRM mAbs increased S. apiospermum conidia germination and reduced conidial phagocytosis by J774.16 macrophages. In a murine infection model, mice treated with antibodies to PRM died prior to control animals. Thus, PRM is involved in morphogenesis and the binding of this glycopeptide by mAbs enhanced the virulence of the fungus. Further insights into the effects of these glycopeptides on the pathobiology of S. apiospermum may lead to new avenues for preventing and treating scedosporiosis.

  20. Regulation of levels of serum antibodies to ryegrass pollen allergen Lol pIV by an internal image anti-idiotypic monoclonal antibody.

    Science.gov (United States)

    Zhou, E M; Kisil, F T

    1995-03-01

    A murine monoclonal anti-idiotypic antibody (anti-Id), designated B1/1, was produced against an idiotope of a murine antibody (mAb91), which recognizes the epitope, site A, of allergen Lol pIV, one of the major groups of allergens in ryegrass (Lolium perenne) pollen. The ability of B1/1 to modulate the antibody responses to Lol pIV was investigated in murine model systems. In the first system, B1/1-keyhole limpet haemocyanin (KLH) conjugate was administered to treat three different strains of mice (C57BL/6, BALB/c and C3H). In the second and third model systems, a solution of B1/1 in phosphate-buffered saline (PBS) was used to treat syngeneic BALB/c mice at various doses and time intervals, respectively. The treatment with either form of B1/1, administered at doses ranging from 100 ng to 100 micrograms mouse, resulted in a reduction of the levels of the antibodies to Lol pIV. In particular, the level of IgE antibodies to Lol pIV was greatly reduced. The administration of a single intravenous (i.v.) injection of a solution of B1/1 8 weeks prior to the challenge with Lol pIV was still effective in reducing the level of antibodies to the allergen. Moreover, the level of antibodies to Lol pIV that expressed the idiotope mAb91 was also markedly decreased. By contrast, it was observed that the level of antibodies to Lol pIV in mice pretreated with B1/1 in PBS at a dose of 10 ng/mouse increased (albeit slightly) compared to that in mice treated with control mAb. These experimental models lend themselves for investigating the mechanism(s) by which an anti-Id modulates antibody responses to a grass pollen allergen.

  1. Cells of the J774 macrophage cell line are primed for antibody-dependent cell-mediated cytotoxicity following exposure to γ-irradiation

    International Nuclear Information System (INIS)

    Duerst, R.; Werberig, K.

    1991-01-01

    Activation of macrophages (M phi) for host defense against tumor cells follows a sequence of priming events followed by an initiating stimulus that results in production and release of cytotoxic molecules that mediate target cell killing. The authors have developed a model to study specific macrophage cytotoxicity in vitro utilizing a cultured murine M phi cell line, J774. Specific cytotoxicity of cultured human gastrointestinal tumor cells is achieved in the presence of murine IgG2a monoclonal antibody (mAb) 17-1-A. The ability of these cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) is greatly enhanced following gamma-irradiation. ADCC can be demonstrated at mAb 17-1-A concentrations greater than or equal to 1 microgram/ml and effector/target cell ratios greater than or equal to 2. Exposure to doses greater than or equal to 10 Gy of gamma-irradiation increases ADCC threefold. Varying the duration from J774 M phi exposure to γ-irradiation until addition of antibody-coated target cells showed that the primed state for ADCC is stable for at least 8 days but approximately 24 hr is required for complete development of the primed state. mAb-dependent target cell death begins 8 hr after addition of mAb and labeled target cells to primed effector cells and is complete by 24 hr. Incubation of unirradiated J774 M phi effector cells with recombinant murine interferon-γ (rmIFN-γ) also results in enhanced ADCC, but the extent of target cell killing achieved is less than that following priming by γ-irradiation. Concomitant priming of γ-irradiated J774 M phi with rmIFN-γ increases the extent of ADCC. Further study of irradiated J774 cells may elucidate the molecular pathways utilized by M phi for achieving and maintaining the primed state for ADCC

  2. IgM but not IgG monoclonal anti-Nocardia brasiliensis antibodies confer protection against experimental actinomycetoma in BALB/c mice.

    Science.gov (United States)

    Gonzalez-Suarez, Maria L; Salinas-Carmona, Mario C; Pérez-Rivera, Isabel

    2009-10-01

    Nocardia brasiliensis is a facultative intracellular microorganism that produces a human chronic infection known as actinomycetoma. Human and mouse anti-N. brasiliensis antibody response identify P24, P26 and P61 immunodominant antigens. In this work, we generated immunoglobulin M (IgM) and IgG monoclonal antibodies (mAbs) specific to immunodominant P61 antigen. The monoclonal IgM (NbM1) and IgG2a (NbG1) antibodies were assessed for their in vitro bactericidal activity, in vivo protective effect and ability to block catalase activity. These mAbs specifically recognized P61, but they did not inhibit its enzyme activity. The in vitro bactericidal effect of NbG1 was higher than the killing ability of the IgM mAb. In vivo experiments with a murine model of experimental infection with N. brasiliensis injected into rear footpads was used to test the effect of NbM1 and NbG1. The negative untreated group developed a chronic actinomycetoma within 4 weeks. IgM mAbs conferred protection to BALB/c mice infected with N. brasiliensis. IgG mAb lacked this protective effect. IgM mAb showed a dose-response correlation between antibody concentration and lesion size. These results demonstrate that humoral immune response mediated by antigen-specific IgM antibody protects against an intracellular bacterial infection.

  3. Interleukin-2 treatment potentiates induction of oral tolerance in a murine model of autoimmunity.

    OpenAIRE

    Rizzo, L V; Miller-Rivero, N E; Chan, C C; Wiggert, B; Nussenblatt, R B; Caspi, R R

    1994-01-01

    The present study addresses the feasibility of potentiating oral tolerance by immunomanipulation, using the murine model of experimental autoimmune uveoretinitis (EAU) induced by immunization with the retinal antigen interphotoreceptor retinoid binding protein (IRBP). Three feedings of 0.2 mg IRBP every other day before immunization did not protect against EAU, whereas a similar regimen of five doses was protective. However, supplementing the nonprotective 3x regimen with as little as one inj...

  4. Murine Models of Gastric Corpus PreneoplasiaSummary

    Directory of Open Access Journals (Sweden)

    Christine P. Petersen

    2017-01-01

    Full Text Available Intestinal-type gastric adenocarcinoma evolves in a field of pre-existing metaplasia. Over the past 20 years, a number of murine models have been developed to address aspects of the physiology and pathophysiology of metaplasia induction. Although none of these models has achieved true recapitulation of the induction of adenocarcinoma, they have led to important insights into the factors that influence the induction and progression of metaplasia. Here, we review the pathologic definitions relevant to alterations in gastric corpus lineages and classification of metaplasia by specific lineage markers. In addition, we review present murine models of the induction and progression of spasmolytic polypeptide (TFF2–expressing metaplasia, the predominant metaplastic lineage observed in murine models. These models provide a basis for the development of a broader understanding of the physiological and pathophysiological roles of metaplasia in the stomach. Keywords: SPEM, Intestinal Metaplasia, Gastric Cancer, TFF2, Chief Cell, Hyperplasia

  5. Murine Typhus: An Important Consideration for the Nonspecific Febrile Illness

    Directory of Open Access Journals (Sweden)

    Gurjot Basra

    2012-01-01

    Full Text Available Murine typhus is a widely distributed flea-borne infection caused by Rickettsia typhi. Symptoms of murine typhus are nonspecific and mimic a variety of other infectious diseases. We herein report a case of murine typhus in an area where the broad use of DDT in the mid-20th century has now made it a rare disease. The patient described presented with headache, fever, and a faint macular rash. Initial laboratory studies revealed a slight transaminase elevation. Further questioning revealed exposure to opossums, prompting the consideration of murine typhus as a diagnosis. Although typhus group antibodies were not present during the patient’s acute illness, empiric therapy with doxycycline was initiated, and the patient defervesced. One month after convalescence, the patient returned to clinic with serum that contained typhus group antibodies with an IgG titer of 1 : 1024. Murine typhus is an important consideration during the workup of a patient with a nonspecific febrile illness. Exposure to reservoir hosts and the flea vector place humans at risk for this disease. Clinician recognition of this entity is required for diagnosis and effective therapy.

  6. Murine strain differences and the effects of zinc on cadmium concentrations in tissues after acute cadmium exposure

    Energy Technology Data Exchange (ETDEWEB)

    King, L.M. [ARS USDA, Germplasm and Gamete Physiology Lab., Beltsville, MD (United States); Anderson, M.B. [Dept. of Anatomy, Tulane Univ. School of Medicine, New Orleans, LA (United States); Sikka, S.C. [Dept. of Urology, Tulane Univ. School of Medicine, New Orleans, LA (United States); George, W.J. [Dept. of Pharmacology, Tulane Univ. School of Medicine, New Orleans, LA (United States)

    1998-10-01

    The role of strain differences in cadmium tissue distribution was studied using sensitive (129/J) and resistant (A/J) mice. These murine strains have previously been shown to differ in their susceptibility to cadmium-induced testicular toxicity. Cadmium concentration was measured in testis, epididymis, seminal vesicle, liver, and kidney at 24 h after cadmium chloride exposure (4, 10, and 20 {mu}mol/kg CdCl{sub 2}). The 129/J mice exhibited a significant increase in cadmium concentration in testis, epididymis, and seminal vesicle at all cadmium doses used, compared to A/J mice. However, cadmium concentrations in liver and kidney were not different between the strains, at any dose, indicating that cadmium uptake is similar in these organs at 24 h. These murine strains demonstrate similar hepatic and renal cadmium uptake but significantly different cadmium accumulation in the reproductive organs at 24 h. The mechanism of the protective effect of zinc on cadmium toxicity was studied by assessing the impact of zinc acetate (ZnAc) treatment on cadmium concentrations in 129/J mice after 24 h. Zinc pretreatment (250 {mu}mol/kg ZnAc), given 24 h prior to 20 {mu}mol/kg CdCl{sub 2} administration, significantly decreased the amount of cadmium in the testis, epididymis, and seminal vesicle of 129/J mice, and significantly increased the cadmium content of the liver after 24 h. Cadmium levels in the kidney were unaffected at this time. Zinc pretreatment also prevented the cadmium-induced decrease in testicular sperm concentration and epididymal sperm motility seen in 129/J mice. These findings suggest that the differences in the two murine strains may be attributed partly to the differential accumulation of cadmium in murine gonads. This may be caused by strain differences in the specificity of cadmium transport mechanisms. The protective role of zinc in cadmium-induced testicular toxicity in the sensitive strain may be due to an interference in the cadmium uptake by susceptible

  7. A novel inexpensive murine model of oral chronic digitalization.

    Science.gov (United States)

    Helber, Izo; Kanashiro, Rosemeire M; Alarcon, Ernesto A; Antonio, Ednei L; Tucci, Paulo J F

    2004-01-01

    A novel inexpensive murine model of oral administration of digitoxin (100 micro g/kg per day) added to routine chow is described. Serum digitoxin levels achieved after oral (n = 5; 116 +/- 14 ng/mL) and subcutaneous (n = 5; 124 +/- 11 ng/mL) administration were similar. A significant increase in the maximal left ventricular pressure rise of treated (n = 9) compared with control (n = 6) rats (dP/dt: 8956 +/- 233 vs 7980 +/- 234 mmHg/s, respectively; P = 0.01) characterized the positive inotropic action of digitoxin. In addition, no differences were observed in treated compared with control rats with regard to the electrocardiogram and systolic and diastolic left ventricular pressures.

  8. Cloning and characterization of murine fanconi anemia group A gene: Fanca protein is expressed in lymphoid tissues, testis, and ovary.

    Science.gov (United States)

    van de Vrugt, H J; Cheng, N C; de Vries, Y; Rooimans, M A; de Groot, J; Scheper, R J; Zhi, Y; Hoatlin, M E; Joenje, H; Arwert, F

    2000-04-01

    Fanconi anemia (FA) is an autosomal recessive disorder in humans characterized by bone marrow failure, cancer predisposition, and cellular hypersensitivity to cross-linking agents such as mitomycin C and diepoxybutane. FA genes display a caretaker function essential for maintenance of genomic integrity. We have cloned the murine homolog of FANCA, the gene mutated in the major FA complementation group (FA-A). The full-length mouse Fanca cDNA consists of 4503 bp and encodes a protein with a predicted molecular weight of 161 kDa. The deduced Fanca mouse protein shares 81% amino acid sequence similarity and 66% identity with the human protein. The nuclear localization signal and partial leucine zipper consensus motifs found in the human FANCA protein were also present in the murine homolog. In spite of the species difference, the murine Fanca cDNA was capable of correcting the cross-linker sensitive phenotype of human FA-A cells, suggesting functional conservation. Based on Northern as well as Western blots, Fanca was mainly expressed in lymphoid tissues, testis, and ovary. This expression pattern correlates with some of the clinical symptoms observed in FA patients. The availability of the murine Fanca cDNA now allows the gene to be studied in experimental mouse models.

  9. Influence of the 3D-conformation, glycan component and microheterogeneity on the epitope structure of Ole e 1, the major olive allergen - Use of recombinant isoforms and specific monoclonal antibodies as immunological tools

    NARCIS (Netherlands)

    González, Eva M.; Villalba, Mayte; Lombardero, Manuel; Aalbers, Marja; van Ree, Ronald; Rodríguez, Rosalía

    2002-01-01

    Ole e 1 is the main allergen of olive pollen, which is a major cause of pollinosis in countries of the Mediterranean area. Nine Ole e 1-specific murine monoclonal antibodies (mAbs), as well as two Ole e 1-isoforms and two Ole e 1-like allergens from lilac and privet, all of them obtained in Pichia

  10. Experimental and clinical analysis of the characteristics of a chimeric monoclonal antibody, MOv18, reactive with an ovarian cancer-associated antigen

    NARCIS (Netherlands)

    Molthoff, C. F.; Buist, M. R.; Kenemans, P.; Pinedo, H. M.; Boven, E.

    1992-01-01

    Monoclonal antibody (Mab) MOv18 preferentially reacts with gynecological carcinomas. We have analyzed the characteristics of murine MOv18 (m-MOv18) and chimeric MOv18 (c-MOv18). We found no differences in affinity and binding to IGROV1 cells between c-MOv18 as IgG and F(ab')2 fragments and m-MOv18.

  11. Cultivation of murine bone marrow macrophages in sponges: a method that permits recovery of viable cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Akporiaye, E T; Stewart, S; Stewart, C C

    1984-01-01

    Various investigators have cultured murine bone marrow or peritoneal cells in vitro on glass or plastic surfaces with the ultimate aim of retrieving adherent macrophages for morphologic and functional evaluation. The removal of these adherent macrophages by conventional techniques has been consistently accompanied by low yield and significant cell damage. The authors report here a simple technique for culturing murine bone marrow cells in gelatin sponges (Spongostan and Gelfoam) in growth medium containing 10% fetal bovine serum and 10% L-cell conditioned medium. Viable cells were retrieved from the sponges in 10 min by digestion with collagenase. The in situ growth kinetics were similar to those found for cells cultured on plastic dishes. The recovered cells were adherent, phagocytic, positive for Fc ..gamma.. receptors, and had esterase activity. 23 references, 1 figure, 1 table.

  12. Production of human anti-HLA monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Walker, M.C.; Mercier, F.; Roger, J.; Varin, M.

    1986-03-01

    Only 40% of the several hundred anti-HLA murine monoclonal antibodies (MAbs) that have been made detect HLA-A,B,C or DR specificities previously defined by human alloantisera, the range of recognized specificities is very narrow, and few of the MAbs have proven useful as tissue typing reagents. In hopes of obtaining HLA typing reagents, the authors are developing a protocol for the production of human anti-HLA MAbs from HLA-antigen (Ag) immunized peripheral blood B cells of volunteering renal patients, immunized to one or more HLA Ags through therapeutic blood transfusions. A simple enrichment of the donor B cells has not been sufficient for anti-HLA MAb production, the authors are currently delineating the conditions necessary for increasing the number of HLA-specific donor B cells by in vitro stimulation with cells expressing the HLA Ag to which the B cell donor is immunized. For the production of MAbs, the stimulated B cells are transformed with Epstein-Barr virus and subsequently fused with KR-4 lymphoblastoid cells. Hybridomas are selected by HAT and Ouabain. Supernatants are screened for anti-HLA activity against lymphocyte targets expressing the original immunizing HLA Ag by complement mediated /sup 51/Cr release assay. Antibody specificity is determined by the complement-dependent microcytotoxicity test used for HLA typing.

  13. Generation of Monoclonal Antibodies against Immunoglobulin Proteins of the Domestic Ferret (Mustela putorius furo)

    Science.gov (United States)

    2017-01-01

    The domestic ferret (Mustela putorius furo) serves as an animal model for the study of several viruses that cause human disease, most notably influenza. Despite the importance of this animal model, characterization of the immune response by flow cytometry (FCM) is severely hampered due to the limited number of commercially available reagents. To begin to address this unmet need and to facilitate more in-depth study of ferret B cells including the identification of antibody-secreting cells, eight unique murine monoclonal antibodies (mAb) with specificity for ferret immunoglobulin (Ig) were generated using conventional B cell hybridoma technology. These mAb were screened for reactivity against ferret peripheral blood mononuclear cells by FCM and demonstrate specificity for CD79β+ B cells. Several of these mAb are specific for the light chain of surface B cell receptor (BCR) and enable segregation of kappa and lambda B cells. Additionally, a mAb that yielded surface staining of nearly all surface BCR positive cells (i.e., pan ferret Ig) was generated. Collectively, these MαF-Ig mAb offer advancement compared to the existing portfolio of polyclonal anti-ferret Ig detection reagents and should be applicable to a wide array of immunologic assays including the identification of antibody-secreting cells by FCM. PMID:28286781

  14. Vitrification by Ultra-fast Cooling at a Low Concentration of Cryoprotectants in a Quartz Microcapillary: A Study Using Murine Embryonic Stem Cells

    Science.gov (United States)

    He, Xiaoming; Park, Eric Y.H.; Fowler, Alex; Yarmush, Martin L.; Toner, Mehmet

    2009-01-01

    Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz microcapillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1–2 M). The cryoprotectants used included 2 M 1,2-propanediol (PROH, cell membrane permeable) and 0.5 M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2 M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells. PMID:18462712

  15. Topical Apigenin Alleviates Cutaneous Inflammation in Murine Models

    Directory of Open Access Journals (Sweden)

    Mao-Qiang Man

    2012-01-01

    Full Text Available Herbal medicines have been used in preventing and treating skin disorders for centuries. It has been demonstrated that systemic administration of chrysanthemum extract exhibits anti-inflammatory properties. However, whether topical applications of apigenin, a constituent of chrysanthemum extract, influence cutaneous inflammation is still unclear. In the present study, we first tested whether topical applications of apigenin alleviate cutaneous inflammation in murine models of acute dermatitis. The murine models of acute allergic contact dermatitis and acute irritant contact dermatitis were established by topical application of oxazolone and phorbol 12-myristate 13-acetate (TPA, respectively. Inflammation was assessed in both dermatitis models by measuring ear thickness. Additionally, the effect of apigenin on stratum corneum function in a murine subacute allergic contact dermatitis model was assessed with an MPA5 physiology monitor. Our results demonstrate that topical applications of apigenin exhibit therapeutic effects in both acute irritant contact dermatitis and allergic contact dermatitis models. Moreover, in comparison with the vehicle treatment, topical apigenin treatment significantly reduced transepidermal water loss, lowered skin surface pH, and increased stratum corneum hydration in a subacute murine allergic contact dermatitis model. Together, these results suggest that topical application of apigenin could provide an alternative regimen for the treatment of dermatitis.

  16. Chest radiographic findings of tsutsugamushi disease and murine typhus in Chunchon

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Heung Chul; Han, Tae Giun; Jang, Won Ho; Hwang, Woo Chul; Park, Man Soo; Lee, Myoung Gu; Kim, Yoon Won [School of Medicine, Hallym University, Chuncheon (Korea, Republic of); Park, Choong Ki [College of Medicine, Hanyang University, Guri (Korea, Republic of)

    1995-06-15

    To evaluate the chest radiographic findings of rickettsial disease including murine typhus and tsutsugamushi disease in Chunchon. Chest radiographic films of 81 cases diagnosed as rickettsial disease(55 cases of tsutsugamushi disease, 26 cases of murine typhus) by immunofluorescence test were retrospectively analyzed. Main serotypes of Rickettsia tsutsugamushi were Gilliam and Karp. Incidence rate of tsutsugamushi disease was 2.1 times greater than that of murine typhus. Chest radiographs were abnormal in 63.6% of tsutsugamushi disease, and in 30.8% of murine typhus. Radiographic findings were Kerly's B line, reticulonodular densities, hilar enlargement, pleural effusion, and splenomegaly in both entities, but pulmonary consolidation was only found in tsutsugamushi disease. The patients with the abnormal radiographic findings were statistically well correlated with cardiomegaly ({rho} < 0.01) and azygos engorgement ({rho} < 0.05), as compared to the patients with normal radiographic findings. Radiographic findings of both murine typhus and tsutsugamushi disease were interstitial pattern. But the chest radiographs in patients with tsutsugamushi disease showed more severe pattern with higher rate of abnormality.

  17. Chest radiographic findings of tsutsugamushi disease and murine typhus in Chunchon

    International Nuclear Information System (INIS)

    Kim, Heung Chul; Han, Tae Giun; Jang, Won Ho; Hwang, Woo Chul; Park, Man Soo; Lee, Myoung Gu; Kim, Yoon Won; Park, Choong Ki

    1995-01-01

    To evaluate the chest radiographic findings of rickettsial disease including murine typhus and tsutsugamushi disease in Chunchon. Chest radiographic films of 81 cases diagnosed as rickettsial disease(55 cases of tsutsugamushi disease, 26 cases of murine typhus) by immunofluorescence test were retrospectively analyzed. Main serotypes of Rickettsia tsutsugamushi were Gilliam and Karp. Incidence rate of tsutsugamushi disease was 2.1 times greater than that of murine typhus. Chest radiographs were abnormal in 63.6% of tsutsugamushi disease, and in 30.8% of murine typhus. Radiographic findings were Kerly's B line, reticulonodular densities, hilar enlargement, pleural effusion, and splenomegaly in both entities, but pulmonary consolidation was only found in tsutsugamushi disease. The patients with the abnormal radiographic findings were statistically well correlated with cardiomegaly (ρ < 0.01) and azygos engorgement (ρ < 0.05), as compared to the patients with normal radiographic findings. Radiographic findings of both murine typhus and tsutsugamushi disease were interstitial pattern. But the chest radiographs in patients with tsutsugamushi disease showed more severe pattern with higher rate of abnormality

  18. Chest radiographic findings of tsutsugamushi disease and murine typhus in Chunchon

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Heung Chul; Han, Tae Giun; Jang, Won Ho; Hwang, Woo Chul; Park, Man Soo; Lee, Myoung Gu; Kim, Yoon Won [School of Medicine, Hallym University, Chuncheon (Korea, Republic of); Park, Choong Ki [College of Medicine, Hanyang University, Guri (Korea, Republic of)

    1995-06-15

    To evaluate the chest radiographic findings of rickettsial disease including murine typhus and tsutsugamushi disease in Chunchon. Chest radiographic films of 81 cases diagnosed as rickettsial disease(55 cases of tsutsugamushi disease, 26 cases of murine typhus) by immunofluorescence test were retrospectively analyzed. Main serotypes of Rickettsia tsutsugamushi were Gilliam and Karp. Incidence rate of tsutsugamushi disease was 2.1 times greater than that of murine typhus. Chest radiographs were abnormal in 63.6% of tsutsugamushi disease, and in 30.8% of murine typhus. Radiographic findings were Kerly's B line, reticulonodular densities, hilar enlargement, pleural effusion, and splenomegaly in both entities, but pulmonary consolidation was only found in tsutsugamushi disease. The patients with the abnormal radiographic findings were statistically well correlated with cardiomegaly ({rho} < 0.01) and azygos engorgement ({rho} < 0.05), as compared to the patients with normal radiographic findings. Radiographic findings of both murine typhus and tsutsugamushi disease were interstitial pattern. But the chest radiographs in patients with tsutsugamushi disease showed more severe pattern with higher rate of abnormality.

  19. Analysis of cardiomyocyte movement in the developing murine heart

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Hisayuki [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Yuasa, Shinsuke, E-mail: yuasa@a8.keio.jp [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Tabata, Hidenori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Tohyama, Shugo; Seki, Tomohisa; Egashira, Toru; Hayashiji, Nozomi; Hattori, Fumiyuki; Kusumoto, Dai; Kunitomi, Akira; Takei, Makoto; Kashimura, Shin; Yozu, Gakuto; Shimojima, Masaya; Motoda, Chikaaki; Muraoka, Naoto [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Nakajima, Kazunori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Sakaue-Sawano, Asako; Miyawaki, Atsushi [Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Fukuda, Keiichi [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan)

    2015-09-04

    The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle. - Highlights: • We directly visualized cardiomyocyte movement inside the developing murine heart. • Cell cycle related genes were upregulated in the proliferating cardiomyocytes. • Time-lapse imaging revealed that proliferating murine cardiomyocytes stayed in place. • Murine ventricular cardiomyocytes proliferate on site during development.

  20. Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus

    International Nuclear Information System (INIS)

    Gromowski, Gregory D.; Barrett, Alan D.T.

    2007-01-01

    The surface of the mature dengue virus (DENV) particle consists of 90 envelope (E) protein dimers that mediate both receptor binding and fusion. The E protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3, of which ED3 contains the critical and dominant virus-specific neutralization sites. In this study the ED3 epitopes recognized by seven, murine, IgG1 DENV-2 type-specific, monoclonal antibodies (MAbs) were determined using site-directed mutagenesis of a recombinant DENV-2 ED3 (rED3) protein. A total of 41 single amino acid substitutions were introduced into the rED3 at 30 different surface accessible residues. The affinity of each MAb with the mutant rED3s was assessed by indirect ELISA and the results indicate that all seven MAbs recognize overlapping epitopes with residues K305 and P384 critical for binding. These residues are conserved among DENV-2 strains and cluster together on the upper lateral face of ED3. A linear relationship was observed between relative occupancy of ED3 on the virion by MAb and neutralization of the majority of virus infectivity (∼ 90%) for all seven MAbs. Depending on the MAb, it is predicted that between 10% and 50% relative occupancy of ED3 on the virion is necessary for virus neutralization and for all seven MAbs occupancy levels approaching saturation were required for 100% neutralization of virus infectivity. Overall, the conserved antigenic site recognized by all seven MAbs is likely to be a dominant DENV-2 type-specific, neutralization determinant

  1. DMPD: The actions of bacterial DNA on murine macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10534106 The actions of bacterial DNA on murine macrophages. Sester DP, Stacey KJ, ... Show The actions of bacterial DNA on murine macrophages. PubmedID 10534106 Title The actions of bacterial DNA on murine macrophage

  2. A 44 bp intestine-specific hermaphrodite-specific enhancer from the C. elegans vit-2 vitellogenin gene is directly regulated by ELT-2, MAB-3, FKH-9 and DAF-16 and indirectly regulated by the germline, by daf-2/insulin signaling and by the TGF-β/Sma/Mab pathway.

    Science.gov (United States)

    Goszczynski, Barbara; Captan, Vasile V; Danielson, Alicia M; Lancaster, Brett R; McGhee, James D

    2016-05-01

    The Caenorhabditis elegans vitellogenin genes are transcribed in the intestine of adult hermaphrodites but not of males. A 44-bp region from the vit-2 gene promoter is able largely to reconstitute this tissue-, stage- and sex-specific-expression. This "enhancer" contains a binding site for the DM-domain factor MAB-3, the male-specific repressor of vitellogenesis, as well as an activator site that we show is the direct target of the intestinal GATA factor ELT-2. We further show that the enhancer is directly activated by the winged-helix/forkhead-factor FKH-9, (whose gene has been shown by others to be a direct target of DAF-16), by an unknown activator binding to the MAB-3 site, and by the full C. elegans TGF-β/Sma/Mab pathway acting within the intestine. The vit-2 gene has been shown by others to be repressed by the daf-2/daf-16 insulin signaling pathway, which so strongly influences aging and longevity in C. elegans. We show that the activity of the 44 bp vit-2 enhancer is abolished by loss of daf-2 but is restored by simultaneous loss of daf-16. DAF-2 acts from outside of the intestine but DAF-16 acts both from outside of the intestine and from within the intestine where it binds directly to the same non-canonical target site that interacts with FKH-9. Activity of the 44 bp vit-2 enhancer is also inhibited by loss of the germline, in a manner that is only weakly influenced by DAF-16 but that is strongly influenced by KRI-1, a key downstream effector in the pathway by which germline loss increases C. elegans lifespan. The complex behavior of this enhancer presumably allows vitellogenin gene transcription to adjust to demands of body size, germline proliferation and nutritional state but we suggest that the apparent involvement of this enhancer in aging and longevity "pathways" could be incidental. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Identification and characterization of a silencer regulatory element in the 3'-flanking region of the murine CD46 gene.

    Science.gov (United States)

    Nomura, M; Tsujimura, A; Begum, N A; Matsumoto, M; Wabiko, H; Toyoshima, K; Seya, T

    2000-01-01

    The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3'-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8 kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein-silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46. PMID:11023821

  4. Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins.

    Science.gov (United States)

    Higel, Fabian; Seidl, Andreas; Demelbauer, Uwe; Sörgel, Fritz; Frieß, Wolfgang

    2014-01-01

    N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.

  5. The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity.

    Directory of Open Access Journals (Sweden)

    Ann R Hunt

    2010-07-01

    Full Text Available Venezuelan equine encephalitis virus (VEEV is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion and E2 (binds receptor and elicits virus neutralizing antibodies. Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs. Six E2 epitopes (E2(c,d,e,f,g,h bound VEEV-neutralizing antibody and mapped to amino acids (aa 182-207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119. Using a 9 A resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration

  6. Preclinical Murine Models for Lung Cancer: Clinical Trial Applications

    Directory of Open Access Journals (Sweden)

    Amelia Kellar

    2015-01-01

    Full Text Available Murine models for the study of lung cancer have historically been the backbone of preliminary preclinical data to support early human clinical trials. However, the availability of multiple experimental systems leads to debate concerning which model, if any, is best suited for a particular therapeutic strategy. It is imperative that these models accurately predict clinical benefit of therapy. This review provides an overview of the current murine models used to study lung cancer and the advantages and limitations of each model, as well as a retrospective evaluation of the uses of each model with respect to accuracy in predicting clinical benefit of therapy. A better understanding of murine models and their uses, as well as their limitations may aid future research concerning the development and implementation of new targeted therapies and chemotherapeutic agents for lung cancer.

  7. L1-mediated retrotransposition of murine B1 and B2 SINEs recapitulated in cultured cells.

    Science.gov (United States)

    Dewannieux, Marie; Heidmann, Thierry

    2005-06-03

    SINEs are short interspersed nucleotide elements with transpositional activity, present at a high copy number (up to a million) in mammalian genomes. They are 80-400 bp long, non-coding sequences which derive either from the 7SL RNA (e.g. human Alus, murine B1s) or tRNA (e.g. murine B2s) polymerase III-driven genes. We have previously demonstrated that Alus very efficiently divert the enzymatic machinery of the autonomous L1 LINE (long interspersed nucleotide element) retrotransposons to transpose at a high rate. Here we show, using an ex vivo assay for transposition, that both B1 and B2 SINEs can be mobilized by murine LINEs, with the hallmarks of a bona fide retrotransposition process, including target site duplications of varying lengths and integrations into A-rich sequences. Despite different phylogenetic origins, transposition of the tRNA-derived B2 sequences is as efficient as that of the human Alus, whereas that of B1s is 20-100-fold lower despite a similar high copy number of these elements in the mouse genome. We provide evidence, via an appropriate nucleotide substitution within the B1 sequence in a domain essential for its intracellular targeting, that the current B1 SINEs are not optimal for transposition, a feature most probably selected for the host sake in the course of evolution.

  8. Biodistribution and pharmacokinetics of monoclonal antibody T1h and variant anti-CD6 murine 10D12 in healthy animals and in experimental arthritis model

    International Nuclear Information System (INIS)

    León, M; Hernández, I; Aldana, L; Ayra, F; Castro, Y; Leyva, R; García, L; Pérez, S.; Casaco, A.

    2016-01-01

    Biodistribution and pharmacokinetic of two radio labeled monoclonal antibodies was performed with the help of imaging techniques. Isotopic labeling was carried out by means of standardized methods. Pharmacokinetic evaluation was performed using the population approach and sparse data design. Introduction: Targeted therapy with monoclonal antibodies (MAb) is an efficient option for the treatment of rheumatoid arthritis. Th1 is a MAb anti human CD6 developed for the treatment of autoimmune disease and 10D12 is its counterpart anti murine CD6 developed as a pharmacological tool to get deep into the response mechanisms in animals models of rheumatoid arthritis.To investigate the behavior of both antibodies in the assay system, molecules were labeled with 125I to evaluate pharmacokinetic in healthy animals and with 99mTc to evaluate the antibody uptake in inflamed area of induced arthritis. Materials and methods: Antibodies were supplied by the Center of Molecular immunology. Iodination was performed by the iodogen method and technetium labeling was carried out directly by Schwarz method. Female C57BL6 from CENPALAB were used for experiments. Biodistribution and pharmacokinetic was performed by a sparse data design using the population approach. Uptake in region of inflammation was quantified by gammagraphy at the same time points of blood sampling. A compartmental model was build to quantify uptake kinetic. Pharmacokinetic profiles were analyzed using MONOLIX software version 4.2. Results: Minor pharmacokinetic differences were found between monoclonal antibodies labeled with 125I and 99mTc. As a humanized antibody, T1h shows a faster clearance than 10D12 and a biodistribution pattern reflecting preference for excretion mechanisms. The arthritis accumulation was not consistent with a targeted mediated uptake. On the other hand, radio labeled 10D12 shows an accumulation profile in arthritis with two peaks of maximum concentration representing an initial transit to

  9. Molecular characterization of two sub-family specific monoclonal antibodies to meningococcal Factor H binding protein

    Directory of Open Access Journals (Sweden)

    C. Lo Passo

    2018-04-01

    Full Text Available Factor H binding protein (FHbp is a component of two licensed vaccines for prevention of sepsis and meningitis caused by serogroup B meningococci. FHbp binds human Factor H (FH, which contributes to evasion of host immunity and FHbp sequence variants can be classified into two sub-families. Antibodies against FHbp elicit complement-mediated killing and can inhibit recruitment of FH to the bacterial surface. We report epitope mapping studies of two murine IgG mAbs, designated JAR 31 and JAR 36, isolated from a mouse immunized with FHbp in sub-family A, which is present in ∼30–40% of invasive isolates. In the present study, we tested the reactivity of mAbs JAR 31 and JAR 36 with seven natural FHbp sequence variants from different phylogenic groups. We screened bacteriophage-displayed peptide libraries to identify amino acid residues contributing to the JAR 36 epitope. Based on the reactivities of mAbs JAR 31 and JAR 36 with the seven FHbp variants, and the frequent occurrences of aspartate (D and lysine (K residues in the JAR 36-bound phage peptides, we selected six residues in the carboxyl-terminal region of FHbp for replacement with alanine (A. The D201A and K203A substitutions respectively eliminated and decreased binding of mAbs JAR 31 and JAR 36 to FHbp. These substitutions did not affect binding of the control mAb JAR 33 or of human FH. JAR 31 or JAR 36 mediated cooperative complement-mediated bactericidal activity with other anti-FHbp mAbs. The identification of two amino acid residues involved in the epitopes recognized by these anti-FHbp mAbs may contribute to a more complete understanding of the spatial requirements for cooperative anti-FHbp mAb bactericidal activity. Keywords: Biochemistry, Immunology, Microbiology, Molecular biology

  10. Engraftment versus immunosuppression: cost-benefit analysis of immunosuppression after intrahepatic murine islet transplantation.

    Science.gov (United States)

    Marzorati, Simona; Melzi, Raffaella; Citro, Antonio; Cantarelli, Elisa; Mercalli, Alessia; Scavini, Marina; Piemonti, Lorenzo

    2014-05-27

    Immunosuppression (IS) in islet transplantation (Tx) is a double-edged sword: it prevents immunoreaction but has the potential to impair islet engraftment. The aim of this study was to identify in murine animal models the IS platform with the best balance between these two opposite effects. To study the impact of IS on islet engraftment diabetic C57BL/6 mice were transplanted with 350 syngeneic islets through the portal vein and treated once-daily with either rapamycin (RAPA; 0.1-0.5-1 mg/kg ip), tacrolimus (FK506; 0.1-0.5-1 mg/kg ip), mycophenolate mofetil (MMF; 60-120-300 mg/kg oral) or vehicle for 14 days. Islet function was evaluated by measuring not-fasting glycemia and by performing an IVGTT on days 15 and 30 post-Tx. RAPA ≥0.5 mg/Kg, FK506 ≥0.5 mg/Kg, and MMF ≥120 mg/kg had detrimental effects on islet engraftment but not on the function of islets already engrafted in the liver. The effect on engraftment was irreversible and persisted even after IS withdrawal. The lower dose of IS that did not affect engraftment was tested for preventing rejection in the full mismatch allogeneic Tx BALB/c to C57BL/6 model. RAPA and/or FK506 were inefficient in preventing rejection, even when anti-IL2R mAb was added to the IS regimen. On the other hand, MMF alone or in association with FK506 significantly prolonged the time to islet rejection. IS showed profound dose-dependent deleterious effects on islet cell engraftment. The MMF/FK506 combination proved the best balance with less toxicity at the time of engraftment and more efficacy in controlling graft rejection.

  11. Adiponectin and plant-derived mammalian adiponectin homolog exert a protective effect in murine colitis

    KAUST Repository

    Arsenescu, Violeta

    2011-04-11

    Background: Hypoadiponectinemia has been associated with states of chronic inflammation in humans. Mesenteric fat hypertrophy and low adiponectin have been described in patients with Crohn\\'s disease. We investigated whether adiponectin and the plant-derived homolog, osmotin, are beneficial in a murine model of colitis. Methods: C57BL/6 mice were injected (i.v.) with an adenoviral construct encoding the full-length murine adiponectin gene (AN+DSS) or a reporter-LacZ (Ctr and V+DSS groups) prior to DSS colitis protocol. In another experiment, mice with DSS colitis received either osmotin (Osm+DSS) or saline (DSS) via osmotic pumps. Disease progression and severity were evaluated using body weight, stool consistency, rectal bleeding, colon lengths, and histology. In vitro experiments were carried out in bone marrow-derived dendritic cells. Results: Mice overexpressing adiponectin had lower expression of proinflammatory cytokines (TNF, IL-1β), adipokines (angiotensin, osteopontin), and cellular stress and apoptosis markers. These mice had higher levels of IL-10, alternative macrophage marker, arginase 1, and leukoprotease inhibitor. The plant adiponectin homolog osmotin similarly improved colitis outcome and induced robust IL-10 secretion. LPS induced a state of adiponectin resistance in dendritic cells that was reversed by treatment with PPARγ agonist and retinoic acid. Conclusion: Adiponectin exerted protective effects during murine DSS colitis. It had a broad activity that encompassed cytokines, chemotactic factors as well as processes that assure cell viability during stressful conditions. Reducing adiponectin resistance or using plant-derived adiponectin homologs may become therapeutic options in inflammatory bowel disease. © 2011 Springer Science+Business Media, LLC.

  12. Lipid peroxidation in microsomes of murine bone marrow after low-dose γ-irradiation

    International Nuclear Information System (INIS)

    Schwenke, K.; Coslar, S.; Muehlensiepen, H.; Altman, K.I.; Feinendegen, L.E.

    1994-01-01

    The principal aim of the study was to investigate the effect of low-dose γ-irradiation on lipid peroxidation (LPO) in murine bone marrow. To this end, the degree of LPO in suspensions of microsomes of murine bone marrow cells (BMC) was determined in terms of malondialdehyde (MDA) formation after whole-body or in vitro exposure to various doses of γ-radiation. These effects were compared to some extent with similar effects in liver and spleen preparations. As to the effect of γ-irradiation on LPO in BMC, the response depends on the dose level and on whether whole-body or in vitro exposures are involved. Whole-body irradiation did not result in an increase in LPO in BMC microsomes, even at such high doses as 15 Gy, although hepatic microsomes showed a marked increase. In contrast, in vitro irradiation of BMC microsomes with 0.1, 10 and 50 Gy brought about an increase in LPO. This increase was already significant (P < 0.05) at 0.1 Gy following a post-irradiation incubation and substantial at 50 Gy, even without subsequent incubation. The results show that low doses of γ-irradiation are able to induce an elevation of LPO in murine BMC microsomes, but only after in vitro irradiation. In the case of whole-body irradiation cellular radical scavengers and other metabolic reactions may prevent a measurable increase in LPO. This is partly illustrated by the case of vitamin-E deficiency, where a substantial increase in LPO in BMC microsomes is observed even without γ-irradiation in comparison with euvitaminotic mice because normally occurring radicals are not scavenged sufficiently. (orig.)

  13. Dosimetric evaluation of anti-CD20 labelled with 188Re

    International Nuclear Information System (INIS)

    Barrio, Graciela; Osso Junior, Joao A.

    2011-01-01

    Radioimmunotherapy has the potential to deliver lethal radiation energy directly to malignant cells via targeting of radioisotope-conjugated monoclonal antibodies (MAbs) to specific antigens. B-cell lymphoma is a particularly good candidate for radioimmunotherapy because the disease is inherently radiosensitive, malignant cells in the blood, bone marrow, spleen and lymphonodes are accessible, and MAbs have been developed to B-cell surface antigens that do not shed or modulate. Rituximab (RTX), the human IgG1-type chimeric form of the parent murine antibody ibritumomab, is specifically targeted against CD20, a surface antigen expressed by pre-B and mature human B lymphocytes. The use of rhenium-188 from a 188 W/ 188 Re generator system represents an attractive alternative radionuclide for therapy. 188 Re is produced from beta decay of the 188 W parent. In addition to the emission of high-energy electrons (Eβ= 2118 keV), 188 Re also decays with emission of a gamma photon with an energy of 155 keV in 15% abundance. Besides the therapeutic usefulness of 188 Re, the emission of gamma photon is an added advantage since the biodistribution of 188 Re-labeled antibodies can be evaluated in vivo with a gamma camera. Also, rhenium has chemical properties similar to technetium. Thus, both can be conjugated to antibodies using similar chemistry methods. The objective of this work is to prove the usefulness of this radiopharmaceutical based on dosimetric studies, that are also required by the Brazilian Regulatory Agency (ANVISA). (author)

  14. Effect of in vitro and in vivo UV irradiation on the production of ETAF activity by human and murine keratinocytes

    International Nuclear Information System (INIS)

    Ansel, J.C.; Luger, T.A.; Green, I.

    1983-01-01

    Cultured epidermal cells and keratinocytes produce a potent hormone-like factor called epidermal cell-derived thymocyte-activating factor (ETAF). ETAF appears to be similar if not identical to a monocyte-derived lymphokine, known as interleukin 1 (IL-1). These two cytokines are able to amplify a diverse number of proliferative and inflammatory processes. Several recent investigations have suggested that UV-induced immunosuppression may be due in part to the inhibition of IL-1/ETAF production by monocytes and keratinocytes, respectively. We therefore decided to directly study the effects of various doses of in vitro and in vivo UV radiation (UVR) on the production of ETAF by normal murine epidermal cells and a murine (Pam 212) and a human (SCC) keratinocyte cell line. Our results surprisingly demonstrated an increase in both the extracellular and the intracellular ETAF activity of the murine epidermal, Pam 212, and SCC after sublethal amounts of in vitro UVR. Likewise, increased ETAF activity of murine epidermal cells was detected after sublethal doses of in vivo UVR. The UV-induced ETAF activity was cycloheximide-sensitive, suggesting that de novo synthesis of ETAF rather than cell membrane leakage was responsible for the increased ETAF activity. The fact that UV irradiation can increase ETAF activity by keratinocytes could have important local and systemic consequences for the host and may provide an efficient, contaminant-free method for generating ETAF activity for further biochemical and immunologic studies

  15. Current Translational Research and Murine Models For Duchenne Muscular Dystrophy

    Science.gov (United States)

    Rodrigues, Merryl; Echigoya, Yusuke; Fukada, So-ichiro; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive muscle degeneration. Mutations in the DMD gene result in the absence of dystrophin, a protein required for muscle strength and stability. Currently, there is no cure for DMD. Since murine models are relatively easy to genetically manipulate, cost effective, and easily reproducible due to their short generation time, they have helped to elucidate the pathobiology of dystrophin deficiency and to assess therapies for treating DMD. Recently, several murine models have been developed by our group and others to be more representative of the human DMD mutation types and phenotypes. For instance, mdx mice on a DBA/2 genetic background, developed by Fukada et al., have lower regenerative capacity and exhibit very severe phenotype. Cmah-deficient mdx mice display an accelerated disease onset and severe cardiac phenotype due to differences in glycosylation between humans and mice. Other novel murine models include mdx52, which harbors a deletion mutation in exon 52, a hot spot region in humans, and dystrophin/utrophin double-deficient (dko), which displays a severe dystrophic phenotype due the absence of utrophin, a dystrophin homolog. This paper reviews the pathological manifestations and recent therapeutic developments in murine models of DMD such as standard mdx (C57BL/10), mdx on C57BL/6 background (C57BL/6-mdx), mdx52, dystrophin/utrophin double-deficient (dko), mdxβgeo, Dmd-null, humanized DMD (hDMD), mdx on DBA/2 background (DBA/2-mdx), Cmah-mdx, and mdx/mTRKO murine models. PMID:27854202

  16. Uptake of radiolabeled anti-CEA antibodies in human colorectal primary tumors as a function of tumor mass

    International Nuclear Information System (INIS)

    Williams, L.E.; Bares, R.B.; Buell, U.; Fass, J.; Schumpelick, V.; Hauptmann, S.

    1993-01-01

    An inverse correlation has been demonstrated between tumor uptake (u, in units of % injected dose/kg) of monoclonal antibody (Mab) and tumor mass (m, in units of g) for colorectal carcinoma in a series of 19 consecutive patients. The correlation (ρ=-0.510), developed using surgical samples was of the form u=ab b and was significant at the 2% level of confidence. All tumors were positive for carcinoembryonic antigen (CEA) and the radiopharmaceutical was in iodine-131 labeled anti-CEA Mab. Such correlations have been predicted earlier from murine and rat tumor uptake data. The slope parameter (b) was -0.362, a number consistent with the previous value (-0.382) found in anti-CEA experiments in mice bearing human xenograft LS174T tumors. (orig.)

  17. Serial passaging of Candida albicans in systemic murine infection suggests that the wild type strain SC5314 is well adapted to the murine kidney.

    Directory of Open Access Journals (Sweden)

    Anja Lüttich

    Full Text Available The opportunistic fungal pathogen Candida albicans has a remarkable ability to adapt to unfavorable environments by different mechanisms, including microevolution. For example, a previous study has shown that passaging through the murine spleen can cause new phenotypic characteristics. Since the murine kidney is the main target organ in murine Candida sepsis and infection of the spleen differs from the kidney in several aspects, we tested whether C. albicans SC5314 could evolve to further adapt to infection and persistence within the kidney. Therefore, we performed a long-term serial passage experiment through the murine kidney of using a low infectious dose. We found that the overall virulence of the commonly used wild type strain SC5314 did not change after eight passages and that the isolated pools showed only very moderate changes of phenotypic traits on the population level. Nevertheless, the last passage showed a higher phenotypic variability and a few individual strains exhibited phenotypic alterations suggesting that microevolution has occurred. However, the majority of the tested single strains were phenotypically indistinguishable from SC5314. Thus, our findings indicate that characteristics of SC5314 which are important to establish and maintain kidney infection over a prolonged time are already well developed.

  18. Differential chemokine responses in the murine brain following lyssavirus infection.

    Science.gov (United States)

    Hicks, D J; Núñez, A; Banyard, A C; Williams, A; Ortiz-Pelaez, A; Fooks, A R; Johnson, N

    2013-11-01

    The hallmark of lyssavirus infection is lethal encephalomyelitis. Previous studies have reported distinct lyssavirus isolate-related differences in severity of cellular recruitment into the encephalon in a murine model of infection following peripheral inoculation with rabies virus (RABV) and European bat lyssavirus (EBLV)-1 and -2. In order to understand the role of chemokines in this process, comparative studies of the chemokine pattern, distribution and production in response to infection with these lyssaviruses were undertaken. Expression of CCL2, CCL5 and CXCL10 was observed throughout the murine brain with a distinct caudal bias in distribution, similar to both inflammatory changes and virus antigen distribution. CCL2 immunolabelling was localized to neuronal and astroglial populations. CCL5 immunolabelling was only detected in the astroglia, while CXCL10 labelling, although present in the astroglia, was more prominent in neurons. Isolate-dependent differences in the amount of chemokine immunolabelling in specific brain regions and chemokine production by neurons in vitro were observed, with a greater expression of CCL5 in vivo and CXCL10 production in vitro after EBLV infection. Additionally, strong positive associations between chemokine immunolabelling and perivascular cuffing and, to a lesser extent, virus antigen score were also observed. These differences in chemokine expression may explain the variation in severity of encephalitic changes observed in animals infected with different lyssavirus isolates. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  19. Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins.

    Science.gov (United States)

    Liu, Liming

    2015-06-01

    Understanding the impact of glycosylation and keeping a close control on glycosylation of product candidates are required for both novel and biosimilar monoclonal antibodies (mAbs) and Fc-fusion protein development to ensure proper safety and efficacy profiles. Most therapeutic mAbs are of IgG class and contain a glycosylation site in the Fc region at amino acid position 297 and, in some cases, in the Fab region. For Fc-fusion proteins, glycosylation also frequently occurs in the fusion partners. Depending on the expression host, glycosylation patterns in mAb or Fc-fusions can be significantly different, thus significantly impacting the pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs. Glycans that have a major impact on PK and PD of mAb or Fc-fusion proteins include mannose, sialic acids, fucose (Fuc), and galactose (Gal). Mannosylated glycans can impact the PK of the molecule, leading to reduced exposure and potentially lower efficacy. The level of sialic acid, N-acetylneuraminic acid (NANA), can also have a significant impact on the PK of Fc-fusion molecules. Core Fuc in the glycan structure reduces IgG antibody binding to IgG Fc receptor IIIa relative to IgG lacking Fuc, resulting in decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activities. Glycoengineered Chinese hamster ovary (CHO) expression systems can produce afucosylated mAbs that have increased ADCC activities. Terminal Gal in a mAb is important in the complement-dependent cytotoxicity (CDC) in that lower levels of Gal reduce CDC activity. Glycans can also have impacts on the safety of mAb. mAbs produced in murine myeloma cells such as NS0 and SP2/0 contain glycans such as Galα1-3Galβ1-4N-acetylglucosamine-R and N-glycolylneuraminic acid (NGNA) that are not naturally present in humans and can be immunogenic when used as therapeutics. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  20. Elucidating the weak protein-protein interaction mechanisms behind the liquid-liquid phase separation of a mAb solution by different types of additives.

    Science.gov (United States)

    Wu, Guoliang; Wang, Shujing; Tian, Zhou; Zhang, Ning; Sheng, Han; Dai, Weiguo; Qian, Feng

    2017-11-01

    Liquid-liquid phase separation (LLPS) has long been observed during the physical stability investigation of therapeutic protein formulations. The buffer conditions and the presence of various excipients are thought to play important roles in the formulation development of monoclonal antibodies (mAbs). In this study, the effects of several small-molecule excipients (histidine, alanine, glycine, sodium phosphate, sodium chloride, sorbitol and sucrose) with diverse physical-chemical properties on LLPS of a model IgG1 (JM2) solutions were investigated by multiple techniques, including UV-vis spectroscopy, circular dichroism, differential scanning calorimetry/fluorimetry, size exclusion chromatography and dynamic light scattering. The LLPS of JM2 was confirmed to be a thermodynamic equilibrium process with no structural changes or irreversible aggregation of proteins. Phase diagrams of various JM2 formulations were constructed, suggesting that the phase behavior of JM2 was dependent on the solution pH, ionic strength and the presence of other excipients such as glycine, alanine, sorbitol and sucrose. Furthermore, we demonstrated that for this mAb, the interaction parameter (k D ) determined at low protein concentration appeared to be a good predictor for the occurrence of LLPS at high concentration. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

    Science.gov (United States)

    Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

    1992-05-15

    A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

  2. Serological and molecular characterization of Syrian Tomato spotted wilt virus isolates

    Directory of Open Access Journals (Sweden)

    Faiz ISMAEIL

    2015-04-01

    Full Text Available Thirty four Syrian isolates of Tomato spotted wilt virus (TSWV collected from tomato and pepper were tested against five specific monoclonal antibodies using TAS-ELISA. The isolates were in two serogroups. Fourteen tomato and sixteen pepper isolates were similar in their reaction with MAb-2, MAb-4, MAb-5 and MAb-6, but did not react with MAb-7 (Serogroup 1. Meanwhile, four isolates collected from pepper reacted with all the MAbs used (Serogroup 2. The expected 620 bp DNA fragment was obtained by RT-PCR from six samples using a specific primer pair designed to amplify the nucleocapsid protein (NP gene of TSWV. The PCR products were sequenced and a phylogenetic tree was constructed. Sequence analysis revealed that the Syrian TSWV isolates were very similar at the nucleotide (97.74 to 99.84% identity and amino acid (96.17 to 99.03% identity sequences levels. The phylogenetic tree showed high similarity of Syrian TSWV isolates with many other representative isolates from different countries.

  3. The murine Cd48 gene: allelic polymorphism in the IgV-like region.

    Science.gov (United States)

    Cabrero, J G; Freeman, G J; Reiser, H

    1998-12-01

    The murine CD48 molecule is a member of the immunoglobulin superfamily which regulates the activation of T lymphocytes. prior cloning experiments using mRNA from two different mouse strains had yielded discrepant sequences within the IgV-like domain of murine CD48. To resolve this issue, we have directly sequenced genomic DNA of 10 laboratory strains and two inbred strains of wild origin. The results of our analysis reveal an allelic polymorphism within the IgV-like domain of murine CD48.

  4. Environmentally determined differences in the murine lung microbiota and their relation to alveolar architecture.

    Directory of Open Access Journals (Sweden)

    Yeojun Yun

    Full Text Available Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect on tissue homeostasis and host defense. In contrast to the upper respiratory tract, the lower respiratory tract of healthy individuals has largely been considered free of microorganisms. To understand airway micro-ecology we studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non-specific-pathogen-free (SPF conditions as well as inbred mice maintained in non-SPF, SPF or germ-free (GF facilities. High-throughput pyrosequencing of reverse transcribed 16S rRNA revealed metabolically active murine lung microbiota in all but GF mice. The overall composition across samples was similar at the phylum and family level. However, species richness was significantly different between lung microbiota from SPF and non-SPF mice. Non-cultivatable Betaproteobacteria such as Ralstonia spp. made up the major constituents and were also confirmed by 16S rRNA gene cloning analysis. Additionally, Pasteurellaceae, Enterobacteria and Firmicutes were isolated from lungs of non-SPF mice. Bacterial communities were detectable by fluorescent in situ hybridization (FISH at alveolar epithelia in the absence of inflammation. Notably, higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae, which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. Our data indicate a common microbial composition of murine lungs, which is diversified through different environmental conditions and affects lung architecture. Identification of the microbiota of murine lungs will pave the path to study their influence on pulmonary immunity to infection and allergens using mouse models.

  5. Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes

    Directory of Open Access Journals (Sweden)

    Merry ZC Ruan

    2016-01-01

    Full Text Available Osteoarthritis (OA is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs. Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab. We show that a10mab-conjugated HDV (a10mabHDV-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4 into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting.

  6. Analysis of the capacity to produce IL-3 in murine AIDS

    DEFF Research Database (Denmark)

    Neuenschwander, A U; Marker, O; Thomsen, Allan Randrup

    1994-01-01

    Adult C57BL/6 mice infected with LP-BM5 murine leukaemia virus represent a model of murine AIDS (MAIDS). In this study we have analysed the capacity of CD4+ T cells from infected mice to produce IL-3 following stimulation with ConA for 24-72 h. In contrast to the position with IL-2, the production...

  7. Generation and characterization of monoclonal antibodies specific for 18 kDa antigen from Taenia solium cysticerci.

    Science.gov (United States)

    Zhang, Shaohua; Luo, Xuenong; Guo, Aijiang; Zhu, Xueliang; Cai, Xuepeng

    2016-07-01

    The gene encoding a mature 18 kDa glycoprotein of Taenia solium cysticerci (Ts18) was cloned and bacterially expressed with a His-tagged fusion protein. Monoclonal antibodies (MAbs) against the recombinant Ts18 antigen were generated in vitro by routine murine hybridoma technique of fusing splenocytes, from BALB/c mice immunized with the vesicular fluid of T. solium cysticerci (TsVF), with mouse myeloma cells (SP2/0). The reactivity and specificity of these MAbs were evaluated by indirect ELISA and immunoblotting techniques. Three stable hybridoma clones, namely 3B11, 6C5, and 6G4, were screened using His-Ts18-based ELISA, and these showed two IgG1 isotypes and one IgM isotype. All MAbs reacted with His-Ts18 at molecular weight (MW) 12.8 kDa and the native antigen at MW 18 kDa in TsVF and whole larval extracts (WLE). In a dot blotting test, MAbs 6C5 and 6G4 showed no obvious cross-reactivity with heterologous vesicular fluids from other taeniid species, including Taenia saginata (TsaVF), Taenia pisiformis (TpVF), Taenia hydatigena (ThVF), Taenia multiceps (TmVF), and Echinococcus granulosus (EgVF). Immunofluorescent assays showed that MAb 6C5 specifically reacted with the Ts18 expressed from pEGFP-N1-Ts18-transfected HeLa cells. Immunolocalization analysis, using MAb 6C5 as a probe, indicated that Ts18 was present at high concentrations in the region of the larval sucker and spiral canal. The results indicate that the Ts18 protein is an abundantly secreted parasite protein and MAbs against it might provide a step forward for improving the diagnosis of porcine cysticercosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools.

    Science.gov (United States)

    Nováková, Zora; Foss, Catherine A; Copeland, Benjamin T; Morath, Volker; Baranová, Petra; Havlínová, Barbora; Skerra, Arne; Pomper, Martin G; Barinka, Cyril

    2017-05-01

    Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. Murine models of H. pylori-induced gastritis and gastric adenocarcinoma.

    Science.gov (United States)

    Krueger, Sabine; Roessner, Albert; Kuester, Doerthe

    2011-10-15

    Laboratory mice have become one of the best animal species for mechanistic studies in gastrointestinal research. Their abundant genetic information, the way of causing carcinogenesis easily by transgenic and gene knockout techniques, limited effort in time and costs, and their practicability provide advantages over other animal models. Meanwhile, several murine practical models have been established for the investigation of the initiation, expansion, and progression of gastritis and gastric carcinoma, for assessing the effects of bacterial, genetic and environmental factors, and for evaluating therapeutic and preventive strategies in gastric diseases. This article gives a review of murine models of gastritis and gastric cancer, placing emphasis on the models associated with Helicobacter pylori infection and techniques used in our laboratory. We discuss matters of murine gastric anatomy, as well as techniques of infection, tissue preparation, and histology. Copyright © 2011 Elsevier GmbH. All rights reserved.

  10. Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies.

    Science.gov (United States)

    Tipton, Thomas R W; Roghanian, Ali; Oldham, Robert J; Carter, Matthew J; Cox, Kerry L; Mockridge, C Ian; French, Ruth R; Dahal, Lekh N; Duriez, Patrick J; Hargreaves, Philip G; Cragg, Mark S; Beers, Stephen A

    2015-03-19

    Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo. © 2015 by The American Society of Hematology.

  11. MEDI4893* Promotes Survival and Extends the Antibiotic Treatment Window in a Staphylococcus aureus Immunocompromised Pneumonia Model.

    Science.gov (United States)

    Hua, L; Cohen, T S; Shi, Y; Datta, V; Hilliard, J J; Tkaczyk, C; Suzich, J; Stover, C K; Sellman, B R

    2015-08-01

    Immunocompromised individuals are at increased risk of Staphylococcus aureus pneumonia. Neutralization of alpha-toxin (AT) with the monoclonal antibody (MAb) MEDI4893* protects normal mice from S. aureus pneumonia; however, the effects of the MAb in immunocompromised mice have not been reported. In this study, passive immunization with MEDI4893* increased survival rates and reduced bacterial numbers in the lungs in an immunocompromised murine S. aureus pneumonia model. Lungs from infected mice exhibited alveolar epithelial damage, protein leakage, and bacterial overgrowth, whereas lungs from mice passively immunized with MEDI4893* retained a healthy architecture, with an intact epithelial barrier. Adjunctive therapy or prophylaxis with a subtherapeutic MEDI4893* dose combined with subtherapeutic doses of vancomycin or linezolid improved survival rates, compared with the monotherapies. Furthermore, coadministration of MEDI4893* with vancomycin or linezolid extended the antibiotic treatment window. These data suggest that MAb-mediated neutralization of AT holds promise in strategies for prevention and adjunctive therapy among immunocompromised patients. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Contemplating the murine test tube: lessons from natural killer cells and Cryptococcus neoformans.

    Science.gov (United States)

    Marr, Kaleb J; Jones, Gareth J; Mody, Christopher H

    2006-06-01

    Murine experimentation has provided many useful tools, including the ability to knockout or over-express genes and to perform experiments that are limited by ethical considerations. Over the past century, mice have imparted valuable insights into the biology of many systems, including human immunity. However, although there are many similarities between the immune response of humans and mice, there are also many differences; none is more prominent than when examining natural killer cell biology. These differences include tissue distribution, effector molecules, receptor repertoire, and cytokine responses, all of which have important implications when extrapolating the studies to the human immune responses to Cryptococcus neoformans.

  13. Heterogeneity in the development of apoptosis in irradiated murine tumours of different histologies

    International Nuclear Information System (INIS)

    Meyn, R.E.; Stephens, L.C.; Ang, K.K.; Hunter, N.R.; Brock, W.A.; Milas, L.; Peters, L.J.

    1993-01-01

    Fifteen murine tumours were evaluated with respect to the degree of apoptosis development that occurs in the tumour tissue in the first few hours following irradiation in vivo. Animals were killed at 3 or 6 h following irradiation with 0, 2.5, 10 or 25 Gy. Apoptotis was scored as percent aberrant nuclei by microscopic examination of histological sections made from tumour specimens. Results showed that three of four mammary adenocarconomas, one ovarian adenocarcinoma, and one lymphoma displayed at least 10% apoptotic cells after 25 Gy, whereas five sarcomas, three squamous cell carcinomas, and a hepatocarcinoma did not. Time courses and dose responses were similar in those tumours that responded. (author)

  14. Heterogeneity in the development of apoptosis in irradiated murine tumours of different histologies

    Energy Technology Data Exchange (ETDEWEB)

    Meyn, R E; Stephens, L C; Ang, K K; Hunter, N R; Brock, W A; Milas, L; Peters, L J [Anderson (M.D.) Cancer Center, Houston, TX (United States)

    1993-11-01

    Fifteen murine tumours were evaluated with respect to the degree of apoptosis development that occurs in the tumour tissue in the first few hours following irradiation in vivo. Animals were killed at 3 or 6 h following irradiation with 0, 2.5, 10 or 25 Gy. Apoptotis was scored as percent aberrant nuclei by microscopic examination of histological sections made from tumour specimens. Results showed that three of four mammary adenocarconomas, one ovarian adenocarcinoma, and one lymphoma displayed at least 10% apoptotic cells after 25 Gy, whereas five sarcomas, three squamous cell carcinomas, and a hepatocarcinoma did not. Time courses and dose responses were similar in those tumours that responded. (author).

  15. Acute Febrile Illness and Complications Due to Murine Typhus, Texas, USA1,2.

    Science.gov (United States)

    Afzal, Zeeshan; Kallumadanda, Sunand; Wang, Feng; Hemmige, Vagish; Musher, Daniel

    2017-08-01

    Murine typhus occurs relatively commonly in southern Texas, as well as in California. We reviewed records of 90 adults and children in whom murine typhus was diagnosed during a 3-year period in 2 hospitals in southern Texas, USA. Most patients lacked notable comorbidities; all were immunocompetent. Initial signs and symptoms included fever (99%), malaise (82%), headache (77%), fatigue (70%), myalgias (68%), and rash (39%). Complications, often severe, in 28% of patients included bronchiolitis, pneumonia, meningitis, septic shock, cholecystitis, pancreatitis, myositis, and rhabdomyolysis; the last 3 are previously unreported in murine typhus. Low serum albumin and elevated procalcitonin, consistent with bacterial sepsis, were observed in >70% of cases. Rash was more common in children; thrombocytopenia, hyponatremia, elevated hepatic transaminases, and complications were more frequent in adults. Murine typhus should be considered as a diagnostic possibility in cases of acute febrile illness in southern and even in more northern US states.

  16. IL-10 down-regulates the expression of survival associated gene hspX of Mycobacterium tuberculosis in murine macrophage

    Directory of Open Access Journals (Sweden)

    Babban Jee

    2017-07-01

    Full Text Available Mycobacterium tuberculosis (MTB adopts a special survival strategy to overcome the killing mechanism(s of host immune system. Amongst the many known factors, small heat shock protein 16.3 (sHSP16.3 of MTB encoded by gene hspX has been reported to be critical for the survival of MTB. In the present study, the effect of recombinant murine interferon-gamma (rmIFN-γ and recombinant murine interleukin-10 (rmIL-10 on the expression of gene hspX of MTB in murine macrophage RAW264.7 has been investigated. By real-time RT-PCR, it was observed that three increasing concentrations (5, 25 and 50 ng/ml of rmIFN-γ significantly up-regulated the expression of hspX whereas similar concentrations of rmIL-10 (5, 25 and 50 ng/ml significantly down-regulated the hspX expression. This effect was not only dependent on the concentration of the stimulus but this was time-dependent as well. A contrasting pattern of hspX expression was observed against combinations of two different concentrations of rmIFN-γ and rmIL-10. The study results suggest that rIL-10 mediated down-regulation of hspX expression, in the presence of low concentration of rIFN-γ, could be used as an important strategy to decrease the dormancy of MTB in its host and thus making MTB susceptible to the standard anti-mycobacterial therapy used for treating tuberculosis. However, as these are only preliminary results in the murine cell line model, this hypothesis needs to be first validated in human cell lines and subsequently in animal models mimicking the latent infection using clinical isolates of MTB before considering the development of modified regimens for humans.

  17. Reemergence of Murine Typhus in the US

    Centers for Disease Control (CDC) Podcasts

    2015-04-21

    Dr. Lucas Blanton discusses the Reemergence of Murine Typhus in Galveston Texas in 2013.  Created: 4/21/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 4/27/2015.

  18. Three-dimensional in vivo imaging of the murine liver: a micro-computed tomography-based anatomical study.

    Directory of Open Access Journals (Sweden)

    Teresa Fiebig

    Full Text Available Various murine models are currently used to study acute and chronic pathological processes of the liver, and the efficacy of novel therapeutic regimens. The increasing availability of high-resolution small animal imaging modalities presents researchers with the opportunity to precisely identify and describe pathological processes of the liver. To meet the demands, the objective of this study was to provide a three-dimensional illustration of the macroscopic anatomical location of the murine liver lobes and hepatic vessels using small animal imaging modalities. We analysed micro-CT images of the murine liver by integrating additional information from the published literature to develop comprehensive illustrations of the macroscopic anatomical features of the murine liver and hepatic vasculature. As a result, we provide updated three-dimensional illustrations of the macroscopic anatomy of the murine liver and hepatic vessels using micro-CT. The information presented here provides researchers working in the field of experimental liver disease with a comprehensive, easily accessable overview of the macroscopic anatomy of the murine liver.

  19. Ly6G-mediated depletion of neutrophils is dependent on macrophages.

    Science.gov (United States)

    Bruhn, Kevin W; Dekitani, Ken; Nielsen, Travis B; Pantapalangkoor, Paul; Spellberg, Brad

    2016-01-01

    Antibody-mediated depletion of neutrophils is commonly used to study neutropenia. However, the mechanisms by which antibodies deplete neutrophils have not been well defined. We noticed that mice deficient in complement and macrophages had blunted neutrophil depletion in response to anti-Ly6G monoclonal antibody (MAb) treatment. In vitro, exposure of murine neutrophils to anti-Ly6G MAb in the presence of plasma did not result in significant depletion of cells, either in the presence or absence of complement. In vivo, anti-Ly6G-mediated neutrophil depletion was abrogated following macrophage depletion, but not complement depletion, indicating a requirement for macrophages to induce neutropenia by this method. These results inform the use and limitations of anti-Ly6G antibody as an experimental tool for depleting neutrophils in various immunological settings.

  20. The Role of Biosphere Reserves in Environmental Education and Training = Le Role des reserves de la biosphere dans l'education et la formation environnementales. Report of the Unesco/MAB Symposium Held During the Unesco/UNEP International Congress on Environmental Education and Training (Moscow, USSR, August 17-21, 1987). Report 20.

    Science.gov (United States)

    Francis, George, Ed.

    Environmental education and training have been key elements of Unesco's Program on Man and the Biosphere (MAB) since its inception in 1971. The MAB Program is an intergovernmental program of research, training, demonstration and distribution of information, aimed at providing the scientific background and the trained personnel to deal with…

  1. Monoclonal antibodies to hyphal exoantigens derived from the opportunistic pathogen Aspergillus terreus.

    Science.gov (United States)

    Nayak, Ajay P; Green, Brett J; Janotka, Erika; Hettick, Justin M; Friend, Sherri; Vesper, Steve J; Schmechel, Detlef; Beezhold, Donald H

    2011-09-01

    Aspergillus terreus has been difficult to identify in cases of aspergillosis, and clinical identification has been restricted to the broad identification of aspergillosis lesions in affected organs or the detection of fungal carbohydrates. As a result, there is a clinical need to identify species-specific biomarkers that can be used to detect invasive A. terreus disease. Monoclonal antibodies (MAbs) were developed to a partially purified preparation of cytolytic hyphal exoantigens (HEA) derived from A. terreus culture supernatant (CSN). Twenty-three IgG1 isotype murine MAbs were developed and tested for cross-reactivity against hyphal extracts of 54 fungal species. Sixteen MAbs were shown to be specific for A. terreus. HEA were detected in conidia, hyphae, and in CSN of A. terreus. HEA were expressed in high levels in the hyphae during early stages of A. terreus growth at 37°C, whereas at room temperature the expression of HEA peaked by days 4 to 5. Expression kinetics of HEA in CSN showed a lag, with peak levels at later time points at room temperature and 37°C than in hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA epitopes by MAb enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation and proteomic analysis demonstrated that MAbs 13E11 and 12C4 immunoprecipitated a putative uncharacterized leucine aminopeptidase (Q0CAZ7), while MAb 19B2 recognized a putative dipeptidyl-peptidase V (DPP5). Studies using confocal laser scanning microscopy showed that the uncharacterized leucine aminopeptidase mostly localized to extracellular matrix structures while dipeptidyl-peptidase V was mostly confined to the cytoplasm.

  2. Monoclonal antibodies and coupling reagents to cell membrane proteins for leukocyte labeling

    International Nuclear Information System (INIS)

    McAfee, J.G.; Gagne, G.; Subramanian, G.; Schneider, R.F.

    1984-01-01

    Current gamma-emitting agents for tagging leukocytes, In-111 oxine or tropolone, label all cell types indiscriminantly, and nuclear localization in lymphocytes results in radiation damage. Coupling reagents and murine monoclonal antibodies (Mab) specific for cell surface antigens of human leukocytes were tried as cell labeling agents to avoid nuclear localization. 10/sup 8/ mixed human leukocytes in Hepes buffer were added to tubes coated with 5 mg of dry cyclic dianhydride of DTPA for 15 minutes at room temperature. After washing, 0.1 ml of In-111 Cl in ACD (pH 6.8) was added. After 30 minutes, a cell labeling yield of 23% was obtained. Washing the cells in an elutriation centrifuge showed that this label was irreversible. Mab for cell surface antigens of human granulocytes were labeled with 300 μCi of I-125 using the Iodobead technic and unbound activity was removed by gel column chromatography. 1-10 μg were added to 10/sup 8/ mixed leukocytes in 0.5 ml plasma or saline for 1 hr. With Mab anti-leu M4 (clone G7 E11), an IgM, the cell labeling yield was 21%, irreversible, and specific for granulocytes. With anti-human leukocyte Mab NEI-042 (clone 9.4), and IgG2a, and anti-granulocyte Mab MAS-065 (clone FMCl1) an IgG1, the cell labeling was relatively unstable. Labeling of leukocyte subpopulations with Mab is feasible, and the binding of multivalent IgM is stronger than that of other immunoglobulins. DTPA cyclic anhydride is firmly bound to cell membranes, but the labeling is non-specific

  3. Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer.

    Directory of Open Access Journals (Sweden)

    Michael H Matho

    2015-09-01

    Full Text Available Vaccinia virus A33 is an extracellular enveloped virus (EEV-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.

  4. Establishment of novel mAb to human ERC/mesothelin useful for study and diagnosis of ERC/mesothelin-expressing cancers.

    Science.gov (United States)

    Ishikawa, Kiyoshi; Segawa, Tatsuya; Hagiwara, Yoshiaki; Maeda, Masahiro; Abe, Masaaki; Hino, Okio

    2009-03-01

    Malignant mesothelioma is a highly aggressive tumor of the serosal cavity that arises from the mesothelial cells of the pleura, peritoneum, or pericardium. The immunohistochemical diagnosis of epithelioid mesothelioma from biopsy or surgically resected specimens has been actively pursued, using markers such as mesothelin. Several markers have indeed been helpful for confirming the diagnosis of mesothelioma and distinguishing between mesothelioma and adenocarcinoma. The authors have developed a novel mAb to human C-ERC/mesothelin, which performed well when used in western blotting, fluorescence-activated cell sorting, immunocytochemistry and immunohistochemistry, and which therefore will be useful in studying the molecular biology of mesothelin, in addition to improving the diagnosis and therapy of mesothelin-expressing cancers.

  5. Expression of biologically active murine interleukin-18 in Lactococcus lactis.

    Science.gov (United States)

    Feizollahzadeh, Sadegh; Khanahmad, Hossein; Rahimmanesh, Ilnaz; Ganjalikhani-Hakemi, Mazdak; Andalib, Alireza; Sanei, Mohammad Hossein; Rezaei, Abbas

    2016-11-01

    The food-grade bacterium Lactococcus lactis is increasingly used for heterologous protein expression in therapeutic and industrial applications. The ability of L. lactis to secrete biologically active cytokines may be used for the generation of therapeutic cytokines. Interleukin (IL)-18 enhances the immune response, especially on mucosal surfaces, emphasizing its therapeutic potential. However, it is produced as an inactive precursor and has to be enzymatically cleaved for maturation. We genetically manipulated L. lactis to secrete murine IL-18. The mature murine IL-18 gene was inserted downstream of a nisin promoter in pNZ8149 plasmid and the construct was used to transform L. lactis NZ3900. The transformants were selected on Elliker agar and confirmed by restriction enzyme digestion and sequencing. The expression and secretion of IL-18 protein was verified by SDS-PAGE, western blotting and ELISA. The biological activity of recombinant IL-18 was determined by its ability to induce interferon (IFN)-γ production in L. lactis co-cultured with murine splenic T cells. The amounts of IL-18 in bacterial lysates and supernatants were 3-4 μg mL -1 and 0.6-0.7 ng mL -1 , respectively. The successfully generated L. lactis strain that expressed biologically active murine IL-18 can be used to evaluate the possible therapeutic effects of IL-18 on mucosal surfaces. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Murine typhus in two travelers returning from Bali, Indonesia: an underdiagnosed disease.

    Science.gov (United States)

    Takeshita, Nozomi; Imoto, Kazuya; Ando, Shuji; Yanagisawa, Kunio; Ohji, Goh; Kato, Yasuyuki; Sakata, Akiko; Hosokawa, Naoto; Kishimoto, Toshio

    2010-01-01

    Two Japanese travelers from Bali were diagnosed with murine typhus in Japan during the same period. Although one had only mild illness, the other experienced liver and kidney dysfunction. Murine typhus may be missed not only in endemic areas around the world, but also in travelers, especially those returning from marine resorts in these areas. © 2010 International Society of Travel Medicine.

  7. Dosimetric evaluation of anti-CD20 labelled with {sup 188}Re

    Energy Technology Data Exchange (ETDEWEB)

    Barrio, Graciela; Osso Junior, Joao A., E-mail: gracielabarrio@usp.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2011-07-01

    Radioimmunotherapy has the potential to deliver lethal radiation energy directly to malignant cells via targeting of radioisotope-conjugated monoclonal antibodies (MAbs) to specific antigens. B-cell lymphoma is a particularly good candidate for radioimmunotherapy because the disease is inherently radiosensitive, malignant cells in the blood, bone marrow, spleen and lymphonodes are accessible, and MAbs have been developed to B-cell surface antigens that do not shed or modulate. Rituximab (RTX), the human IgG1-type chimeric form of the parent murine antibody ibritumomab, is specifically targeted against CD20, a surface antigen expressed by pre-B and mature human B lymphocytes. The use of rhenium-188 from a {sup 188}W/{sup 188}Re generator system represents an attractive alternative radionuclide for therapy. {sup 188}Re is produced from beta decay of the {sup 188}W parent. In addition to the emission of high-energy electrons (E{beta}= 2118 keV), {sup 188}Re also decays with emission of a gamma photon with an energy of 155 keV in 15% abundance. Besides the therapeutic usefulness of {sup 188}Re, the emission of gamma photon is an added advantage since the biodistribution of {sup 188}Re-labeled antibodies can be evaluated in vivo with a gamma camera. Also, rhenium has chemical properties similar to technetium. Thus, both can be conjugated to antibodies using similar chemistry methods. The objective of this work is to prove the usefulness of this radiopharmaceutical based on dosimetric studies, that are also required by the Brazilian Regulatory Agency (ANVISA). (author)

  8. DMPD: Toll-like receptor 9 in murine lupus: more friend than foe! [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18241699 Toll-like receptor 9 in murine lupus: more friend than foe! Yu P, Musette ...us: more friend than foe! PubmedID 18241699 Title Toll-like receptor 9 in murine lupus...P, Peng SL. Immunobiology. 2008;213(2):151-7. Epub 2007 Sep 21. (.png) (.svg) (.html) (.csml) Show Toll-like receptor 9 in murine lup

  9. Murine Models of Sepsis and Trauma: Can We Bridge the Gap?

    Science.gov (United States)

    Stortz, Julie A; Raymond, Steven L; Mira, Juan C; Moldawer, Lyle L; Mohr, Alicia M; Efron, Philip A

    2017-07-01

    Sepsis and trauma are both leading causes of death in the United States and represent major public health challenges. Murine models have largely been used in sepsis and trauma research to better understand the pathophysiological changes that occur after an insult and to develop potential life-saving therapeutic agents. Mice are favorable subjects for this type of research given the variety of readily available strains including inbred, outbred, and transgenic strains. In addition, they are relatively easy to maintain and have a high fecundity. However, pharmacological therapies demonstrating promise in preclinical mouse models of sepsis and trauma often fail to demonstrate similar efficacy in human clinical trials, prompting considerable criticism surrounding the capacity of murine models to recapitulate complex human diseases like sepsis and traumatic injury. Fundamental differences between the two species include, but are not limited to, the divergence of the transcriptomic response, the mismatch of temporal response patterns, differences in both innate and adaptive immunity, and heterogeneity within the human population in comparison to the homogeneity of highly inbred mouse strains. Given the ongoing controversy, this narrative review aims to not only highlight the historical importance of the mouse as an animal research model but also highlight the current benefits and limitations of the model as it pertains to sepsis and trauma. Lastly, this review will propose future directions that may promote further use of the model. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  10. Biodistribution of 99mTc-labeled anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody h-R3 in a xenograft model of human lung adenocarcinoma

    International Nuclear Information System (INIS)

    Morales-Morales, Alejo; Duconge, Jorge; Caballero-Torres, Idania; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Iznaga-Escobar, Normando

    1999-01-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody (MAb) h-R3 is an (IgG 1 ), which binds to an extracellular domain of EGF-R. It was used to evaluate the biodistribution on nude mice xenografted with H-125 human lung adenocarcinoma cell line. Results were compared with its murine version of the MAb ior-egf/r3. Twenty-one athymic female 4NMRI nu/nu mice were injected intraperitoneally with 10 μg/100 μCi of 99m Tc-labeled MAbs. Immunoreactivity of 99m Tc-labeled MAbs were measured by enzyme-linked immunosorbent assay (ELISA) on H-125 cell line and the immunoreactive fractions was determined by the Lindmo method. Among all organs, significant accumulation was found in serum (27.05 ± 2.08 %ID/g) and tumor (3.903 ± 0.89 %ID/g) at 4 h after injection. These values decreased to 5.03 ± 0.50 %ID/g and 2.19 ± 0.56 %ID/g for serum and tumor, respectively. The immunoreactive fraction was found to be 0.70, with a correlation coefficient r=0.9984. With the good biodistribution and tumor uptake of the 99m Tc-labeled humanized antibody h-R3, a phase I diagnostic clinical trial of tumor with epithelial origin should be pursued

  11. Biodistribution of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody h-R3 in a xenograft model of human lung adenocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Morales-Morales, Alejo; Duconge, Jorge; Caballero-Torres, Idania; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Iznaga-Escobar, Normando E-mail: normando@ict.cim.sld.cu

    1999-04-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody (MAb) h-R3 is an (IgG{sub 1}), which binds to an extracellular domain of EGF-R. It was used to evaluate the biodistribution on nude mice xenografted with H-125 human lung adenocarcinoma cell line. Results were compared with its murine version of the MAb ior-egf/r3. Twenty-one athymic female 4NMRI nu/nu mice were injected intraperitoneally with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled MAbs. Immunoreactivity of {sup 99m}Tc-labeled MAbs were measured by enzyme-linked immunosorbent assay (ELISA) on H-125 cell line and the immunoreactive fractions was determined by the Lindmo method. Among all organs, significant accumulation was found in serum (27.05 {+-} 2.08 %ID/g) and tumor (3.903 {+-} 0.89 %ID/g) at 4 h after injection. These values decreased to 5.03 {+-} 0.50 %ID/g and 2.19 {+-} 0.56 %ID/g for serum and tumor, respectively. The immunoreactive fraction was found to be 0.70, with a correlation coefficient r=0.9984. With the good biodistribution and tumor uptake of the {sup 99m}Tc-labeled humanized antibody h-R3, a phase I diagnostic clinical trial of tumor with epithelial origin should be pursued.

  12. Biomechanical Properties of Murine Meniscus Surface via AFM-based Nanoindentation

    Science.gov (United States)

    Li, Qing; Doyran, Basak; Gamer, Laura W.; Lu, X. Lucas; Qin, Ling; Ortiz, Christine; Grodzinsky, Alan J.; Rosen, Vicki; Han, Lin

    2015-01-01

    This study aimed to quantify the biomechanical properties of murine meniscus surface. Atomic force microscopy (AFM)-based nanoindentation was performed on the central region, proximal side of menisci from 6- to 24-week old male C57BL/6 mice using microspherical tips (Rtip ≈ 5 μm) in PBS. A unique, linear correlation between indentation depth, D, and response force, F, was found on menisci from all age groups. This non-Hertzian behavior is likely due to the dominance of tensile resistance by the collagen fibril bundles on meniscus surface that are mostly aligned along the circumferential direction observed on 12-week old menisci. The indentation resistance was calculated as both the effective stiffness, Sind = dF/dD, and the effective modulus, Eind, via the isotropic Hertz model. Values of Sind and Eind were found to depend on indentation rate, suggesting the existence of poro-viscoelasticity. These values do not significantly vary with anatomical sites, lateral versus medial compartments, or mouse age. In addition, Eind of meniscus surface (e.g., 6.1 ± 0.8 MPa for 12 weeks of age, mean ± SEM, n = 13) was found to be significantly higher than those of meniscus surfaces in other species, and of murine articular cartilage surface (1.4 ± 0.1 MPa, n = 6). In summary, these results provided the first direct mechanical knowledge of murine knee meniscus tissues. We expect this understanding to serve as a mechanics-based benchmark for further probing the developmental biology and osteoarthritis symptoms of meniscus in various murine models. PMID:25817332

  13. Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide γ-chain-specific monoclonal antibody

    International Nuclear Information System (INIS)

    Ioannides, C.G.; Itoh, K.; Fox, F.E.; Pahwa, R.; Good, R.A.; Platsoucas, C.D.

    1987-01-01

    The authors developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the λ chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, they identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125 I-labeled denatured lysates of T3 + WT31 - lymphocytes expanded in culture from a SCID patient. Chemical crosslinking of 125 I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These experiments were done with polyclonal cell populations. Cloned T3 + WT31 - cell populations are required to determine whether the TCR contains two λ polypeptide chains. Using the same 9D7 anti-P18K mAb and immunoblotting analysis, they identified a 35 kDa γ-chain polypeptide under reducing conditions expressed on purified L3T4 - Lyt2 - BALB/c mouse thymocytes. This γ-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions

  14. Shared epitopes of glycoprotein A and protein 4.1 defined by antibody NaM10-3C10.

    Science.gov (United States)

    Rasamoelisolo, M; Czerwinski, M; Willem, C; Blanchard, D

    1998-06-01

    We have produced the murine monoclonal antibody (MAb) NaM70-3C10 (IgM) from splenocytes of mice immunized with human red blood cells (RBCs). The MAb agglutinated untreated as well as trypsin, chymotrypsin, neuraminidase, or ficin-treated RBCs from controls. In contrast, control RBCs treated with papaine or bromelaine were not agglutinated. On immunoblots, the MAb bound to glycophorin A (GPA) and to a 80 kDa protein identified as protein 4.1. Analysis by agglutination of variant RBCs carrying hybrid glycophorins made of the N-terminus (amino acids 1-58) of GPA and of the C-terminus (amino acids 27-72) of glycophorin B (GPB) and competition-inhibition test using purified GPA and a synthetic peptide corresponding to the amino acid sequence 48-58 of GPA demonstrated that the epitope is located within residues 48-58 of GPA. Epitope analysis with immobilized peptides showed that the MAb recognizes the sequence 53Pro-Pro-Glu-Glu-GIu58 of GPA. A homologous sequence is also present within amino acids 395 to 405 of protein 4.1. Finally, the MAb bound to 16 kDa chymotryptic peptide of protein 4.1, which carries the above amino acid sequence. In conclusion, it may be assumed that NaM70-3C10 specifically recognizes a common epitope on the extracellular domain of GPA and on the intracellular protein 4.1; this specificity explains the persistence of the 80 kDa band on blots when RBCs are treated with papain.

  15. Isolation of Blastomyces dermatitidis yeast from lung tissue during murine infection for in vivo transcriptional profiling.

    Science.gov (United States)

    Marty, Amber J; Wüthrich, Marcel; Carmen, John C; Sullivan, Thomas D; Klein, Bruce S; Cuomo, Christina A; Gauthier, Gregory M

    2013-07-01

    Blastomyces dermatitidis belongs to a group of thermally dimorphic fungi that grow as sporulating mold in the soil and convert to pathogenic yeast in the lung following inhalation of spores. Knowledge about the molecular events important for fungal adaptation and survival in the host remains limited. The development of high-throughput analytic tools such as RNA sequencing (RNA-Seq) has potential to provide novel insight on fungal pathogenesis especially if applied in vivo during infection. However, in vivo transcriptional profiling is hindered by the low abundance of fungal cells relative to mammalian tissue and difficulty in isolating fungal cells from the tissues they infect. For the purpose of obtaining B. dermatitidis RNA for in vivo transcriptional analysis by RNA-Seq, we developed a simple technique for isolating yeast from murine lung tissue. Using a two-step approach of filtration and centrifugation following lysis of murine lung cells, 91% of yeast cells causing infection were isolated from lung tissue. B. dermatitidis recovered from the lung yielded high-quality RNA with minimal murine contamination and was suitable for RNA-Seq. Approximately 87% of the sequencing reads obtained from the recovered yeast aligned with the B. dermatitidis genome. This was similar to 93% alignment for yeast grown in vitro. The use of near-freezing temperature along with short ex vivo time minimized transcriptional changes that would have otherwise occurred with higher temperature or longer processing time. In conclusion, we have developed a technique that recovers the majority of yeast causing pulmonary infection and yields high-quality fungal RNA with minimal contamination by mammalian RNA. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Multivariate modelling with 1H NMR of pleural effusion in murine cerebral malaria

    Directory of Open Access Journals (Sweden)

    Ghosh Soumita

    2011-11-01

    Full Text Available Abstract Background Cerebral malaria is a clinical manifestation of Plasmodium falciparum infection. Although brain damage is the predominant pathophysiological complication of cerebral malaria (CM, respiratory distress, acute lung injury, hydrothorax/pleural effusion are also observed in several cases. Immunological parameters have been assessed in pleural fluid in murine models; however there are no reports of characterization of metabolites present in pleural effusion. Methods 1H NMR of the sera and the pleural effusion of cerebral malaria infected mice were analyzed using principal component analysis, orthogonal partial least square analysis, multiway principal component analysis, and multivariate curve resolution. Results It has been observed that there was 100% occurrence of pleural effusion (PE in the mice affected with CM, as opposed to those are non-cerebral and succumbing to hyperparasitaemia (NCM/HP. An analysis of 1H NMR and SDS-PAGE profile of PE and serum samples of each of the CM mice exhibited a similar profile in terms of constituents. Multivariate analysis on these two classes of biofluids was performed and significant differences were detected in concentrations of metabolites. Glucose, creatine and glutamine contents were high in the PE and lipids being high in the sera. Multivariate curve resolution between sera and pleural effusion showed that changes in PE co-varied with that of serum in CM mice. The increase of glucose in PE is negatively correlated to the glucose in serum in CM as obtained from the result of multiway principal component analysis. Conclusions This study reports for the first time, the characterization of metabolites in pleural effusion formed during murine cerebral malaria. The study indicates that the origin of PE metabolites in murine CM may be the serum. The loss of the components like glucose, glutamine and creatine into the PE may worsen the situation of patients, in conjunction with the enhanced

  17. [Virulence of Sporothrix globosa in murine models].

    Science.gov (United States)

    Cruz Choappa, Rodrigo; Pérez Gaete, Salomón; Rodríguez Badilla, Valentina; Vieille Oyarzo, Peggy; Opazo Sanchez, Héctor

    The sporothricosis disease is an infection caused by species included in Sporothrix schenkii complex. Verify the virulence of a strain of S. globosa using two different concentrations of inoculum by intraperitoneally and subcutaneously, into a mouse model. Nonrandomized pilot study, in murine inoculated with a strain of S. globosa (CBS 14.076M) by intraperitoneally and subcutaneously with inoculum concentrations of 0.5 and 4 McFarland. For this purpose 18 rodents CF-1 (ISP, Santiago, Chile) were used. The studied strain did not induce illness or injury on animals, they all survived and neither the tissue culture nor the histopathological analysis showed fungal growth or suggestive infection by organ abnormalities. The S. globosa strain did not present any virulence enough to cause disease at 0.5 and 4.0 McFarland concentration inoculum when inoculated in both intraperitoneally and subcutaneously, in murine models. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  18. Enhanced detection and study of murine norovirus-1 using a more efficient microglial cell line

    Directory of Open Access Journals (Sweden)

    Lu Yuanan

    2009-11-01

    Full Text Available Abstract Background Human Noroviruses are the predominant cause of non-bacterial gastroenteritis worldwide. To facilitate prevention and control, a norovirus isolated from mice can provide a model to understand human noroviruses. To establish optimal viral infectivity conditions for murine noroviruses, several cell lines of hematopoietic lineage, including murine BV-2, RAW 264.7, and TIB, as well as human CHME-5, were tested comparatively for their sensitivity to murine norovirus-1. Results Except for CHME-5, all three murine-derived cell lines were susceptible to MNV infection. Viral infection of these cells was confirmed by RT-PCR. Using both viral plaque and replication assays, BV-2 and RAW 264.7 cells were determined to have comparable sensitivities to MNV-1 infection. Comparisons of cell growth characteristics, general laboratory handling and potential in-field applications suggest the use of BV-2 to be more advantageous. Conclusion Results obtained from these studies demonstrate that an immortalized microglial cell line can support MNV-1 replication and provides a more efficient method to detect and study murine noroviruses, facilitating future investigations using MNV-1 as a model to study, detect, and control Human Norovirus.

  19. Three-dimensional alginate spheroid culture system of murine osteosarcoma.

    Science.gov (United States)

    Akeda, Koji; Nishimura, Akinobu; Satonaka, Haruhiko; Shintani, Ken; Kusuzaki, Katsuyuki; Matsumine, Akihiko; Kasai, Yuichi; Masuda, Koichi; Uchida, Atsumasa

    2009-11-01

    Osteosarcoma (OS) is the most common primary malignant tumor of the bone and often forms pulmonary metastases, which are the most important prognostic factor. For further elucidation of the mechanism underlying the progression and metastasis of human OS, a culture system mimicking the microenvironment of the tumor in vivo is needed. We report a novel three-dimensional (3D) alginate spheroid culture system of murine osteosarcoma. Two different metastatic clones, the parental Dunn and its derivative line LM8, which has a higher metastatic potential to the lungs, were encapsulated in alginate beads to develop the 3D culture system. The beads containing murine OS cells were also transplanted into mice to determine their metastatic potential in vivo. In this culture system, murine OS cells encapsulated in alginate beads were able to grow in a 3D structure with cells detaching from the alginate environment. The number of detaching cells was higher in the LM8 cell line than the Dunn cell line. In the in vivo alginate bead transplantation model, the rate of pulmonary metastasis was higher with LM8 cells compared with that of Dunn cells. The cell characteristics and kinetics in this culture system closely reflect the original malignant potential of the cells in vivo.

  20. Lymphocyte targeting with /sup 111/In-labelled monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Loutfi, I.; Batchelor, J.R.; Lavender, J.P.; Epenetos, A.A.

    1988-01-01

    In vitro tests were conducted using human T and B cell lines, as well as whole blood, to establish the usefulness of 2 murine monoclonal antibodies (MAbs), an anti-CD5 (Pan T) and a Pan B, for potential radioimmunolocalization and therapy. Both MAbs showed specificity for the cell line in question as tested by indirect immunofluorescence and radioimmunoassay. Assays carried out on whole blood showed 40-70% of the added activity of /sup 111/In-labelled Pan B antibody binding to B cells and 20-24% of /sup 111/In-Pan T antibody binding to T cells. The amount of internalised /sup 111/In-labelled Pan B was 6% of total amount at 24 hr indicating a slow internalization process. These results should allow for in vivo targeting of normal and neoplastic B and T cells.

  1. Identification of factors for physicians to facilitate early differential diagnosis of scrub typhus, murine typhus, and Q fever from dengue fever in Taiwan.

    Science.gov (United States)

    Chang, Ko; Lee, Nan-Yao; Ko, Wen-Chien; Tsai, Jih-Jin; Lin, Wei-Ru; Chen, Tun-Chieh; Lu, Po-Liang; Chen, Yen-Hsu

    2017-02-01

    Dengue fever, rickettsial diseases, and Q fever are acute febrile illnesses with similar manifestations in tropical areas. Early differential diagnosis of scrub typhus, murine typhus, and Q fever from dengue fever may be made by understanding the distinguishing clinical characteristics and the significance of demographic and weather factors. We conducted a retrospective study to identify clinical, demographic, and meteorological characteristics of 454 dengue fever, 178 scrub typhus, 143 Q fever, and 81 murine typhus cases in three Taiwan hospitals. Case numbers of murine typhus and Q fever correlated significantly with temperature and rainfall; the scrub typhus case number was only significantly related with temperature. Neither temperature nor rainfall correlated with the case number of dengue fever. The rarity of dengue fever cases from January to June in Taiwan may be a helpful clue for diagnosis in the area. A male predominance was observed, as the male-to-female rate was 2.1 for murine typhus and 7.4 for Q fever. Multivariate analysis revealed the following six important factors for differentiating the rickettsial diseases and Q fever group from the dengue fever group: fever ≥8 days, alanine aminotransferase > aspartate aminotransferase, platelets >63,000/mL, C-reactive protein >31.9 mg/L, absence of bone pain, and absence of a bleeding syndrome. Understanding the rarity of dengue in the first half of a year in Taiwan and the six differentiating factors may help facilitate the early differential diagnosis of rickettsial diseases and Q fever from dengue fever, permitting early antibiotic treatment. Copyright © 2015. Published by Elsevier B.V.

  2. Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors.

    Science.gov (United States)

    Lobos-González, Lorena; Silva, Verónica; Araya, Mariela; Restovic, Franko; Echenique, Javiera; Oliveira-Cruz, Luciana; Fitzpatrick, Christopher; Briones, Macarena; Villegas, Jaime; Villota, Claudio; Vidaurre, Soledad; Borgna, Vincenzo; Socias, Miguel; Valenzuela, Sebastián; Lopez, Constanza; Socias, Teresa; Varas, Manuel; Díaz, Jorge; Burzio, Luis O; Burzio, Verónica A

    2016-09-06

    We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy.

  3. Magnetization transfer from macromolecules to water protons in murine dental tissues as revealed by 500 MHz 1H-NMR

    International Nuclear Information System (INIS)

    Nakamura, Koji; Era, Seiichi; Nagai, Naoki; Sogami, Masaru; Takasaki, Akihiko; Kato, Kazuo.

    1997-01-01

    Although much is known about magnetization transfer phenomena in biological soft tissues, little is known about those in hard tissues. Using a 500 MHz 1 H-NMR spectrometer, we studied the spin-lattice relaxation time (T 1 (H 2 O)) and the intermolecular cross-relaxation times (T IS (H 2 O)) from irradiated macromolecular protons to observed water protons in murine lower incisors (hard tissue) and compared with those in murine lens tissue (soft tissue). Mean values for the water content (%) of murine lower incisors and lens tissue were 16.02±2.39 (n=14) and 67.20±4.60 (n=15), respectively. These findings were consistent with the large different in water content between soft tissues and hard tissues. T IS (H 2 O) values obtained by f 2 -irradiation at 7.13 or -4.00 ppm showed no significant difference between lower incisors and lens tissue. Plots of 1/T IS (H 2 O) values vs. tissue dry weight (W(%)) for lower incisor tissue approximated a straight line with slope approximately equal for that obtained for lens tissue. These results suggest that the state of water in hard tissue may be similar to that in soft tissues, in spite of the significant difference in water content. Thus, saturation transfer NMR techniques such as measurement of T IS (H 2 O) values may be applicable to the study of water-macromolecule interactions in both biological soft and hard tissues. (author)

  4. Quantitation of Murine Stroma and Selective Purification of the Human Tumor Component of Patient-Derived Xenografts for Genomic Analysis.

    Directory of Open Access Journals (Sweden)

    Valentina E Schneeberger

    Full Text Available Patient-derived xenograft (PDX mouse models are increasingly used for preclinical therapeutic testing of human cancer. A limitation in molecular and genetic characterization of PDX tumors is the presence of integral murine stroma. This is particularly problematic for genomic sequencing of PDX models. Rapid and dependable approaches for quantitating stromal content and purifying the malignant human component of these tumors are needed. We used a recently developed technique exploiting species-specific polymerase chain reaction (PCR amplicon length (ssPAL differences to define the fractional composition of murine and human DNA, which was proportional to the fractional composition of cells in a series of lung cancer PDX lines. We compared four methods of human cancer cell isolation: fluorescence-activated cell sorting (FACS, an immunomagnetic mouse cell depletion (MCD approach, and two distinct EpCAM-based immunomagnetic positive selection methods. We further analyzed DNA extracted from the resulting enriched human cancer cells by targeted sequencing using a clinically validated multi-gene panel. Stromal content varied widely among tumors of similar histology, but appeared stable over multiple serial tumor passages of an individual model. FACS and MCD were superior to either positive selection approach, especially in cases of high stromal content, and consistently allowed high quality human-specific genomic profiling. ssPAL is a dependable approach to quantitation of murine stromal content, and MCD is a simple, efficient, and high yield approach to human cancer cell isolation for genomic analysis of PDX tumors.

  5. V-cbl, an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas

    International Nuclear Information System (INIS)

    Langdon, W.Y.; Klinken, S.P.; Hartley, J.W.; Morse, H.C. III; Ruscetti, S.K.

    1989-01-01

    Cas NS-1 is an acutely transforming murine retrovirus that induces pre-B and pro-B cell lymphomas. Molecular cloning showed it was generated from the ecotropic Cas-Br-M virus by sequential recombinations with endogenous retroviral sequences and a cellular oncogene. The oncogene sequence shows no homology with known oncogenes but some similarity to the yeast transcriptional activator GCN4. A 100-kDa gag-cbl fusion protein, with no detectable kinase activity, is responsible for the cellular transformation. The cellular homologue of v-cbl, present in mouse and human DNA, is expressed in a range of hemopoietic lineages

  6. Azithromycin prophylaxis and treatment of murine toxoplasmosis.

    Science.gov (United States)

    Tabbara, Khalid F; Hammouda, Ehab; Tawfik, Abdulkader; Al-Omar, Othman M; Abu El-Asrar, Ahmed M

    2005-03-01

    To evaluate the azithromycin effects alone and in combination with other agents in the prophylaxis and treatment of murine toxoplasmosis. A total of 280 BALB/c mice were included, and 2 x 103 Toxoplasma organisms of the RH strain Toxoplasma gondii strain ATCC50174 were given intraperitoneally to each mouse. In experiment one, 40 animals were given azithromycin 200 milligram/kilogram/daily for 3 days starting the day of inoculation, 40 mice were control. In experiment 2, the treatment was started 48 hours after inoculation and given daily for 3 days: one group received azithromycin 200 milligram/kilogram/day, the second group received pyrimethamine 25 milligram/kilogram/day, and the sulfadiazine 100 milligram/kilogram/day. The third group was control. In experiment 3, 7 groups of animals received one of the following (1) none, (2) azithromycin 200 milligram/kilogram/day, (3) pyrimethamine 25 milligram/kilogram/day and sulfadiazine 100 milligram/kilogram/day, (4) azithromycin and sulfadiazine, (5) azithromycin and pyrimethamine, (6) azithromycin with sulfadiazine and pyrimethamine, (7) sulfadiazine alone. Treatment was initiated 72 hours after inoculation for 3 days. The study was conducted at the Animal Care Facility of King Saud University, Riyadh, Kingdom of Saudi Arabia. Animals that received azithromycin simultaneously with inoculation survived, and all control animals died. All animals died in groups receiving single drug therapy. Animals treated with azithromycin and sulfadiazine showed a survival rate of 40%, sulfadiazine and pyrimethamine 40%, or azithromycin with sulfadiazine and pyrimethamine 95% (p<0.0001). Azithromycin alone was found to be effective in the prophylaxis of murine toxoplasmosis. Combination therapy was effective in the treatment of murine toxoplasmosis.

  7. Adenoviral vaccination combined with CD40 stimulation and CTLA-4 blockage can lead to complete tumor regression in a murine melanoma model

    DEFF Research Database (Denmark)

    Sørensen, Maria Rathmann; Holst, Peter J; Steffensen, Maria Abildgaard

    2010-01-01

    that the delay in tumor growth can be converted to complete regression and long-term survival in 30-40% of the mice by a booster vaccination plus combinational treatment with agonistic anti-CD40 monoclonal antibodies (mAb) and anti-CTLA-4 mAb. Regarding the mechanism underlying the improved clinical effect......, analysis of the tumor-specific response revealed a significantly prolonged tumor-specific CD8 T cell response in spleens of the mice receiving the combinational treatment compared with mice receiving either treatment individually. Matching this, CD8 T cell depletion completely prevented tumor control...

  8. The kunitz protease inhibitor domain of protease nexin-2 inhibits factor XIa and murine carotid artery and middle cerebral artery thrombosis

    Science.gov (United States)

    Wu, Wenman; Li, Hongbo; Navaneetham, Duraiswamy; Reichenbach, Zachary W.; Tuma, Ronald F.

    2012-01-01

    Coagulation factor XI (FXI) plays an important part in both venous and arterial thrombosis, rendering FXIa a potential target for the development of antithrombotic therapy. The kunitz protease inhibitor (KPI) domain of protease nexin-2 (PN2) is a potent, highly specific inhibitor of FXIa, suggesting its possible role in the inhibition of FXI-dependent thrombosis in vivo. Therefore, we examined the effect of PN2KPI on thrombosis in the murine carotid artery and the middle cerebral artery. Intravenous administration of PN2KPI prolonged the clotting time of both human and murine plasma, and PN2KPI inhibited FXIa activity in both human and murine plasma in vitro. The intravenous administration of PN2KPI into WT mice dramatically decreased the progress of FeCl3-induced thrombus formation in the carotid artery. After a similar initial rate of thrombus formation with and without PN2KPI treatment, the propagation of thrombus formation after 10 minutes and the amount of thrombus formed were significantly decreased in mice treated with PN2KPI injection compared with untreated mice. In the middle cerebral artery occlusion model, the volume and fraction of ischemic brain tissue were significantly decreased in PN2KPI-treated compared with untreated mice. Thus, inhibition of FXIa by PN2KPI is a promising approach to antithrombotic therapy. PMID:22674803

  9. A monoclonal antibody to inclusion body disease of cranes virus enabling specific immunohistochemistry and competitive ELISA

    Science.gov (United States)

    Letchworth, G.J.; Fishel, J.R.; Hansen, W.R.

    1997-01-01

    Inclusion body disease of cranes (IBDC) herpesvirus kills some infected cranes and persists in convalescent animals. To enable further study and rapid identification of carrier animals, we developed a monoclonal antibody (MAb) to IBDC virus and used it in immunohistochemistry and a competitive enzyme-linked immunosorbent assay (ELISA). We used conventional techniques to make murine MAbs directed against IBDC virus purified from infected duck embryo cells. Hybridomas reacting in an ELISA with IBDC virus but not uninfected duck embryo cells were characterized by radioimmunoprecipitation, in situ immunohistochemistry, and competitive ELISA with neutralizing and nonneutralizing crane sera. MAb 2C11 immunoprecipitated 59-, 61-, and 110-kD proteins from IBDC virus-infected but not uninfected cells and stained glutaraldehyde-fixed IBDC virus plaques but not surrounding uninfected duck embryo cells in vitro. Antibody 2C11 did not react with duck embryo cells infected with falcon herpesvirus, psittacine herpesvirus, infectious laryngotracheitis, pigeon herpesvirus, or duck plague virus. A competitive ELISA using antibody 2C11 identified most sera that were positive in the neutralization test. This antibody will be useful in further characterizing IBDC virus, its pathogenesis, and its natural history.

  10. System visualization of integrated biofuels and high value chemicals developed within the MacroAlgaeBiorefinery (MAB3) project

    DEFF Research Database (Denmark)

    Seghetta, Michele; Hasler, Berit; Bastianoni, Simone

    MacroAlgaeBiorefinery (MAB3) may functions as production platform and raw material supplier for future sustainable production chains of biofuels and high value chemicals. Biofuels are interesting energy source but challenges in terms of the composition of the biomass and resulting energy...... efficiencies has to be compensated for to make the biofuel prices competitive in replacing fossil fuel. Since it is difficult to increase the yield of the single biorefinery, the overall system productivity can be improved integrating different sub-systems. In this study, macroalgae cultivation in Denmark...... is integrated with a biogas biorefinery, a bioethanol biorefinery and a fish feed industry. The modeled system is able to adapt itself to different amount and quality of feedstock and to maximize valuable outputs (e.g. bio-fuels and chemical). Macroalgae are harvested and utilized as feedstock in bioethanol...

  11. Chimeric anti-tenascin antibody 81C6: Increased tumor localization compared with its murine parent

    International Nuclear Information System (INIS)

    Zalutsky, Michael R.; Archer, Gary E.; Garg, Pradeep K.; Batra, Surinder K.; Bigner, Darell D.

    1996-01-01

    When labeled using the Iodogen method, a chimeric antibody composed of the human IgG 2 constant region and the variable regions of murine anti-tenascin 81C6 exhibited superior uptake in human glioma xenografts compared with its murine parent. In the current study, three paired-label experiments were performed in athymic mice with subcutaneous D-54 MG human glioma xenografts to evaluate further the properties of radioiodinated chimeric 81C6. These studies demonstrated that (a) the enhanced tumor uptake of chimeric 81C6 is specific; (b) when labeling was performed using N-succinimidyl 3-iodobenzoate, chimeric 81C6 again showed preferential accumulation in tumor compared with murine 81C6; and (c) the tumor uptake advantage observed previously with murine 81C6 for N-succinimidyl 3-iodobenzoate compared with Iodogen labeling did not occur with chimeric 81C6

  12. Influenza human monoclonal antibody 1F1 interacts with three major antigenic sites and residues mediating human receptor specificity in H1N1 viruses.

    Directory of Open Access Journals (Sweden)

    Tshidi Tsibane

    Full Text Available Most monoclonal antibodies (mAbs to the influenza A virus hemagglutinin (HA head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates. The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca(2. The 1F1 heavy chain also reaches into the receptor binding site (RBS and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.

  13. A comparison of targeting of neuroblastoma with mIBG and anti L1-CAM antibody mAb chCE7: therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients

    NARCIS (Netherlands)

    Hoefnagel, C. A.; Rutgers, M.; Buitenhuis, C. K.; Smets, L. A.; de Kraker, J.; Meli, M.; Carrel, F.; Amstutz, H.; Schubiger, P. A.; Novak-Hofer, I.

    2001-01-01

    Iodine-131 labelled anti L1-CAM antibody mAb chCE7 was compared with the effective neuroblastoma-seeking agent 131I-labelled metaiodobenzylguanidine (MIBG) with regard to (a) its therapeutic efficacy in treating nude mice with neuroblastoma xenografts and (b) its tumour targeting ability in

  14. Novel Application of Micro-Computerized Tomography for Morphologic Characterization of the Murine Penis.

    Science.gov (United States)

    O'Neill, Marisol; Huang, Gene O; Lamb, Dolores J

    2017-12-01

    The murine penis model has enriched our understanding of anomalous penile development. The morphologic characterization of the murine penis using conventional serial sectioning methods is labor intensive and prone to errors. To develop a novel application of micro-computerized tomography (micro-CT) with iodine staining for rapid, non-destructive morphologic study of murine penis structure. Penises were dissected from 10 adult wild-type mice and imaged using micro-CT with iodine staining. Images were acquired at 5-μm spatial resolution on a Bruker SkyScan 1272 micro-CT system. After images were acquired, the specimens were washed of any remaining iodine and embedded in paraffin for conventional histologic examination. Histologic and micro-CT measurements for all specimens were made by 2 independent observers. Measurements of penile structures were made on virtual micro-CT sections and histologic slides. The Lin concordance correlation coefficient demonstrated almost perfect strength of agreement for interobserver variability for histologic section (0.9995, 95% CI = 0.9990-0.9997) and micro-CT section (0.9982, 95% CI = 0.9963-0.9991) measurements. Bland-Altman analysis for agreement between the 2 modalities of measurement demonstrated mean differences of -0.029, 0.022, and -0.068 mm for male urogenital mating protuberance, baculum, and penile glans length, respectively. There did not appear to be a bias for overestimation or underestimation of measured lengths and limits of agreement were narrow. The enhanced ability offered by micro-CT to phenotype the murine penis has the potential to improve translational studies examining the molecular pathways contributing to anomalous penile development. The present study describes the first reported use of micro-CT with iodine staining for imaging the murine penis. Producing repeated histologic sections of identical orientation was limited by inherent imperfections in mounting and tissue sectioning, but this was

  15. [Expression of homeobox gene Msx-1, Msx-2 and Dlx-2 during murine mandibular first molar development].

    Science.gov (United States)

    Ma, Li; Chen, Zhi; Song, Guang-tai; Fan, Ming-wen; Zhang, Qi; Wang, Zhi-feng

    2003-11-01

    To observe the expression of homeobox gene Msx-1, Msx-2 and Dlx-2 during murine mandibular first molar development. The murine heads or mandibles on embryonic days 11-18 (E11-18) and postnatal day 1-3 (P1-3) were removed, fixed and embedded, 5 micro m serial sections were cut in the coronal plane. Msx-1, Msx-2 and Dlx-2 RNA probes were synthesized by in vitro transcription and labeled with digoxigenin. Msx-1, Msx-2 and Dlx-2 mRNA expression was observed after in situ hybridization. During molar development Msx-1 transcripts appeared only in mesenchymal cells, not in epithelial cells. Msx-2 and Dlx-2 both expressed in the epithelial and mesenchymal cells. At the initiation stage of the molar development Msx-2 and Dlx-2 had similar expression. At the bud stage (E13-14) Msx-2 mRNA signaling was intensive in the enamel organ and slight in the dental mesenchyme; Dlx-2 signaling was stronger in the dental papilla. At cap stage (E15-16) Msx-2 showed prominent mRNA signaling in enamel knot and Dlx-2 was maximal in the dental papilla. At the late bell stage (P2-3) Msx-2 transcripts were observed in odontoblasts but not labeled in ameloblasts, and Dlx-2 transcripts appeared in ameloblasts but no labeling was seen in odontoblasts. Msx-1, Msx-2 and Dlx-2 are expressed in various patterns during murine mandibular first molar development, suggesting they possibly play a role in the interaction between the epithelium and mesenchyme during the molar development.

  16. Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells.

    Science.gov (United States)

    Xuan, X; Maeda, K; Mikami, T; Otsuka, H

    1996-12-01

    The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.

  17. Immunoelectron microscopy of lipopolysaccharide in Chlamydia trachomatis

    DEFF Research Database (Denmark)

    Birkelund, Svend; Lundemose, AG; Christiansen, Gunna

    1989-01-01

    Monoclonal antibodies (MAb) specific for Chlamydia trachomatis lipopolysaccharide (LPS) and major outer membrane protein (MOMP) were used for immunoelectron microscopy analysis. MAb specific for MOMP showed strong reaction with the chlamydial surface, whereas MAb specific for LPS showed strong...... association of gold particles with the periphery of the chlamydial body. After fixation of the chlamydia cells, the reactivity was, however, similar to the anti-MOMP reactivity. Enzyme-linked immunosorbent assay showed that MAb specific for LPS could remove LPS from the chlamydial membrane....

  18. Reversal of Liver Fibrosis in Chronic Murine Schistosomiasis ...

    African Journals Online (AJOL)

    NO Al-Harbi, SA Bahashwan, MS Aboonq, MA Ramadan, AA Bahashwan. Abstract. Purpose: To evaluate the safety, pharmacological effect and mechanism of action of an antifibrotic compound, safironil (SAF)/praziquantel (PZQ) combination on reversal of liver fibrogenesis in chronic murine Schistosomiasis mansoni.

  19. Automated segmentation of murine lung tumors in x-ray micro-CT images

    Science.gov (United States)

    Swee, Joshua K. Y.; Sheridan, Clare; de Bruin, Elza; Downward, Julian; Lassailly, Francois; Pizarro, Luis

    2014-03-01

    Recent years have seen micro-CT emerge as a means of providing imaging analysis in pre-clinical study, with in-vivo micro-CT having been shown to be particularly applicable to the examination of murine lung tumors. Despite this, existing studies have involved substantial human intervention during the image analysis process, with the use of fully-automated aids found to be almost non-existent. We present a new approach to automate the segmentation of murine lung tumors designed specifically for in-vivo micro-CT-based pre-clinical lung cancer studies that addresses the specific requirements of such study, as well as the limitations human-centric segmentation approaches experience when applied to such micro-CT data. Our approach consists of three distinct stages, and begins by utilizing edge enhancing and vessel enhancing non-linear anisotropic diffusion filters to extract anatomy masks (lung/vessel structure) in a pre-processing stage. Initial candidate detection is then performed through ROI reduction utilizing obtained masks and a two-step automated segmentation approach that aims to extract all disconnected objects within the ROI, and consists of Otsu thresholding, mathematical morphology and marker-driven watershed. False positive reduction is finally performed on initial candidates through random-forest-driven classification using the shape, intensity, and spatial features of candidates. We provide validation of our approach using data from an associated lung cancer study, showing favorable results both in terms of detection (sensitivity=86%, specificity=89%) and structural recovery (Dice Similarity=0.88) when compared against manual specialist annotation.

  20. A novel murine model for evaluating bovine papillomavirus prophylactics/therapeutics for equine sarcoid-like tumours.

    Science.gov (United States)

    Bogaert, Lies; Woodham, Andrew W; Da Silva, Diane M; Martens, Ann; Meyer, Evelyne; Kast, W Martin

    2015-09-01

    Equine sarcoids are highly recurrent bovine papillomavirus (BPV)-induced fibroblastic neoplasms that are the most common skin tumours in horses. In order to facilitate the study of potential equine sarcoid prophylactics or therapeutics, which can be a slow and costly process in equines, a murine model for BPV-1 protein-expressing equine sarcoid-like tumours was developed in mice through stable transfection of BPV-1 E5 and E6 in a murine fibroblast tumour cell line (K-BALB). Like equine sarcoids, these murine tumour cells (BPV-KB) were of fibroblast origin, were tumorigenic and expressed BPV-1 proteins. As an initial investigation of the preclinical potential of this tumour model for equine sarcoids prophylactics, mice were immunized with BPV-1 E5E6 Venezuelan equine encephalitis virus replicon particles, prior to BPV-KB challenge, which resulted in an increased tumour-free period compared with controls, indicating that the BPV-KB murine model may be a valuable preclinical alternative to equine clinical trials.

  1. Amelioration of murine passive immune thrombocytopenia by IVIg and a therapeutic monoclonal CD44 antibody does not require the Myd88 signaling pathway.

    Directory of Open Access Journals (Sweden)

    Andrew R Crow

    Full Text Available Immune thrombocytopenia (ITP is an autoimmune bleeding disorder characterized by a low platelet count and the production of anti-platelet antibodies. The majority of ITP patients have antibodies to platelet integrin α(IIbβ₃ (GPIIbIIIa which can direct platelet phagocytosis by macrophages. One effective treatment for patients with ITP is intravenous immunoglobulin (IVIg which rapidly reverses thrombocytopenia. The exact mechanism of IVIg action in human patients is unclear, although in mouse models of passive ITP, IVIg can rapidly increase platelet counts in the absence of adaptive immunity. Another antibody therapeutic that can similarly increase platelet counts independent of adaptive immunity are CD44 antibodies. Toll-like receptors (TLRs are pattern recognition receptors which play a central role in helping direct the innate immune system. Dendritic cells, which are notable for their expression of TLRs, have been directly implicated in IVIg function as an initiator cell, while CD44 can associate with TLR2 and TLR4. We therefore questioned whether IVIg, or the therapeutic CD44 antibody KM114, mediate their ameliorative effects in a manner dependent upon normal TLR function. Here, we demonstrate that the TLR4 agonist LPS does not inhibit IVIg or KM114 amelioration of antibody-induced thrombocytopenia, and that these therapeutics do not ameliorate LPS-induced thrombocytopenia. IVIg was able to significantly ameliorate murine ITP in C3H/HeJ mice which have defective TLR4. All known murine TLRs except TLR3 utilize the Myd88 adapter protein to drive TLR signaling. Employing Myd88 deficient mice, we found that both IVIg and KM114 ameliorate murine ITP in Myd88 deficient mice to the same extent as normal mice. Thus both IVIg and anti-CD44 antibody can mediate their ameliorative effects in murine passive ITP independent of the Myd88 signaling pathway. These data help shed light on the mechanism of action of IVIg and KM114 in the amelioration of

  2. Murine HPV16 E7-expressing transgenic skin effectively emulates the cellular and molecular features of human high-grade squamous intraepithelial lesions

    Directory of Open Access Journals (Sweden)

    Z.K. Tuong

    2018-06-01

    Full Text Available Currently available vaccines prevent HPV infection and development of HPV-associated malignancies, but do not cure existing HPV infections and dysplastic lesions. Persistence of infection(s in immunocompetent patients may reflect induction of local immunosuppressive mechanisms by HPV, providing a target for therapeutic intervention. We have proposed that a mouse, expressing HPV16 E7 oncoprotein under a Keratin 14 promoter (K14E7 mice, and which develops epithelial hyperplasia, may assist with understanding local immune suppression mechanisms that support persistence of HPV oncogene-induced epithelial hyperplasia. K14E7 skin grafts recruit immune cells from immunocompetent hosts, but consistently fail to be rejected. Here, we review the literature on HPV-associated local immunoregulation, and compare the findings with published observations on the K14E7 transgenic murine model, including comparison of the transcriptome of human HPV-infected pre-malignancies with that of murine K14E7 transgenic skin. We argue from the similarity of i the literature findings and ii the transcriptome profiles that murine K14E7 transgenic skin recapitulates the cellular and secreted protein profiles of high-grade HPV-associated lesions in human subjects. We propose that the K14E7 mouse may be an appropriate model to further study the immunoregulatory effects of HPV E7 expression, and can facilitate development and testing of therapeutic vaccines.

  3. Importance of Neutralizing Monoclonal Antibodies Targeting Multiple Antigenic Sites on the Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein To Avoid Neutralization Escape

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lingshu; Shi, Wei; Chappell, James D.; Joyce, M. Gordon; Zhang, Yi; Kanekiyo, Masaru; Becker, Michelle M.; van Doremalen, Neeltje; Fischer, Robert; Wang, Nianshuang; Corbett, Kizzmekia S.; Choe, Misook; Mason, Rosemarie D.; Van Galen, Joseph G.; Zhou, Tongqing; Saunders, Kevin O.; Tatti, Kathleen M.; Haynes, Lia M.; Kwong, Peter D.; Modjarrad, Kayvon; Kong, Wing-Pui; McLellan, Jason S.; Denison, Mark R.; Munster, Vincent J.; Mascola, John R.; Graham, Barney S.; Gallagher, Tom

    2018-03-07

    structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts.

  4. Development and characterization of antiserum to murine granulocyte-macrophage colony-stimulating factor

    International Nuclear Information System (INIS)

    Mochizuki, D.Y.; Eisenman, J.R.; Conlon, P.J.; Park, L.S.; Urdal, D.L.

    1986-01-01

    The expression in yeast of a cDNA clone encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) has made possible the purification of large quantities of this recombinant protein. Rabbits immunized with pure recombinant GM-CSF generated antibodies that were shown to be specific for both recombinant GM-CSF and GM-CSF isolated from natural sources. Other lymphokines, including IL 1β, IL 2, IL 3, and recombinant human GM-CSF did not react with the antiserum. The antiserum together with recombinant GM-CSF that had been radiolabeled with 125 I to high specific activity, formed the foundation for a rapid, sensitive, and quantitative radioimmunoassay specific for murine GM-CSF. Furthermore, the antiserum was found to inhibit the biologic activities of GM-CSF as measured in both a bone marrow proliferation assay and a colony assay, and thus should prove to be a useful reagent for dissecting the complex growth factor activities involved in murine hematopoiesis

  5. Protective effects of astaxanthin from Paracoccus carotinifaciens on murine gastric ulcer models.

    Science.gov (United States)

    Murata, Kenta; Oyagi, Atsushi; Takahira, Dai; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Ishibashi, Takashi; Hara, Hideaki

    2012-08-01

    The purpose of this study was to investigate the effect of astaxanthin extracted from Paracoccus carotinifaciens on gastric mucosal damage in murine gastric ulcer models. Mice were pretreated with astaxanthin for 1 h before ulcer induction. Gastric ulcers were induced in mice by oral administration of hydrochloride (HCl)/ethanol or acidified aspirin. The effect of astaxanthin on lipid peroxidation in murine stomach homogenates was also evaluated by measuring the level of thiobarbituric acid reactive substance (TBARS). The free radical scavenging activities of astaxanthin were also measured by electron spin resonance (ESR) measurements. Astaxanthin significantly decreased the extent of HCl/ethanol- and acidified aspirin-induced gastric ulcers. Astaxanthin also decreased the level of TBARS. The ESR measurement showed that astaxanthin had radical scavenging activities against the 1,1-diphenyl-2-picrylhydrazyl radical and the superoxide anion radical. These results suggest that astaxanthin has antioxidant properties and exerts a protective effect against ulcer formation in murine models. Copyright © 2011 John Wiley & Sons, Ltd.

  6. Single Amino Acid Insertion in Loop 4 Confers Amphotropic Murine Leukemia Virus Receptor Function upon Murine Pit1

    DEFF Research Database (Denmark)

    Lundorf, Mikkel D.; Pedersen, Finn Skou; O'Hara, Bryan

    1998-01-01

    Pit1 is the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related human protein Pit2 is a receptor for amphotropic murine leukemia virus (A-MuLV). The A-MuLV-related isolate 10A1 can utilize both Pit1 and Pit2 as receptors. A stretch...

  7. Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza.

    Directory of Open Access Journals (Sweden)

    Cameron P Simmons

    2007-05-01

    Full Text Available New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs and tested their efficacy for prophylaxis and therapy in a murine model of infection.Using Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1 in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1. mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1.These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1

  8. Differentiation of the emerging human pathogens Trichosporon asahii and Trichosporon asteroides from other pathogenic yeasts and moulds by using species-specific monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Genna E Davies

    Full Text Available The fungal genus Trichosporon contains emerging opportunistic pathogens of humans, and is the third most commonly isolated non-candidal yeast from humans. Trichosporon asahii and T. asteroides are the most important species causing disseminated disease in immunocompromised patients, while inhalation of T. asahii spores is the most important cause of summer-type hypersensitivity pneumonitis in healthy individuals. Trichosporonosis is misdiagnosed as candidiasis or cryptococcosis due to a lack of awareness and the ambiguity of diagnostic tests for these pathogens. In this study, hybridoma technology was used to produce two murine monoclonal antibodies (MAbs, CA7 and TH1, for detection and differentiation of Trichosporon from other human pathogenic yeasts and moulds. The MAbs react with extracellular antigens from T. asahii and T. asteroides, but do not recognise other related Trichosporon spp., or unrelated pathogenic yeasts and moulds including Candida, Cryptococcus, Aspergillus, Fusarium, and Scedosporium spp., or the etiologic agents of mucormycosis. Immunofluorescence and Western blotting studies show that MAb CA7, an immunoglobulin G1 (IgG1, binds to a major 60 kDa glycoprotein antigen produced on the surface of hyphae, while TH1, an immunoglobulin M (IgM, binds to an antigen produced on the surface of conidia. The MAbs were used in combination with a standard mycological growth medium (Sabouraud Dextrose Agar to develop an enzyme-linked immunosorbent assay (ELISA for differentiation of T. asahii from Candida albicans and Cryptococcus neoformans in single and mixed species cultures. The MAbs represent a major advance in the identification of T. asahii and T. asteroides using standard mycological identification methods.

  9. Enhancement of tumor radioresponse by combined chemotherapy in murine hepatocarcinoma

    International Nuclear Information System (INIS)

    Seong, Jin Sil; Kim, Sung Hee; Suh, Chang Ok

    2000-01-01

    The purpose of this study was to identify drugs that can enhance radioresponse of murine hepatocarcinoma. C3H/HeJ mice bearing 8 mm tumors of murine hepatocarcinoma, HCa-l, were treated with 25 Gy radiation and one of the following drugs: 5-Fu, 150 mg/kg; adriamycin, 8 mg/kg; cisplatin, 6 mg/kg; paclitaxel, 40 mg/kg; and gemcitabine, 50 mg/kg. Tumor response to the treatment was determined by tumor growth delay assay and by enhancement factor. Apoptotic level was assessed in tissue sections. Expression of regulating molecules was analyzed by western blotting for p53, 8c1-2, Sax, Bel-XL, Bd-XS, and p21 WAF1/CIP1 . Among the drugs tested, only gemcitabine enhanced the antitumor effect of radiation, with enhancement factor of 1.6. Induction of apoptosis by a combination of gerncitabine and radiation was shown as only additive level. In analysis of radiation-induced expression of regulating molecules, the most significant change by combining gemcitabine was activation of p21 WAF1/CIP1 . Gemcitabine is the first drug showing an enhancement of radioresponse in murine hepatocarcinoma, when combined with radiation. The key element of enhancement is thought to be p21 WAF1/CIP1

  10. Enhancement of tumor radioresponse by combined chemotherapy in murine hepatocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Seong, Jin Sil; Kim, Sung Hee; Suh, Chang Ok [College of Medicine, Yonsei Univ., Seoul (Korea, Republic of)

    2000-12-01

    The purpose of this study was to identify drugs that can enhance radioresponse of murine hepatocarcinoma. C3H/HeJ mice bearing 8 mm tumors of murine hepatocarcinoma, HCa-l, were treated with 25 Gy radiation and one of the following drugs: 5-Fu, 150 mg/kg; adriamycin, 8 mg/kg; cisplatin, 6 mg/kg; paclitaxel, 40 mg/kg; and gemcitabine, 50 mg/kg. Tumor response to the treatment was determined by tumor growth delay assay and by enhancement factor. Apoptotic level was assessed in tissue sections. Expression of regulating molecules was analyzed by western blotting for p53, 8c1-2, Sax, Bel-XL, Bd-XS, and p21{sup WAF1/CIP1}. Among the drugs tested, only gemcitabine enhanced the antitumor effect of radiation, with enhancement factor of 1.6. Induction of apoptosis by a combination of gerncitabine and radiation was shown as only additive level. In analysis of radiation-induced expression of regulating molecules, the most significant change by combining gemcitabine was activation of p21 {sup WAF1/CIP1}. Gemcitabine is the first drug showing an enhancement of radioresponse in murine hepatocarcinoma, when combined with radiation. The key element of enhancement is thought to be p21{sup WAF1/CIP1}.

  11. Chromosomal mechanisms in murine radiation acute myeloid leukemogenesis

    International Nuclear Information System (INIS)

    Bouffler, S.D.; Breckon, G.; Cox, R.

    1996-01-01

    Chromosome 2 abnormalities, particularly interstitial deletions, characterize murine radiation-induced acute myeloid leukaemias (AMLs). Here, G-band analyses in CBA/H mice of early (1-6 month) post 3 Gy X-radiation events in bone marrow cells in vivo and karyotype evolution in one unusual AML are presented. The early event analysis showed that all irradiated animals carry chromosome 2 abnormalities, that chromosome 2 abnormalities are more frequent than expected and that interstitial deletions are more common in chromosome 2 than in the remainder of the genome. On presentation AML case N122 carried a t(2; 11) terminal translocation which, with passaging, evolved into a del2(C3F3). Therefore two pathways in leukaemogenesis might exist, one deletion-driven, the other terminal tranlocation-driven involving interstitial genes and terminal genes respectively of chromosome 2. As all irradiated individuals carried chromosome 2 abnormalities, the formation of these aberrations does not determine individual leukaemogenic sensitivity as only 20-25% of animals would be expected to develop AML. Similar lines of argument suggest that chromosome 2 abnormalities are necessary but not sufficient for radiation leukaemogenesis in CBA/H nor are they rate limiting in leukaemogenesis. (Author)

  12. Adipocytes enhance murine pancreatic cancer growth via a hepatocyte growth factor (HGF)-mediated mechanism.

    Science.gov (United States)

    Ziegler, Kathryn M; Considine, Robert V; True, Eben; Swartz-Basile, Deborah A; Pitt, Henry A; Zyromski, Nicholas J

    2016-04-01

    Obesity accelerates the development and progression of pancreatic cancer, though the mechanisms underlying this association are unclear. Adipocytes are biologically active, producing factors such as hepatocyte growth factor (HGF) that may influence tumor progression. We therefore sought to test the hypothesis that adipocyte-secreted factors including HGF accelerate pancreatic cancer cell proliferation. Murine pancreatic cancer cells (Pan02 and TGP-47) were grown in a) conditioned medium (CM) from murine F442A preadipocytes, b) HGF-knockdown preadipocyte CM, c) recombinant murine HGF at increasing doses, and d) CM plus HGF-receptor (c-met) inhibitor. Cell proliferation was measured using the MTT assay. ANOVA and t-test were applied; p TGP-47 cell proliferation relative to control (59 ± 12% and 34 ± 12%, p TGP-47 cells remained unchanged. Recombinant HGF dose-dependently increased Pan02, but not TGP-47, proliferation (p TGP-47 cells. These experiments demonstrate that adipocyte-derived factors accelerate murine pancreatic cancer proliferation. In the case of Pan02 cells, HGF is responsible, in part, for this proliferation. Copyright © 2016 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.

  13. Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

    International Nuclear Information System (INIS)

    Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

    1986-01-01

    In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [ 3 H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

  14. Gp96 Peptide Antagonist gp96-II Confers Therapeutic Effects in Murine Intestinal Inflammation

    Directory of Open Access Journals (Sweden)

    Claudia A. Nold-Petry

    2017-12-01

    96-II peptide reduced IL–1β, IL-6, TNF and GM-CSF in human peripheral blood mononuclear cells to a similar degree without affecting cell viability, whereas RANTES, IL-25 and MIF were twofold to threefold increased.Conclusiongp96-II peptide protects against murine intestinal inflammation by regulating inflammation in vivo and in vitro, pointing to its promise as a novel treatment for inflammatory bowel disease.

  15. A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria.

    Directory of Open Access Journals (Sweden)

    Ryan W Carroll

    Full Text Available BACKGROUND: Cerebral malaria (CM is a neurological syndrome that includes coma and seizures following malaria parasite infection. The pathophysiology is not fully understood and cannot be accounted for by infection alone: patients still succumb to CM, even if the underlying parasite infection has resolved. To that effect, there is no known adjuvant therapy for CM. Current murine CM (MCM models do not allow for rapid clinical identification of affected animals following infection. An animal model that more closely mimics the clinical features of human CM would be helpful in elucidating potential mechanisms of disease pathogenesis and evaluating new adjuvant therapies. METHODOLOGY/PRINCIPAL FINDINGS: A quantitative, rapid murine coma and behavior scale (RMCBS comprised of 10 parameters was developed to assess MCM manifested in C57BL/6 mice infected with Plasmodium berghei ANKA (PbA. Using this method a single mouse can be completely assessed within 3 minutes. The RMCBS enables the operator to follow the evolution of the clinical syndrome, validated here by correlations with intracerebral hemorrhages. It provides a tool by which subjects can be identified as symptomatic prior to the initiation of trial treatment. CONCLUSIONS/SIGNIFICANCE: Since the RMCBS enables an operator to rapidly follow the course of disease, label a subject as affected or not, and correlate the level of illness with neuropathologic injury, it can ultimately be used to guide the initiation of treatment after the onset of cerebral disease (thus emulating the situation in the field. The RMCBS is a tool by which an adjuvant therapy can be objectively assessed.

  16. The Role of the MHV Receptor and Related Glycoproteins in Murine Hepatitis Virus Infection of Murine Cell Lines

    Science.gov (United States)

    1995-04-13

    vaccinia virus-T7 RNA polymerase s y stem for e xpression of target genes . Mol . Cell . BioI . 7 : 2538-2544 . Gagneten , S ., Gout , 0 ., Dubois-Dalcq...glycoprotein. These results showed f or the first time that two murine CEA- related genes can be co-expressed in some cell lines from inbred mice...49 Southern Hybridization ................ . ............ 50 Subcloning of PCR Products and Gene Cloning ........ 51 Growth

  17. Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model.

    LENUS (Irish Health Repository)

    Monk, Ian R

    2010-01-01

    Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells.

  18. Molecular basis of a high affinity murine interleukin-5 receptor.

    OpenAIRE

    Devos, R; Plaetinck, G; Van der Heyden, J; Cornelis, S; Vandekerckhove, J; Fiers, W; Tavernier, J

    1991-01-01

    The mouse interleukin-5 receptor (mIL-5R) consists of two components one of which, the mIL-5R alpha-chain, binds mIL-5 with low affinity. Recently we demonstrated that monoclonal antibodies (Mabs) recognizing the second mIL-5R beta-chain, immunoprecipitate a p130-140 protein doublet which corresponds to the mIL-3R and the mIL-3R-like protein, the latter chain for which so far no ligand has been identified. In this study we show that a high affinity mIL-5R can be reconstituted on COS1 cells by...

  19. Effects of replacing fishmeal with animal by-products meal supplementation in diets on the growth and nutrient utilization of mangrove red snapper

    Science.gov (United States)

    Jamil, Khalid; Abbas, Ghulam; Akhtar, Rukhsana; Lin, Hong; Li, Zhenxing

    2007-07-01

    A feeding trial was conducted for 75 d to evaluate the nutritive value of a mixture of animal by-products (MAB) as a possible protein source in diets for juvenile mangrove red snapper, Lutjanus argentimaculatus (mean initial body weight, 30 g). Fish were fed one of five isonitrogenous diets (40% crude protein) replacing 0, 25% (MAB25), 50% (MAB50), 75% (MAB75) and 100% (MAB100) of fish meal protein with similar percentages of MAB. The MAB consisted of 25% cow liver meal, 20% leather meal, 20% meat and bone meal, 15% blood meal, 10% APC (poultry feather meal), 8% poultry manure dried, 1.5% choline and 0.5% chromic oxide. After 75 d of feeding, fish fed with diets MAB50, MAB75 and MAB100 exhibited significantly lower growth performance than that of fish fed with control and MAB25 diets. The optimum level of MAB was estimated to be 23%. Replacement of fish meal by MAB23% showed the following performance: maximum weight gain, 510%; SGR, 2.39% and FCE, 2.83%. The MAB substitution up to 75% of fish meal protein in diets did not show differences in apparent protein digestibility (83.6% for MAB25, 79.2% for MAB50, 78.7% for MAB75) compared with control (83.4%), whereas in MAB100 group digestibility (65.3%) was significantly lower than in other groups. The apparent phosphorus absorption of test diet groups was significantly higher (37.1% for MAB25, 28.5% for MAB50, 55.6% for MAB75 and 54.5% for MAB100) than that of control (11.2%). The levels of protein and ash in the whole body, carcass and viscera increased as MAB substitution in diets increased, whereas lipids and moisture remained consistent among all treatment groups. These results showed that approximately 23% of fish meal protein could be replaced by a mixture of animal by-products for juvenile snapper growing from 30 g to 167 g in 75 d without compromising growth performance and feed efficiency.

  20. Murine alpha1-adrenoceptor subtypes. I. Radioligand binding studies

    NARCIS (Netherlands)

    Yang, M.; Reese, J.; Cotecchia, S.; Michel, M. C.

    1998-01-01

    Alpha1-adrenoceptors were identified in murine tissues by [3H]prazosin saturation binding studies, with a rank order of cerebral cortex > cerebellum > liver > lung > kidney > heart > spleen, with the spleen not exhibiting detectable expression. Competition binding studies were performed with

  1. Validation of diffuse correlation spectroscopy sensitivity to nicotinamide-induced blood flow elevation in the murine hindlimb using the fluorescent microsphere technique

    Science.gov (United States)

    Proctor, Ashley R.; Ramirez, Gabriel A.; Han, Songfeng; Liu, Ziping; Bubel, Tracy M.; Choe, Regine

    2018-03-01

    Nicotinamide has been shown to affect blood flow in both tumor and normal tissues, including skeletal muscle. Intraperitoneal injection of nicotinamide was used as a simple intervention to test the sensitivity of noninvasive diffuse correlation spectroscopy (DCS) to changes in blood flow in the murine left quadriceps femoris skeletal muscle. DCS was then compared with the gold-standard fluorescent microsphere (FM) technique for validation. The nicotinamide dose-response experiment showed that relative blood flow measured by DCS increased following treatment with 500- and 1000-mg / kg nicotinamide. The DCS and FM technique comparison showed that blood flow index measured by DCS was correlated with FM counts quantified by image analysis. The results of this study show that DCS is sensitive to nicotinamide-induced blood flow elevation in the murine left quadriceps femoris. Additionally, the results of the comparison were consistent with similar studies in higher-order animal models, suggesting that mouse models can be effectively employed to investigate the utility of DCS for various blood flow measurement applications.

  2. Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains.

    Science.gov (United States)

    Lusso, Paolo; Earl, Patricia L; Sironi, Francesca; Santoro, Fabio; Ripamonti, Chiara; Scarlatti, Gabriella; Longhi, Renato; Berger, Edward A; Burastero, Samuele E

    2005-06-01

    The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on

  3. Temporal Regulation of fim Genes in Uropathogenic Escherichia coli during Infection of the Murine Urinary Tract

    Directory of Open Access Journals (Sweden)

    William R. Schwan

    2017-01-01

    Full Text Available Uropathogenic Escherichia coli (UPEC adhere to cells in the human urinary tract via type 1 pili that undergo phase variation where a 314-bp fimS DNA element flips between Phase-ON and Phase-OFF orientations through two site-specific recombinases, FimB and FimE. Three fim-lux operon transcriptional fusions were created and moved into the clinical UPEC isolate NU149 to determine their temporal regulation in UPEC growing in the urinary tract. Within murine urinary tracts, the UPEC strains demonstrated elevated transcription of fimA and fimB early in the infection, but lower transcription by the fifth day in murine kidneys. In contrast, fimE transcription was much lower than either fimA or fimB early, increased markedly at 24 h after inoculation, and then dropped five days after inoculation. Positioning of fimS was primarily in the Phase-ON position over the time span in UPEC infected bladders, whereas in UPEC infected murine kidneys the Phase-OFF orientation was favored by the fifth day after inoculation. Hemagglutination titers with guinea pig erythrocytes remained constant in UPEC growing in infected murine bladders but fell substantially in UPEC infected kidneys over time. Our results show temporal in vivo regulation of fim gene expression in different environmental niches when UPEC infects the murine urinary tract.

  4. Characterization of an immunodominant cancer-specific O-glycopeptide epitope in murine podoplanin (OTS8)

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Schjoldager, Katrine T; Cló, Emiliano

    2010-01-01

    antibody 237, developed to a spontaneous murine fibrosarcoma, was shown to be directed to murine podoplanin (OTS8) with truncated Tn O-glycans. Our understanding of such cancer-specific auto-antibodies to truncated glycoforms of glycoproteins is limited. Here we have investigated immunogenicity...... of a chemoenzymatically produced Tn-glycopeptide derived from the putative murine podoplanin O-glycopeptide epitope. We found that the Tn O-glycopeptide was highly immunogenic in mice and produced a Tn-glycoform specific response with no reactivity against unglycosylated peptides or the O-glycopeptide with extended O......-glycan (STn and T glycoforms). The immunodominant epitope was strictly dependent on the peptide sequence, required Tn at a specific single Thr residue (Thr(77)), and antibodies to the epitope were not found in naive mice. We further tested a Tn O-glycopeptide library derived from human podoplanin...

  5. Murine scid cells complement ataxia-telangiectasia cells and show a normal port-irradiation response of DNA synthesis

    International Nuclear Information System (INIS)

    Komatsu, K.; Yoshida, M.; Okumura, Y.

    1993-01-01

    The murine severe combined immunodeficient mutation (scid) is characterized by a lack of both B and T cells, due to a deficit in lymphoid variable-(diversity)-joining (V(D)J) rearrangement. Scid cells are highly sensitive to both radiation-induced killing and chromosomal aberrations. Significantly reduced D 0 and n values were demonstrated in scid cells and were similar to ataxia-telangiectasia (AT) cells (a unique human disease conferring whole body radiosensitivity). However, the kinetics of DNA synthesis after irradiation were different between the two cell types. In contrast with the radioresistant DNA synthesis of AT cells, DNA synthesis of scid cells was markedly inhibited after irradiation. The existence of different mutations was also supported by evidence of complementation in somatic cell hybrids between scid cells and AT cells. Results indicate that the radiobiological character of scid is similar to AT but is presumably caused by different mechanisms. (author)

  6. Hemagglutinin-specific neutralization of subacute sclerosing panencephalitis viruses.

    Directory of Open Access Journals (Sweden)

    Miguel Ángel Muñoz-Alía

    Full Text Available Subacute sclerosing panencephalitis (SSPE is a progressive, lethal complication of measles caused by particular mutants of measles virus (MeV that persist in the brain despite high levels of neutralizing antibodies. We addressed the hypothesis that antigenic drift is involved in the pathogenetic mechanism of SSPE by analyzing antigenic alterations in the MeV envelope hemagglutinin protein (MeV-H found in patients with SSPE in relation to major circulating MeV genotypes. To this aim, we obtained cDNA for the MeV-H gene from tissue taken at brain autopsy from 3 deceased persons with SSPE who had short (3-4 months, SMa79, average (3.5 years, SMa84, and long (18 years, SMa94 disease courses. Recombinant MeVs with a substituted MeV-H gene were generated by a reverse genetic system. Virus neutralization assays with a panel of anti-MeV-H murine monoclonal antibodies (mAbs or vaccine-immunized mouse anti-MeV-H polyclonal sera were performed to determine the antigenic relatedness. Functional and receptor-binding analysis of the SSPE MeV-H showed activity in a SLAM/nectin-4-dependent manner. Similar to our panel of wild-type viruses, our SSPE viruses showed an altered antigenic profile. Genotypes A, G3, and F (SSPE case SMa79 were the exception, with an intact antigenic structure. Genotypes D7 and F (SSPE SMa79 showed enhanced neutralization by mAbs targeting antigenic site IIa. Genotypes H1 and the recently reported D4.2 were the most antigenically altered genotypes. Epitope mapping of neutralizing mAbs BH015 and BH130 reveal a new antigenic site on MeV-H, which we designated Φ for its intermediate position between previously defined antigenic sites Ia and Ib. We conclude that SSPE-causing viruses show similar antigenic properties to currently circulating MeV genotypes. The absence of a direct correlation between antigenic changes and predisposition of a certain genotype to cause SSPE does not lend support to the proposed antigenic drift as a

  7. Discovery of a new strain of murine rotavirus that is consistently shed in large quantities after oral inoculation of adult mice

    International Nuclear Information System (INIS)

    McNeal, Monica M.; Belli, Janine; Basu, Mitali; Choi, Anthony H.-C.; Ward, Richard L.

    2004-01-01

    In 1990, we developed the adult mouse model for studies on active immunity against shedding of the EDIM strain of murine rotavirus. Low and inconsistent levels of EDIM shedding in some strains of adult mice, particularly those on C57BL/6 backgrounds, established the need for an alternative murine rotavirus strain for these studies. Fortuitously, such a rotavirus strain was obtained from mice housed within the conventional colony at Children's Hospital. This strain, named EMcN, was clearly distinguishable from EDIM based on electropherotype. Furthermore, sequence analyses of VP4 and VP7 genes of EMcN revealed non-identities in 5% of the amino acids of both proteins relative to EDIM but established EMcN as another G3P[16] strain of murine rotavirus. Subgroup analysis showed EMcN belonged to SG1 while EDIM was found to be non-SG1/SG2. Similarly, unlike EDIM, the EMcN strain was identified as serotype G3 based on neutralization by hyperimmune antisera developed against prototype human and simian G3 rotavirus strains. Although EDIM produced more days of diarrhea and was shed in greater quantities in neonatal BALB/c mice, EMcN was shed in much greater quantities in adult BALB/c mice. More importantly, in contrast to the EDIM strain, EMcN was shown to be consistently shed in large quantities in adult C57BL/6 mice and ko mice on this background. Therefore, it is recommended that the EMcN strain be used for future challenge studies with mice on this background

  8. Tc-99m direct radiolabeling of monoclonal antibody ior egf/r3: quality control and image studies in mice

    International Nuclear Information System (INIS)

    Dias, Carla Roberta; Marczewski, Barbara; Moraes, Vanessa; Barboza, Marycel Figols de; Osso Junior, Joao Alberto

    2005-01-01

    Monoclonal antibodies (Mabs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in the practice of nuclear medicine. The ior egf/r3 (Centis, Cuba) is a murine monoclonal antibody against epidermal growth factor receptor (EGF-R) and has been widely used in the radioimmunodiagnosis of tumors of epithelial origin. Labeled with 99m Tc, its main application in Nuclear Medicine is the follow up, detection and evaluation of tumor recurrences. The objective of this work is to describe the preparation of a lyophilized formulation (kit) for radiolabeling the Mab ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, image studies and stability of the formulation are reported. The study demonstrated that the kit formulation can be labeled with 99m Tc at high yields and can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies.(author)

  9. Tc-{sup 99m} direct radiolabeling of monoclonal antibody ior egf/r3: quality control and image studies in mice

    Energy Technology Data Exchange (ETDEWEB)

    Dias, Carla Roberta; Marczewski, Barbara; Moraes, Vanessa; Barboza, Marycel Figols de; Osso Junior, Joao Alberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN-CNEN/SP), SP (Brazil). Centro de Radiofarmacia]. E-mail: crdias@ipen.br

    2005-10-15

    Monoclonal antibodies (Mabs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in the practice of nuclear medicine. The ior egf/r3 (Centis, Cuba) is a murine monoclonal antibody against epidermal growth factor receptor (EGF-R) and has been widely used in the radioimmunodiagnosis of tumors of epithelial origin. Labeled with 99m Tc, its main application in Nuclear Medicine is the follow up, detection and evaluation of tumor recurrences. The objective of this work is to describe the preparation of a lyophilized formulation (kit) for radiolabeling the Mab ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, image studies and stability of the formulation are reported. The study demonstrated that the kit formulation can be labeled with 99m Tc at high yields and can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies.(author)

  10. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

    Energy Technology Data Exchange (ETDEWEB)

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Oshita, Masatoshi; Ideno, Shoji [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Yunoki, Mikihiro [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Kuhara, Motoki [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano 396-0002 (Japan); Yamamoto, Naomasa [Department of Biochemistry, School of Pharmaceutical Sciences, Ohu University, Koriyama, Fukushima 963-8611 (Japan); Okuno, Yoshinobu [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa 768-0061 (Japan); Ikuta, Kazuyoshi, E-mail: ikuta@biken.osaka-u.ac.jp [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan)

    2009-09-11

    Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

  11. In vivo neutralization of hepatitis B virus infection by an anti-preS1 humanized antibody in chimpanzees

    International Nuclear Information System (INIS)

    Hong, Hyo Jeong; Ryu, Chun Jeih; Hur, Hyangsuk; Kim, Seho; Oh, Han Kyu; Oh, Mee Sook; Park, Song Yong

    2004-01-01

    Previously, we generated a murine monoclonal antibody (mAb), KR127, that recognizes amino acids (aa) 37-45 of the preS1 of hepatitis B virus (HBV). In this study, we have constructed a humanized version of KR127 and evaluated its HBV-neutralizing activity in chimpanzees. A study chimpanzee was given a single intravenous dose of the humanized antibody, followed by intravenous challenge with adr subtype of wild type HBV, while a control chimpanzee was only challenged with the virus. The result showed that the study chimpanzee did not develop HBV infection during 1 year, while the control chimpanzee was infected, indicating that the humanized antibody exhibited in vivo virus-neutralizing activity and thus protected the chimpanzee from HBV infection. In addition, the humanized antibody bound to the preS1 of all subtypes of HBV. We first demonstrate that an anti-preS1 mAb can neutralize HBV infection in vivo. This humanized antibody will be useful for the immunoprophylaxis of HBV infection

  12. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

    International Nuclear Information System (INIS)

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki; Oshita, Masatoshi; Ideno, Shoji; Yunoki, Mikihiro; Kuhara, Motoki; Yamamoto, Naomasa; Okuno, Yoshinobu; Ikuta, Kazuyoshi

    2009-01-01

    Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

  13. A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease.

    Science.gov (United States)

    Calvert, Amanda E; Dixon, Kandice L; Piper, Joseph; Bennett, Susan L; Thibodeaux, Brett A; Barrett, Alan D T; Roehrig, John T; Blair, Carol D

    2016-07-01

    The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Effects of the murine skull in optoacoustic brain microscopy.

    Science.gov (United States)

    Kneipp, Moritz; Turner, Jake; Estrada, Héctor; Rebling, Johannes; Shoham, Shy; Razansky, Daniel

    2016-01-01

    Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small-animal neuroimaging methods, a variety of acoustic and light-related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high-resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically- and optically-determined resolution scenarios. It is shown that strong low-pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis.

    Science.gov (United States)

    Godfrey, Charlotte L; Mead, Emma J; Daramola, Olalekan; Dunn, Sarah; Hatton, Diane; Field, Ray; Pettman, Gary; Smales, C Mark

    2017-08-01

    mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilise available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localised to the translationally active heavy polysomes. The highest titre cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5' untranslated regions that might be utilised to enhance recombinant transcript translation. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells.

    Directory of Open Access Journals (Sweden)

    Guiyu Lou

    Full Text Available GPBAR1/TGR5 is a novel plasma membrane-bound G protein-coupled bile acid (BA receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β and tumor necrosis factor-α (TNF-α in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA diet was blunted in JNK-/- mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1 expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation.

  17. IL-17A influences essential functions of the monocyte/macrophage lineage and is involved in advanced murine and human atherosclerosis.

    Science.gov (United States)

    Erbel, Christian; Akhavanpoor, Mohammadreza; Okuyucu, Deniz; Wangler, Susanne; Dietz, Alex; Zhao, Li; Stellos, Konstantinos; Little, Kristina M; Lasitschka, Felix; Doesch, Andreas; Hakimi, Maani; Dengler, Thomas J; Giese, Thomas; Blessing, Erwin; Katus, Hugo A; Gleissner, Christian A

    2014-11-01

    Atherosclerosis is a chronic inflammatory disease. Lesion progression is primarily mediated by cells of the monocyte/macrophage lineage. IL-17A is a proinflammatory cytokine, which modulates immune cell trafficking and is involved inflammation in (auto)immune and infectious diseases. But the role of IL-17A still remains controversial. In the current study, we investigated effects of IL-17A on advanced murine and human atherosclerosis, the common disease phenotype in clinical care. The 26-wk-old apolipoprotein E-deficient mice were fed a standard chow diet and treated either with IL-17A mAb (n = 15) or irrelevant Ig (n = 10) for 16 wk. Furthermore, essential mechanisms of IL-17A in atherogenesis were studied in vitro. Inhibition of IL-17A markedly prevented atherosclerotic lesion progression (p = 0.001) by reducing inflammatory burden and cellular infiltration (p = 0.01) and improved lesion stability (p = 0.01). In vitro experiments showed that IL-17A plays a role in chemoattractance, monocyte adhesion, and sensitization of APCs toward pathogen-derived TLR4 ligands. Also, IL-17A induced a unique transcriptome pattern in monocyte-derived macrophages distinct from known macrophage types. Stimulation of human carotid plaque tissue ex vivo with IL-17A induced a proinflammatory milieu and upregulation of molecules expressed by the IL-17A-induced macrophage subtype. In this study, we show that functional blockade of IL-17A prevents atherosclerotic lesion progression and induces plaque stabilization in advanced lesions in apolipoprotein E-deficient mice. The underlying mechanisms involve reduced inflammation and distinct effects of IL-17A on monocyte/macrophage lineage. In addition, translational experiments underline the relevance for the human system. Copyright © 2014 by The American Association of Immunologists, Inc.

  18. Differential activation of murine herpesvirus 68- and Kaposi's sarcoma-associated herpesvirus-encoded ORF74 G protein-coupled receptors by human and murine chemokines

    NARCIS (Netherlands)

    Verzijl, D.; Fitzsimons, C.P.; Van Dijk, M.; Stewart, J.P.; Timmerman, H.; Smit, M.J.; Leurs, R.

    2004-01-01

    Infection of mice with murine gammaherpesvirus 68 (MHV-68) is a well-characterized small animal model for the study of gammaherpesvirus infection. MHV-68 belongs to the same herpesvirus family as herpesvirus saimiri (HVS) of New World squirrel monkeys and human herpesvirus 8 (HHV-8) (also referred

  19. Noninvasive murine glioma detection improved following photobleaching of skin PpIX fluorescence

    Science.gov (United States)

    Gibbs-Strauss, Summer L.; Davis, Scott C.; O'Hara, Julia A.; Hoopes, P. Jack; Hasan, Tayyaba; Pogue, Brian W.

    2008-02-01

    Aminolevulinic Acid (ALA) is a prodrug which can be administered to cells, animals or patients after which it is transformed via the Heme synthesis pathway into the fluorescent molecule Protoporphyrin IX (PpIX). PpIX has been shown to be useful as both a photosensitizer for photodynamic therapy (PDT) and as a fluorescence imaging contrast agent. The ALA-PpIX system not only provides contrast for fluorescence imaging but also gives information about the metabolic activity of the imaged tissue and thus could be useful for monitoring cancer therapy. In the current study skin photobleaching was examined to determine if PpIX fluorescence contrast in malignant brain tumors could be better visualized noninvasively. Red light photobleaching decreased skin PpIX fluorescence and increased the ability to noninvasively quantify PpIX fluorescence in murine gliomas, as in vivo measurements of mean PpIX fluorescence more closely matched ex vivo quantification following skin photobleaching. Three doses of blue light photobleaching (4 J/cm2, 8 J/cm2 and 12 J/cm2) were tested and determined to give similar levels of skin photobleaching as well as a similar window of decreased skin PpIX fluorescence for noninvasive fluorescence imaging following the photobleaching dose administration.

  20. Absence of hypoxanthine:guanine phosphoribosyltransferase activity in murine Dunn osteosarcoma

    International Nuclear Information System (INIS)

    Abelson, H.T.; Gorka, C.

    1983-01-01

    The transplantable murine Dunn osteosarcoma has no detectable hypoxanthine:guanine phosphoribosyltransferase (EC 2.4.2.8) activity. This was established from the tumors directly and from tissue culture cell lines derived from the tumor using a variety of assays: e.g., no [3H]hypoxanthine uptake into tumor or tissue culture cells, no conversion of [3H]hypoxanthine to [3H]IMP by cell extracts from tumors or tissue culture cells, no growth of tissue culture cells in hypoxanthine:aminopterin:thymidine medium, and normal growth of these cells in 10 microM 6-mercaptopurine. Ten human osteosarcomas have been assayed, and two have no apparent hypoxanthine:guanine phosphoribosyltransferase enzyme activity. After high-dose methotrexate treatment in vivo, murine tumors could be selectively killed and normal tissues could be spared by using a rescue regimen of hypoxanthine-thymidine-allopurinol

  1. Evaluation of the effect of radiation levels and dose rates in irradiation of murine fibroblasts used as a feeder layer in the culture of human keratinocytes

    International Nuclear Information System (INIS)

    Yoshito, Daniele; Almeida, Tiago L.; Santin, Stefany Plumeri; Somessari, Elizabeth S.R.; Silveira, Carlos G. da; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

    2009-01-01

    In 1975, Rheinwald and Green published an effective methodology for obtaining and cultivating human keratinocytes. This methodology consisted of seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate of which is then controlled through the action of ionizing radiation. The presence of the feeder layer encourages the development of keratinocyte colonies and their propagation in similar cultures, becoming possible several clinical applications as skin substitutes or wound dressings in situations such as post burn extensive skin loss and other skin disorders. However, good development of these keratinocytes depends on a high quality feeder layer among other factors. In the present work, we evaluated the relationship between radiation levels and dose rates applied to fibroblasts used in construction of feeder layers and the radiation effect on keratinocytes colonies forming efficiency. Results indicate 3T3 lineage murine fibroblasts irradiated with doses varying between 60 and 100 Gy can be used as a feeder layer immediately after irradiation or storage of the irradiated cells in suspension at 4 g C for 24 hours with similar results. The exception is when the irradiation dose rate is 2.75 Gyh -1 ; in this case, results suggested that the fibroblasts should be used immediately after irradiation. (author)

  2. Therapeutic effects of anti-CD115 monoclonal antibody in mouse cancer models through dual inhibition of tumor-associated macrophages and osteoclasts.

    Directory of Open Access Journals (Sweden)

    Laetitia Fend

    Full Text Available Tumor progression is promoted by Tumor-Associated Macrophages (TAMs and metastasis-induced bone destruction by osteoclasts. Both myeloid cell types depend on the CD115-CSF-1 pathway for their differentiation and function. We used 3 different mouse cancer models to study the effects of targeting cancer host myeloid cells with a monoclonal antibody (mAb capable of blocking CSF-1 binding to murine CD115. In mice bearing sub-cutaneous EL4 tumors, which are CD115-negative, the anti-CD115 mAb depleted F4/80(+ CD163(+ M2-type TAMs and reduced tumor growth, resulting in prolonged survival. In the MMTV-PyMT mouse model, the spontaneous appearance of palpable mammary tumors was delayed when the anti-CD115 mAb was administered before malignant transition and tumors became palpable only after termination of the immunotherapy. When administered to mice already bearing established PyMT tumors, anti-CD115 treatment prolonged their survival and potentiated the effect of chemotherapy with Paclitaxel. As shown by immunohistochemistry, this therapeutic effect correlated with the depletion of F4/80(+CD163(+ M2-polarized TAMs. In a breast cancer model of bone metastasis, the anti-CD115 mAb potently blocked the differentiation of osteoclasts and their bone destruction activity. This resulted in the inhibition of cancer-induced weight loss. CD115 thus represents a promising target for cancer immunotherapy, since a specific blocking antibody may not only inhibit the growth of a primary tumor through TAM depletion, but also metastasis-induced bone destruction through osteoclast inhibition.

  3. Comparison of Adipose-Derived and Bone Marrow Mesenchymal Stromal Cells in a Murine Model of Crohn's Disease.

    Science.gov (United States)

    Xie, Minghao; Qin, Huabo; Luo, Qianxin; He, Xiaosheng; He, Xiaowen; Lan, Ping; Lian, Lei

    2017-01-01

    Mesenchymal stromal cells (MSCs) have been used in the treatment of Crohn's disease (CD) because of the immunomodulatory ability. The aim of this study was to investigate the therapeutic effect of adipose-derived MSCs (AD-MSCs) and to compare the therapeutic effect of AD-MSCs with that of bone marrow MSCs (BM-MSCs) in a murine model of CD. Murine colitis model of CD was created by trinitrobenzene sulfonic acid (TNBS). Twelve hours after treatment with TNBS, the mouse model was injected with MSCs intraperitoneally. Real-time polymerase chain reaction and immunohistochemistry staining were used to measure the expression levels of inflammatory cytokines in colonic tissues to investigate the therapeutic effect of AD-MSCs. The ten-day survival was recorded after infusion of MSCs. Intraperitoneal injection of MSCs alleviated the clinical and histopathologic severity of intestinal inflammation, and improved the survival of the TNBS-induced mouse model of CD. AD-MSCs could effectively increase the expression of interleukin-10 and reduce the secretion of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-12, and vascular endothelial growth factor. The mucosal injury was repaired by AD-MSCs. These effects were comparable between AD-MSCs and BM-MSCs. The therapeutic effect appears similar between AD-MSCs and BM-MSCs in treating CD. AD-MSCs may be a potential alternative of cell-based therapy for CD.

  4. Phage-display libraries of murine and human antibody Fab fragments

    DEFF Research Database (Denmark)

    Engberg, J; Andersen, P S; Nielsen, L K

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...

  5. L1 cell adhesion molecule as a potential therapeutic target in murine models of endometriosis using a monoclonal antibody approach.

    Directory of Open Access Journals (Sweden)

    Cássia G T Silveira

    Full Text Available BACKGROUND/AIMS: The neural cell adhesion molecule L1CAM is a transmembrane glycoprotein abnormally expressed in tumors and previously associated with cell proliferation, adhesion and invasion, as well as neurite outgrowth in endometriosis. Being an attractive target molecule for antibody-based therapy, the present study assessed the ability of the monoclonal anti-L1 antibody (anti-L1 mAb to impair the development of endometriotic lesions in vivo and endometriosis-associated nerve fiber growth. METHODS AND RESULTS: Endometriosis was experimentally induced in sexually mature B6C3F1 (n=34 and CD-1 nude (n=21 mice by autologous and heterologous transplantation, respectively, of endometrial fragments into the peritoneal cavity. Transplantation was confirmed four weeks post-surgery by in vivo magnetic resonance imaging and laparotomy, respectively. Mice were then intraperitoneally injected with anti-L1 mAb or an IgG isotype control antibody twice weekly, over a period of four weeks. Upon treatment completion, mice were sacrificed and endometrial implants were excised, measured and fixed. Endometriosis was histologically confirmed and L1CAM was detected by immunohistochemistry. Endometriotic lesion size was significantly reduced in anti-L1-treated B6C3F1 and CD-1 nude mice compared to mice treated with control antibody (P<0.05. Accordingly, a decreased number of PCNA positive epithelial and stromal cells was detected in autologously and heterologously induced endometriotic lesions exposed to anti-L1 mAb treatment. Anti-L1-treated mice also presented a diminished number of intraperitoneal adhesions at implantation sites compared with controls. Furthermore, a double-blind counting of anti-neurofilament L stained nerves revealed significantly reduced nerve density within peritoneal lesions in anti-L1 treated B6C3F1 mice (P=0.0039. CONCLUSIONS: Local anti-L1 mAb treatment suppressed endometriosis growth in B6C3F1 and CD-1 nude mice and exerted a potent

  6. Comparison of vectorial ion transport in primary murine airway and human sinonasal air-liquid interface cultures, models for studies of cystic fibrosis, and other airway diseases.

    Science.gov (United States)

    Zhang, Shaoyan; Fortenberry, James A; Cohen, Noam A; Sorscher, Eric J; Woodworth, Bradford A

    2009-01-01

    The purpose of this study was to compare vectorial ion transport within murine trachea, murine nasal septa, and human sinonasal cultured epithelium. Our hypothesis is that murine septal epithelium, rather than trachea, will more closely mimic the electrophysiology properties of human sinonasal epithelium. Epithelium from murine trachea, murine septa, and human sinonasal tissue were cultured at an air-liquid interface to confluence and full differentiation. A limited number of homozygous dF508 epithelia were also cultured. Monolayers were mounted in modified Ussing chambers to investigate pharmacologic manipulation of ion transport. The change in forskolin-stimulated current (delta-I(SC), expressed as micro-A/cm(2)) in murine septal (n = 19; 16.84 +/- 2.09) and human sinonasal (n = 18; 12.15 +/- 1.93) cultures was significantly increased over murine tracheal cultures (n = 15; 6.75 +/- 1.35; p = 0.035 and 0.0005, respectively). Forskolin-stimulated I(SC) was inhibited by the specific cystic fibrosis transmembrane regulator (CFTR) inhibitor INH-172 (5 microM). No forskolin-stimulated I(SC) was shown in cultures of dF508 homozygous murine septal epithelium (n = 3). Murine septal I(SC) was largely inhibited by amiloride (12.03 +/- 0.66), whereas human sinonasal cultures had a very limited response (0.70 +/- 0.47; p < 0.0001). The contribution of CFTR to stimulated chloride current as measured by INH-172 was highly significantly different between all groups (murine septa, 19.51 +/- 1.28; human sinonasal, 11.12 +/- 1.58; murine trachea, 4.85 +/- 0.49; p < 0.0001). Human sinonasal and murine septal epithelial cultures represent a useful model for studying CFTR activity and may provide significant advantages over lower airway tissues for investigating upper and lower respiratory pathophysiology.

  7. Friend and Moloney murine leukemia viruses specifically recombine with different endogenous retroviral sequences to generate mink cell focus-forming viruses.

    Science.gov (United States)

    Evans, L H; Cloyd, M W

    1985-01-01

    A group of mink cell focus-forming (MCF) viruses was derived by inoculation of NFS/N mice with Moloney murine leukemia virus (Mo-MuLV 1387) and was compared to a similarly derived group of MCF viruses from mice inoculated with Friend MuLV (Fr-MuLV 57). Antigenic analyses using monoclonal antibodies specific for MCF virus and xenotropic MuLV envelope proteins and genomic structural analyses by RNase T1-resistant oligonucleotide finger-printing indicated that the Moloney and Friend MCF viruses arose by recombination of the respective ecotropic MuLVs with different endogenous retrovirus sequences of NFS mice.

  8. Tropism, Cytotoxicity, and Inflammatory Properties of Two Envelope Genes of Murine Leukemia Virus Type-Endogenous Retroviruses of C57BL/6J Mice

    Directory of Open Access Journals (Sweden)

    Young-Kwan Lee

    2011-01-01

    Full Text Available Envelope (env proteins of certain endogenous retroviruses (ERVs participate in various pathophysiological processes. In this study, we characterized pathophysiologic properties of two murine leukemia virus-type ERV (MuLV-ERV env genes cloned from the ovary of C57BL/6J mice. The two env genes (named ENVOV1 and ENVOV2, with 1,926\\,bp coding region, originated from two MuLV-ERV loci on chromosomes 8 and 18, respectively. ENVOV1 and ENVOV2 were ~75 kDa and predominantly expressed on the cell membrane. They were capable of producing pseudotype murine leukemia virus virions. Tropism trait and infectivity of ENVOV2 were similar to the polytropic env; however, ENVOV1 had very low level of infectivity. Overexpression of ENVOV2, but not ENVOV1, exerted cytotoxic effects and induced expression of COX-2, IL-1β, IL-6, and iNOS. These findings suggest that the ENVOV1 and ENVOV2 are capable of serving as an env protein for virion assembly, and they exert differential cytotoxicity and modulation of inflammatory mediators.

  9. Biological markers as predictors of radiosensitivity in syngeneic murine tumors

    International Nuclear Information System (INIS)

    Chang, Sei Kyung; Shin, Hyun Soo; Seong, Jin Sil; Kim, Sung Hee

    2006-01-01

    We investigated whether a relationship exists between tumor control dose 50 (TCD 50 ) or tumor growth delay (TGD) and radiation induced apoptosis (RIA) in syngeneic murine tumors. Also we investigated the biological markers that can predict radiosensitivity in murine tumor system through analysis of relationship between TCD 50 , TGD, RIA and constitutive expression levels of the genetic products regulating RIA. Syngeneic murine tumors such as ovarian adenocarcinoma, mammary carcinoma, squamous cell carcinoma, fibrosarcoma, hepatocarcinoma were used in this study. C3H/HeJ mice were bred and maintained in our specific pathogen free mouse colony and were 8 ∼ 12 weeks old when used for the experiments. The tumors, growing in the right hind legs of mice, were analyzed for TCD 50 , TGD, and RIA at 8 mm in diameter. The tumors were also analyzed for the constitutive expression levels of p53, p21 WAF1/CIP1 , BAX, Bcl-2, Bcl-x L , Bcl-x S , and p34. Correlation analysis was performed whether the level of RIA were correlated with TCD 50 or TGD, and the constitutive expression levels of genetic products regulating RIA were correlated with TCD 50 , TGD, RIA. The level of RIA showed a significant positive correlation (R = 0.922, ρ = 0.026) with TGD, and showed a trend to correlation (R = -0.848), marginally significant correlation with TCD 50 (ρ = 0.070). It indicates that tumors that respond to radiation with high percentage of apoptosis were more radiosensitive. The constitutive expression levels of p21 WAF1/CIP1 and p34 showed a significant correlation either with TCD 50 (R = 0.893, ρ = 0.041 and R = 0.904, ρ = 0.035) or with TGD (R = -0.922, ρ 0.026 and R = -0.890, ρ = 0.043). The tumors with high constitutive expression levels of p21 WAF1/CIP1 or p34 were less radiosensitive than those with low expression. Radiosensitivity may be predicted with the level of RIA in murine tumors. The constitutive expression levels of p21 WAF1/CIP1 or p34 can be used as biological

  10. Biological markers as predictors of radiosensitivity in syngeneic murine tumors

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Sei Kyung; Shin, Hyun Soo [Bundang CHA General Hospital, Seongnam (Korea, Republic of); Seong, Jin Sil; Kim, Sung Hee [Yonsei Cancer Center, Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2006-06-15

    We investigated whether a relationship exists between tumor control dose 50 (TCD{sub 50}) or tumor growth delay (TGD) and radiation induced apoptosis (RIA) in syngeneic murine tumors. Also we investigated the biological markers that can predict radiosensitivity in murine tumor system through analysis of relationship between TCD{sub 50}, TGD, RIA and constitutive expression levels of the genetic products regulating RIA. Syngeneic murine tumors such as ovarian adenocarcinoma, mammary carcinoma, squamous cell carcinoma, fibrosarcoma, hepatocarcinoma were used in this study. C3H/HeJ mice were bred and maintained in our specific pathogen free mouse colony and were 8 {approx} 12 weeks old when used for the experiments. The tumors, growing in the right hind legs of mice, were analyzed for TCD{sub 50}, TGD, and RIA at 8 mm in diameter. The tumors were also analyzed for the constitutive expression levels of p53, p21{sup WAF1/CIP1}, BAX, Bcl-2, Bcl-x{sub L}, Bcl-x{sub S}, and p34. Correlation analysis was performed whether the level of RIA were correlated with TCD{sub 50} or TGD, and the constitutive expression levels of genetic products regulating RIA were correlated with TCD{sub 50}, TGD, RIA. The level of RIA showed a significant positive correlation (R = 0.922, {rho} = 0.026) with TGD, and showed a trend to correlation (R = -0.848), marginally significant correlation with TCD{sub 50} ({rho} = 0.070). It indicates that tumors that respond to radiation with high percentage of apoptosis were more radiosensitive. The constitutive expression levels of p21{sup WAF1/CIP1} and p34 showed a significant correlation either with TCD{sub 50} (R = 0.893, {rho} = 0.041 and R = 0.904, {rho} = 0.035) or with TGD (R = -0.922, {rho} 0.026 and R = -0.890, {rho} = 0.043). The tumors with high constitutive expression levels of p21{sup WAF1/CIP1} or p34 were less radiosensitive than those with low expression. Radiosensitivity may be predicted with the level of RIA in murine tumors. The

  11. Turnover of T cells in murine gammaherpesvirus 68-infected mice

    DEFF Research Database (Denmark)

    Hamilton-Easton, A M; Christensen, Jan Pravsgaard; Doherty, P C

    1999-01-01

    Respiratory challenge of C57BL/6 mice with murine gammaherpesvirus 68 induces proliferation of T lymphocytes early after infection, as evidenced by incorporation of the DNA precursor bromodeoxyuridine. Using pulse-chase analysis, splenic and peripheral blood activated T lymphocytes were found...

  12. Local IL-23 expression in murine vaginal candidiasis and its relationship with infection and immune status.

    Science.gov (United States)

    Wu, Yan; Tan, Zhijian; Liu, Zhixiang; Xia, Dechao; Li, Jiawen

    2006-01-01

    To investigate the expression of vaginal IL-23 and its role in experimental murine vaginal candidiasis and its relationship with infection and immune status, immuno-competent (group A) and immuno-suppressed (group B) murine models of vaginal candidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls (group C). The level of IL-23 p19 mRNA in murine vaginal tissue was determined by RT-PCR. Significantly increased levels of IL-23p19mRNA were observed on the 4th, the 7th and 14th day after inoculation in immuno-competent group when compared with that in control group (Pvaginal candidiasis and has a protective function during infection. Low vaginal IL-23 level may correlate with the increased susceptibility to Candida albicans in immuno-suppressed group.

  13. [Evaluation of Fusarium spp. pathogenicity in plant and murine models].

    Science.gov (United States)

    Forero-Reyes, Consuelo M; Alvarado-Fernández, Angela M; Ceballos-Rojas, Ana M; González-Carmona, Lady C; Linares-Linares, Melva Y; Castañeda-Salazar, Rubiela; Pulido-Villamarín, Adriana; Góngora-Medina, Manuel E; Cortés-Vecino, Jesús A; Rodríguez-Bocanegra, María X

    The genus Fusarium is widely recognized for its phytopathogenic capacity. However, it has been reported as an opportunistic pathogen in immunocompetent and immunocompromised patients. Thus, it can be considered a microorganism of interest in pathogenicity studies on different hosts. Therefore, this work evaluated the pathogenicity of Fusarium spp. isolates from different origins in plants and animals (murine hosts). Twelve isolates of Fusarium spp. from plants, animal superficial mycoses, and human superficial and systemic mycoses were inoculated in tomato, passion fruit and carnation plants, and in immunocompetent and immunosuppressed BALB/c mice. Pathogenicity tests in plants did not show all the symptoms associated with vascular wilt in the three plant models; however, colonization and necrosis of the vascular bundles, regardless of the species and origin of the isolates, showed the infective potential of Fusarium spp. in different plant species. Moreover, the pathogenicity tests in the murine model revealed behavioral changes. It was noteworthy that only five isolates (different origin and species) caused mortality. Additionally, it was observed that all isolates infected and colonized different organs, regardless of the species and origin of the isolates or host immune status. In contrast, the superficial inoculation test showed no evidence of epidermal injury or colonization. The observed results in plant and murine models suggest the pathogenic potential of Fusarium spp. isolates in different types of hosts. However, further studies on pathogenicity are needed to confirm the multihost capacity of this genus. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  14. Sex differences in the MB49 syngeneic, murine model of bladder cancer.

    Science.gov (United States)

    White-Gilbertson, Shai; Davis, Megan; Voelkel-Johnson, Christina; Kasman, Laura M

    The MB49 syngeneic, murine model of bladder cancer has been widely used for more than 35 years. In humans, bladder cancer is one third as prevalent in women as in men, with a trend toward lower prevalence in parous compared to nulliparous women. Our objective was to determine if the MB49 bladder cancer model reproduces the sex differences observed in humans, and to determine its sensitivity to testosterone and the pregnancy hormone, human chorionic gonadotropin (hCG). Male and female C57BL/6 mice were implanted with MB49 murine bladder cancer cells, and observed for tumor growth. MB49 dose responses to hCG and dihydrotestosterone were determined in vitro . MB49 tumor growth was significantly greater in male mice than female mice. Pregnancy did not affect MB49 tumor growth in female mice. MB49 cells did not proliferate in response to hCG in vitro and the functional receptor for gonadotropins was absent. Dihydrotestosterone strongly stimulated growth of MB49 cells in vitro . The MB49 murine model of bladder cancer reproduced some aspects of the sex differences observed in humans. Our results suggest that testosterone may stimulate MB49 cell proliferation, which may explain the more rapid MB49 tumor growth observed in male mice.

  15. Development of a murine model of acute radiation encephalopathy

    International Nuclear Information System (INIS)

    Xing Yigang; Tang Yamei; Liu Jun; Sun Ying

    2003-01-01

    Objective: To develop a murine model of acute radiation encephalopathy. Methods: A total of 40 rats were subjected to local γ-irradiation to the brain with the dosage of 7 Gy/d for 6 consecutive days. The amount of food intake, hairs and skin of irradiated field, body weight, general activities, CNS symptoms and signs were examined and recorded after irradiation. On day 3, 7, 14 and 30, the brain tissue was removed to observe histopathologic changes. Results: During the first two days after irradiation, the irradiated rats were agitated, and the amount of food intake decreased from day 2 onwards. No serious skin reaction to irradiation was observed. Survived rats had normal activities without any abnormal nervous signs. Histopathologic changes showed slight neuronal degeneration, smaller cell body, red-colored cytoplasm, disappearance of Nissl body, vacuolation, typical cell shrinkage, chromatin condensation and nuclear divergence. On the 14th and 30th days, hypochromatism, loose and reticular necrotic foci were found in some samples. Conclusion: The murine model of acute radiation encephalopathy is useful and practical in radiobiological studies

  16. Evidence for differential changes of junctional complex proteins in murine neurocysticercosis dependent upon CNS vasculature.

    Science.gov (United States)

    Alvarez, Jorge I; Teale, Judy M

    2007-09-12

    The delicate balance required to maintain homeostasis of the central nervous system (CNS) is controlled by the blood-brain barrier (BBB). Upon injury, the BBB is disrupted compromising the CNS. BBB disruption has been represented as a uniform event. However, our group has shown in a murine model of neurocysticercosis (NCC) that BBB disruption varies depending upon the anatomical site/vascular bed analyzed. In this study further understanding of the mechanisms of BBB disruption was explored in blood vessels located in leptomeninges (pial vessels) and brain parenchyma (parenchymal vessels) by examining the expression of junctional complex proteins in murine brain infected with Mesocestoides corti. Both pial and parenchymal vessels from mock infected animals showed significant colocalization of junctional proteins and displayed an organized architecture. Upon infection, the patterned organization was disrupted and in some cases, particular tight junction and adherens junction proteins were undetectable or appeared to be undergoing proteolysis. The extent and timing of these changes differed between both types of vessels (pial vessel disruption within days versus weeks for parenchymal vessels). To approach potential mechanisms, the expression and activity of matrix metalloproteinase-9 (MMP-9) were evaluated by in situ zymography. The results indicated an increase in MMP-9 activity at sites of BBB disruption exhibiting leukocyte infiltration. Moreover, the timing of MMP activity in pial and parenchymal vessels correlated with the timing of permeability disruption. Thus, breakdown of the BBB is a mutable process despite the similar structure of the junctional complex between pial and parenchymal vessels and involvement of MMP activity.

  17. JST Thesaurus Headwords and Synonyms: murine hepatitis virus [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term murine hepatitis virus 名詞 一般 * * * * マウス肝炎...ウイルス マウスカンエンウイルス マウスカンエンウイルス Thesaurus2015 200906024862381907 C LS07 UNKNOWN_2 murine hepatitis virus

  18. Flow Cytometry Assessment of In Vitro Generated CD138+ Human Plasma Cells

    Directory of Open Access Journals (Sweden)

    Rayelle Itoua Maïga

    2014-01-01

    Full Text Available The in vitro CD40-CD154 interaction promotes human B lymphocytes differentiation into plasma cells. Currently, CD138 is the hallmark marker enabling the detection of human plasma cells, both in vitro and in vivo; its presence can be monitored by flow cytometry using a specific antibody. We have developed a culture system allowing for the differentiation of memory B lymphocytes. In order to detect the newly formed plasma cells, we have compared their staining using five anti-CD138 monoclonal antibodies (mAbs. As a reference, we also tested human cell lines, peripheral blood mononuclear cells, and bone marrow samples. The five anti-CD138 mAbs stained RPMI-8226 cells (>98% with variable stain index (SI. The highest SI was obtained with B-A38 mAb while the lowest SI was obtained with DL-101 and 1D4 mAbs. However, the anti-CD138 mAbs were not showing equivalent CD138+ cells frequencies within the generated plasma cells. B-A38, B-B4, and MI-15 were similar (15–25% while DL-101 mAb stained a higher proportion of CD138-positive cells (38–42%. DL-101 and B-A38 mAbs stained similar populations in bone marrow samples but differed in their capacity to bind to CD138high and CD138lo cell lines. In conclusion, such cellular fluctuations suggest heterogeneity in human plasma cell populations and/or in CD138 molecules.

  19. Prevention of murine cerebral malaria by low-dose cyclosporin A.

    Science.gov (United States)

    Grau, G E; Gretener, D; Lambert, P H

    1987-08-01

    The effects of cyclosporin A (CsA) were investigated in an experimental model of cerebral malaria. In this model, Plasmodium berghei ANKA-infected CBA/Ca mice develop a clinically and histologically characterized neurological syndrome which is considered to be the result of immunopathological reactions mediated by L3T4+ T cells. It was shown that CsA displayed a strong protective effect on neurological complications when given at a dose 1 mg/kg/day for 5 consecutive days (Days 4-8), which had no effect on the parasite. Paradoxically, this protection against neurological complications was not seen when parasiticidal doses were used during this limited 5-day period. A similar protective effect was observed with two CsA derivatives, C5-34 and H7-94. The mechanisms by which CsA and the two derivatives could prevent murine cerebral malaria are unknown but can be related to exquisite effects on some lymphocyte functions. In view of these results, it might be conceivable to investigate the benefits of using low doses of CsA in man, in conjunction with the classical antiparasite therapy, for the management of cerebral malaria.

  20. Major immunogenic proteins of phocid herpes-viruses and their relationships to proteins of canine and feline herpesviruses.

    Science.gov (United States)

    Harder, T C; Harder, M; de Swart, R L; Osterhaus, A D; Liess, B

    1998-04-01

    The immunogenic proteins of cells infected with the alpha- or the gamma-herpesvirus of seals, phocid herpesvirus-1 and -2 (PhHV-1, -2), were examined in radioimmunoprecipitation assays as a further step towards the development of a PhHV-1 vaccine. With sera obtained from convalescent seals of different species or murine monoclonal antibodies (Mabs), at least seven virus-induced glycoproteins were detected in lysates of PhHV-1-infected CrFK cells. A presumably disulphide-linked complex composed of glycoproteins of 59, 67 and 113/120 kDa, expressed on the surface of infected cells, was characterized as a major immunogenic infected cell protein of PhHV-1. This glycoprotein complex has previously been identified as the proteolytically cleavable glycoprotein B homologue of PhHV-1 (14). At least three distinct neutralization-relevant epitopes were operationally mapped, by using Mabs, on the glycoprotein B of PhHV-1. Among the infected cell proteins of the antigenically closely related feline and canine herpesvirus, the glycoprotein B equivalent proved to be the most highly conserved glycoprotein. Sera obtained from different seal species from Arctic, Antarctic, and European habitats did not precipitate uniform patterns of infected cell proteins from PhHV-1-infected cell lysates although similar titres of neutralizing antibodies were displayed. Thus, antigenic differences among the alphaherpesvirus species prevalent in the different pinniped populations cannot be excluded. PhHV-2 displayed a different pattern of infected cell proteins and only limited cross-reactivity to PhHV-1 at the protein level was detected, which is in line with its previous classification as a distinct species, based on nucleotide sequence analysis, of the gammaherpesvirus linenge. A Mab raised against PhHV-2 and specific for a major glycoprotein of 117 kDa, cross reacted with the glycoprotein B of PhHV-1. The 117-kDa glycoprotein could represent the uncleaved PhHV-2 glycoprotein B homologue.

  1. [Nuclear matrix organization of the chromocenters in cultured murine fibroblasts].

    Science.gov (United States)

    Sheval', E V; Poliakov, V Iu

    2010-01-01

    In the current work, the structural organization of nuclear matrix of pericentromeric heterochromatin blocks (chromocenters) inside cultured murine fibroblasts was investigated. After 2 M NaCl extraction without DNase I treatment, chromocenters were extremely swelled, and it was impossible to detect them using conventional electron microscopy. Using immunogolding with anti-topoisomerase IIalpha antibody, we demonstrated that residual chromocenters were subdivided into numerous discrete aggregates. After 2 M NaCl extraction with DNase I treatment, the residual chromocenters appeared as a dense meshwork of thin fibers, and using this feature, the residual chromocenters were easily distinguished from the rest of nuclear matrix. After extraction with dextran sulfate and heparin, the chromocenters were decondensed, and chromatin complexes having rosette organization (central core from which numerous DNA fibers radiated) were seen. Probably, the appearance of these rosettes was a consequence of incomplete chromatin extraction. Thus, the nuclear matrix of pericentromeric chromosome regions in cultured murine fibroblasts differs morphologically from the rest of nuclear matrix.

  2. Cytoplasmic superoxide dismutase and catalase activity and resistance to radiation lethality in murine tumor cells

    International Nuclear Information System (INIS)

    Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Rosenberg, S.O.

    1986-01-01

    Reduced species of molecular oxygen are produced by the interaction of ionizing radiation with aqueous solutions containing molecular oxygen. The enzymes catalase and superoxide dismutase (SOD) are thought to function in vivo as scavengers of metabolically produced peroxide and superoxide respectively. SOD has been shown to protect against the lethal effects of ionizing radiation in vitro and in vivo. The authors have investigated the relationship between the cytosolic SOD catalase content and the sensitivity to radiation lethality of a number of murine cell lines (402AX, EL-4, MB-2T3, MB-4, MEL, P-815, SAI, SP-2, and SV-3T3). K/sub i/(CN - ) for murine Cu-Zn-SOD was determined to be 6.8 x 10 -6 M. No cytosolic Mn-SOD activity was found in any of the cell lines studied. No correlation was found between the cytosolic Cu-Zn-SOD or cytosolic catalase activity and the resistance to radiation lethality or the murine cell lines studied

  3. Characterization of immortalized MARCO and SR-AI/II-deficient murine alveolar macrophage cell lines

    Directory of Open Access Journals (Sweden)

    Imrich Amy

    2008-05-01

    Full Text Available Abstract Background Alveolar macrophages (AM avidly bind and ingest unopsonized inhaled particles and bacteria through class A scavenger receptors (SRAs MARCO and SR-AI/II. Studies to characterize the function of these SRAs have used AMs from MARCO or SR-AI/II null mice, but this approach is limited by the relatively low yield of AMs. Moreover, studies using both MARCO and SR-AI/II-deficient (MS-/- mice have not been reported yet. Hence, we sought to develop continuous cell lines from primary alveolar macrophages from MS-/- mice. Results We used in vitro infection of the primary AMs with the J2 retrovirus carrying the v-raf and v-myc oncogenes. Following initial isolation in media supplemented with murine macrophage colony-stimulating factor (M-CSF, we subcloned three AM cell lines, designated ZK-1, ZK-2 and ZK-6. These cell lines grow well in RPMI-1640-10% FBS in the absence of M-CSF. These adherent but trypsin-sensitive cell lines have a doubling time of approximately 14 hours, exhibit typical macrophage morphology, and express macrophage-associated cell surface Mac-1 (CD11b and F4/80 antigens. The cell lines show robust Fc-receptor dependent phagocytosis of opsonized red blood cells. Similar to freshly isolated AMs from MS-/- mice, the cell lines exhibit decreased phagocytosis of unopsonized titanium dioxide (TiO2, fluorescent latex beads and bacteria (Staphylococcus aureus compared with the primary AMs from wild type (WT C57BL/6 mice. Conclusion Our results indicated that three contiguous murine alveolar macrophage cell lines with MS-/- (ZK1, ZK2 and ZK6 were established successfully. These cell lines demonstrated macrophage morphology and functional activity. Interestingly, similar to freshly isolated AMs from MS-/- mice, the cell lines have a reduced, but not absent, ability to bind and ingest particles, with an altered pattern of blockade by scavenger receptor inhibitors. These cell lines will facilitate in vitro studies to further define

  4. MC148 encoded by human molluscum contagiosum poxvirus is an antagonist for human but not murine CCR8

    DEFF Research Database (Denmark)

    Lüttichau, H R; Gerstoft, J; Schwartz, T W

    2001-01-01

    The viral CC chemokines MC148, encoded by the poxvirus molluscum contagiosum, and viral macrophage inflammatory protein (vMIP)-I and vMIP-II, encoded by human herpesvirus 8, were probed on the murine CC receptor (CCR) 8 in parallel with human CCR8. In calcium mobilization assays, vMIP-I acted...... as a high-affinity agonist, whereas vMIP-II acted as a low-affinity antagonist on the murine CCR8 as well as the human CCR8. MC148 was found to bind and block responses through the human CCR8 with high affinity, but surprisingly MC148 was unable to bind and block responses through the murine CCR8. Because...

  5. Radiobiological studies on target cell populations in murine bone marrow transplantation recipients.

    NARCIS (Netherlands)

    van Os, Ronald Peter

    1994-01-01

    The experiments presented in this thesis were designed to investigate the role of total body irradiation (TBI) in conditioning murine recipients of syngeneic and allogeneic bone marrow transplantation (BMT). ... Zie: Summary

  6. Production of Autoantibodies by Murine B-1a Cells Stimulated with Helicobacter pylori Urease through Toll-Like Receptor 2 Signaling ▿ †

    Science.gov (United States)

    Kobayashi, Fumiko; Watanabe, Eri; Nakagawa, Yohko; Yamanishi, Shingo; Norose, Yoshihiko; Fukunaga, Yoshitaka; Takahashi, Hidemi

    2011-01-01

    Helicobacter pylori infection is associated with several autoimmune diseases, in which autoantibody-producing B cells must be activated. Among these B cells, CD5-positive B-1a cells from BALB/c mice were confirmed to secrete autoantibodies when cocultured with purified H. pylori urease in the absence of T cells. To determine the mechanisms for autoantibody production, CD5-positive B-1a cells were sorted from murine spleen cells and stimulated with either purified H. pylori urease or H. pylori coated onto plates (referred to hereafter as plate-coated H. pylori), and autoantibody production was measured by enzyme-linked immunosorbent assay (ELISA). Complete urease was not secreted from H. pylori but was visually expressed over the bacterium-like endotoxin. Urease-positive plated-coated H. pylori stimulated B-1a cells to produce autoantibodies, although urease-deficient isotype-matched H. pylori did not. Autoantibody secretion by B-1a cells was inhibited when bacteria were pretreated with anti-H. pylori urease-specific antibody having neutralizing ability against urease enzymatic activity but not with anti-H. pylori urease-specific antibody without neutralizing capacity. The B-1a cells externally express various Toll-like receptors (TLRs): TLR1, TLR2, TLR4, and TLR6. Among the TLRs, blocking of TLR2 on B-1a cells with a specific monoclonal antibody (MAb), T2.5, inhibited autoantibody secretion when B-1a cells were stimulated with plate-coated H. pylori or H. pylori urease. Moreover, B-1a cells from TLR2-knockout mice did not produce those autoantibodies. The present study provides evidence that functional urease expressed on the surface of H. pylori will directly stimulate B-1a cells via innate TLR2 to produce various autoantibodies and may induce autoimmune disorders. PMID:21947775

  7. Bystander-mediated genomic instability after high LET radiation in murine primary haemopoietic stem cells

    International Nuclear Information System (INIS)

    Bowler, Deborah A.; Moore, Stephen R.; Macdonald, Denise A.; Smyth, Sharon H.; Clapham, Peter; Kadhim, Munira A.

    2006-01-01

    Communication between irradiated and unirradiated (bystander) cells can result in responses in unirradiated cells that are similar to responses in their irradiated counterparts. The purpose of the current experiment was to test the hypothesis that bystander responses will be similarly induced in primary murine stem cells under different cell culture conditions. The experimental systems used here, co-culture and media transfer, are similar in that they both restrict communication between irradiated and bystander cells to media borne factors, but are distinct in that with the media transfer technique, cells can only communicate after irradiation, and with co-culture, cells can communication before, during and after irradiation. In this set of parallel experiments, cell type, biological endpoint, and radiation quality and dose, were kept constant. In both experimental systems, clonogenic survival was significantly decreased in all groups, whether irradiated or bystander, suggesting a substantial contribution of bystander effects (BE) to cell killing. Genomic instability (GI) was induced under all radiation and bystander conditions in both experiments, including a situation where unirradiated cells were incubated with media that had been conditioned for 24 h with irradiated cells. The appearance of delayed aberrations (genomic instability) 10-13 population doublings after irradiation was similar to the level of initial chromosomal damage, suggesting that the bystander factor is able to induce chromosomal alterations soon after irradiation. Whether these early alterations are related to those observed at later timepoints remains unknown. These results suggest that genomic instability may be significantly induced in a bystander cell population whether or not cells communicate during irradiation

  8. Responses of the Murine Myeloid Colony-Forming Cell to Ansamycin Antibiotics

    Science.gov (United States)

    Horoszewicz, Julius S.; Carter, William A.

    1974-01-01

    The in vitro susceptibility of murine myeloid colony-forming cells to the antiproliferative activities of three ansamycin antibiotics was determined. These cells were found to be 10- to 40-fold more susceptible than the corresponding human ones. PMID:4151701

  9. JST Thesaurus Headwords and Synonyms: murine leukemia virus [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term murine leukemia virus 名詞 一般 * * *... * マウス白血病ウイルス マウスハッケツビョウウイルス マウスハッケツビョーウイルス Thesaurus2015 200906060491156251 C LS07 UNKNOWN_2 murine leukemia virus

  10. Forced recombination of psi-modified murine leukaemia virus-based vectors with murine leukaemia-like and VL30 murine endogenous retroviruses

    DEFF Research Database (Denmark)

    Mikkelsen, J G; Lund, Anders Henrik; Duch, M

    1999-01-01

    Co-encapsidation of retroviral RNAs into virus particles allows for the generation of recombinant proviruses through events of template switching during reverse transcription. By use of a forced recombination system based on recombinational rescue of replication- defective primer binding site-imp....... We note that recombination-based rescue of primer binding site knock-out retroviral vectors may constitute a sensitive assay to register putative genetic interactions involving endogenous retroviral RNAs present in cells of various species.......-impaired Akv-MLV-derived vectors, we here examine putative genetic interactions between vector RNAs and copackaged endogenous retroviral RNAs of the murine leukaemia virus (MLV) and VL30 retroelement families. We show (i) that MLV recombination is not blocked by nonhomology within the 5' untranslated region...... harbouring the supposed RNA dimer-forming cis -elements and (ii) that copackaged retroviral RNAs can recombine despite pronounced sequence dissimilarity at the cross-over site(s) and within parts of the genome involved in RNA dimerization, encapsidation and strand transferring during reverse transcription...

  11. Transgene stability for three replication competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Duch, M.; Carrasco, M.L.; Jespersen, T.

    2004-01-01

    cassette consisting of an internal ribosome entry site followed by the enhanced green fluorescent protein coding sequence inserted in different configurations into murine leukemia virus genomes. In two of the constructs, the insert was located in the upstream part of the U3 region while in the third...

  12. Intestinal IgA responses to Giardia muris in mice depleted of helper T lymphocytes and in immunocompetent mice.

    Science.gov (United States)

    Heyworth, M F

    1989-04-01

    Immunocompetent mice infected with Giardia muris generate an intestinal antibody response to this parasite and clear G. muris infection. Previous work has shown that G. muris infection is prolonged in mice that have been depleted of helper (CD4+) T lymphocytes by treatment with a monoclonal antibody (mAb) directed against the murine CD4 antigen. The aim of the present study was to compare the intestinal anti-Giardia antibody response in immunocompetent mice and in mice depleted of helper T (Th) lymphocytes by treatment with anti-CD4 mAb. Immunocompetent mice generated an IgA response to G. muris, as judged by the presence of IgA on Giardia trophozoites harvested from the intestine of these animals more than 10 days after the start of the infection. The anti-Giardia IgA response was impaired in mice depleted of Th lymphocytes, as judged by virtual absence of immunofluorescent staining of trophozoites from these animals for surface-bound IgA. Clearance of G. muris infection was impaired by treatment of mice with anti-CD4 mAb. The results suggest that Th (CD4+) lymphocytes are important for the generation of a local IgA response against G. muris trophozoites in the mouse intestine and that IgA anti-trophozoite antibody may contribute to the clearance of G. muris from the intestine of immunocompetent mice.

  13. Stability of monoclonal antibodies at high-concentration

    DEFF Research Database (Denmark)

    Neergaard, Martin S; Nielsen, Anders D; Parshad, Henrik

    2014-01-01

    Few studies have so far directly compared the impact of antibody subclass on protein stability. This case study investigates two mAbs (one IgG1 and one IgG4 ) with identical variable region. Investigations of mAbs that recognize similar epitopes are necessary to identify possible differences betw...

  14. Adherence of murine lymphocytes to high endothelial venules in vitro and its radiation effect

    Energy Technology Data Exchange (ETDEWEB)

    Lixin, Liu; Zijun, Mao; Zhiwei, Yin [Suzhou Medical Coll., JS (China). Dept. of Pathophysiology

    1991-02-01

    Using the assay of specific adhesion of lymphocytes to high endothelial venules (HEV) on cryostat sections of mesenteric lymph nodes (MLN), the effects of different doses (0, 1, 2, 4, 8 Gy) of {sup 60}Co {gamma}-ray irradiation of murine MLN lymphocytes in vitro on adhesion to normal HEV was observed. The results showed that in the irradiated murine MLN lymphocytes the ability to adhere to HEV of normal MLN was reduced. Statistical significance was revealed at 2, 4, 8 Gy irradiations. This results suggests that irradiation can inhibit the specific recognition and adhesion of lymphocytes to HEV to a certain extent.

  15. Production, Characterization and Antioxidant Potential of Protease from Streptomyces sp. MAB18 Using Poultry Wastes

    Directory of Open Access Journals (Sweden)

    Panchanathan Manivasagan

    2013-01-01

    Full Text Available Poultry waste is an abundant renewable source for the recovery of several value-added metabolites with potential industrial applications. This study describes the production of protease on poultry waste, with the subsequent use of the same poultry waste for the extraction of antioxidants. An extracellular protease-producing strain was isolated from Cuddalore coast, India, and identified as Streptomyces sp. MAB18. Its protease was purified 17.13-fold with 21.62% yield with a specific activity of 2398.36 U/mg and the molecular weight was estimated as 43 kDa. The enzyme was optimally active at pH 8–10 and temperature 50–60°C and it was most stable up to pH 12 and 6–12% of NaCl concentration. The enzyme activity was reduced when treated with Hg2+, Pb2+, and SDS and stimulated by Fe2+, Mg2+, Triton X-100, DMSO (dimethyl sulfoxide, sodium sulphite, and β-mercaptoethanol. Furthermore, the antioxidant activities of protease were evaluated using in vitro antioxidant assays, such as DPPH radical-scavenging activity, O2 scavenging activity, NO scavenging activity, Fe2+ chelating activity, and reducing power. The enzyme showed important antioxidant potential with an IC50 value of 78±0.28 mg/mL. Results of the present study indicate that the poultry waste-derived protease may be useful as supplementary protein and antioxidant in the animal feed formulations.

  16. Analysis of the epitope structure of Plum pox virus coat protein.

    Science.gov (United States)

    Candresse, Thierry; Saenz, Pilar; García, Juan Antonio; Boscia, Donato; Navratil, Milan; Gorris, Maria Teresa; Cambra, Mariano

    2011-05-01

    Typing of the particular Plum pox virus (PPV) strain responsible in an outbreak has important practical implications and is frequently performed using strain-specific monoclonal antibodies (MAbs). Analysis in Western blots of the reactivity of 24 MAbs to a 112-amino-acid N-terminal fragment of the PPV coat protein (CP) expressed in Escherichia coli showed that 21 of the 24 MAbs recognized linear or denaturation-insensitive epitopes. A series of eight C-truncated CP fragments allowed the mapping of the epitopes recognized by the MAbs. In all, 14 of them reacted to the N-terminal hypervariable region, defining a minimum of six epitopes, while 7 reacted to the beginning of the core region, defining a minimum of three epitopes. Sequence comparisons allowed the more precise positioning of regions recognized by several MAbs, including those recognized by the 5B-IVIA universal MAb (amino acids 94 to 100) and by the 4DG5 and 4DG11 D serogroup-specific MAbs (amino acids 43 to 64). A similar approach coupled with infectious cDNA clone mutagenesis showed that a V74T mutation in the N-terminus of the CP abolished the binding of the M serogroup-specific AL MAb. Taken together, these results provide a detailed positioning of the epitopes recognized by the most widely used PPV detection and typing MAbs.

  17. Puerarin Facilitates T-Tubule Development of Murine Embryonic Stem Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Lu Wang

    2014-07-01

    Full Text Available Aims: The embryonic stem cell-derived cardiomyocytes (ES-CM is one of the promising cell sources for repopulation of damaged myocardium. However, ES-CMs present immature structure, which impairs their integration with host tissue and functional regeneration. This study used murine ES-CMs as an in vitro model of cardiomyogenesis to elucidate the effect of puerarin, the main compound found in the traditional Chinese medicine the herb Radix puerariae, on t-tubule development of murine ES-CMs. Methods: Electron microscope was employed to examine the ultrastructure. The investigation of transverse-tubules (t-tubules was performed by Di-8-ANEPPS staining. Quantitative real-time PCR was utilized to study the transcript level of genes related to t-tubule development. Results: We found that long-term application of puerarin throughout cardiac differentiation improved myofibril array and sarcomeres formation, and significantly facilitated t-tubules development of ES-CMs. The transcript levels of caveolin-3, amphiphysin-2 and junctophinlin-2, which are crucial for the formation and development of t-tubules, were significantly upregulated by puerarin treatment. Furthermore, puerarin repressed the expression of miR-22, which targets to caveolin-3. Conclusion: Our data showed that puerarin facilitates t-tubule development of murine ES-CMs. This might be related to the repression of miR-22 by puerarin and upregulation of Cav3, Bin1 and JP2 transcripts.

  18. Establishment of a method of murine obestatin RIA and its primary application

    International Nuclear Information System (INIS)

    Yan Guangtao Lin Ji; Hao Xiuhua; Xue Hui; Den Zihui; Di Dongdong

    2009-01-01

    Objective: To develop a method of murine obestatin RIA and study the role obestatin played in traumatic stress responses. Methods: New Zealand white rabbits were immunized with synthetic murine obestatin to obtain anti-serum, while chloramines-T method was used to iodinate obestatin antigen and a method of RIA for obestatin was established. A mouse sepsis model made with cecal ligation and puncture was established. Serum obestatin levels in the sacrificed models were determined with this method of RIA at 3h, 6h, 9h after the cecum puncture respectively (5 animals each time) as well as in 5 animals after sham operation. Results: Both the shape of standard curve and metrical results of the obestatin RIA were satisfactory. Serum obstatin levels at 6h after injury were significantly higher than those in animals with sham operation out the levels at 3h and 9h were not much different from those in sham operation animals. Meanwhile, the levels at 6h after injury expressed a trend to be significantly higher than that at 3h or 9h after injury. Conclusion: The established method for murine obestatin RIA is highly sensitive, simple and reliable, and it can be used to detect samples from rats and mice. Obestatin may be a traumatic stress factor participating in the modulation of homeostasis after sepsis. (authors)

  19. The pharmacokinetics and metabolism of lumiracoxib in chimeric humanized and murinized FRG mice.

    Science.gov (United States)

    Dickie, A P; Wilson, C E; Schreiter, K; Wehr, R; Wilson, E M; Bial, J; Scheer, N; Wilson, I D; Riley, R J

    2017-07-01

    The pharmacokinetics and metabolism of lumiracoxib were studied, after administration of single 10mg/kg oral doses to chimeric liver-humanized and murinized FRG mice. In the chimeric humanized mice, lumiracoxib reached peak observed concentrations in the blood of 1.10±0.08μg/mL at 0.25-0.5h post-dose with an AUC inf of 1.74±0.52μgh/mL and an effective half-life for the drug of 1.42±0.72h (n=3). In the case of the murinized animals peak observed concentrations in the blood were determined as 1.15±0.08μg/mL at 0.25h post-dose with an AUC inf of 1.94±0.22μgh/mL and an effective half-life of 1.28±0.02h (n=3). Analysis of blood indicated only the presence of unchanged lumiracoxib. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles obtained in humanized mice were different compared to murinized animals with e.g., a higher proportion of the dose detected in the form of acyl glucuronide metabolites and much reduced amounts of taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57bl/6J mice and humans, revealed a greater though not complete match between chimeric humanized mice and humans, such that the liver-humanized FRG model may represent a useful approach to assessing the biotransformation of such compounds in humans. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. CD44 antibodies and immune thrombocytopenia in the amelioration of murine inflammatory arthritis.

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    Patrick J Mott

    Full Text Available Antibodies to CD44 have been used to successfully ameliorate murine models of autoimmune disease. The most often studied disease model has been murine inflammatory arthritis, where a clear mechanism for the efficacy of CD44 antibodies has not been established. We have recently shown in a murine passive-model of the autoimmune disease immune thrombocytopenia (ITP that some CD44 antibodies themselves can induce thrombocytopenia in mice, and the CD44 antibody causing the most severe thrombocytopenia (IM7, also is known to be highly effective in ameliorating murine models of arthritis. Recent work in the K/BxN serum-induced model of arthritis demonstrated that antibody-induced thrombocytopenia reduced arthritis, causing us to question whether CD44 antibodies might primarily ameliorate arthritis through their thrombocytopenic effect. We evaluated IM7, IRAWB14.4, 5035-41.1D, KM201, KM114, and KM81, and found that while all could induce thrombocytopenia, the degree of protection against serum-induced arthritis was not closely related to the length or severity of the thrombocytopenia. CD44 antibody treatment was also able to reverse established inflammation, while thrombocytopenia induced by an anti-platelet antibody targeting the GPIIbIIIa platelet antigen, could not mediate this effect. While CD44 antibody-induced thrombocytopenia may contribute to some of its therapeutic effect against the initiation of arthritis, for established disease there are likely other mechanisms contributing to its efficacy. Humans are not known to express CD44 on platelets, and are therefore unlikely to develop thrombocytopenia after CD44 antibody treatment. An understanding of the relationship between arthritis, thrombocytopenia, and CD44 antibody treatment remains critical for continued development of CD44 antibody therapeutics.

  1. Noncanonical microRNAs and endogenous siRNAs in lytic infection of murine gammaherpesvirus.

    Directory of Open Access Journals (Sweden)

    Jing Xia

    Full Text Available MicroRNA (miRNA and endogenous small interfering RNA (endo-siRNA are two essential classes of small noncoding RNAs (sncRNAs in eukaryotes. The class of miRNA is diverse and there exist noncanonical miRNAs that bypass the canonical miRNA biogenesis pathway. In order to identify noncanonical miRNAs and endo-siRNAs responding to virus infection and study their potential function, we sequenced small-RNA species from cells lytically infected with murine gammaherpesvirus 68 (MHV68. In addition to three novel canonical miRNAs in mouse, two antisense miRNAs in virus and 25 novel noncanonical miRNAs, including miRNAs derived from transfer RNAs, small nucleolar RNAs and introns, in the host were identified. These noncanonical miRNAs exhibited features distinct from that of canonical miRNAs in lengths of hairpins, base pairings and first nucleotide preference. Many of the novel miRNAs are conserved in mammals. Besides several known murine endo-siRNAs detected by the sequencing profiling, a novel locus in the mouse genome was identified to produce endo-siRNAs. This novel endo-siRNA locus is comprised of two tandem inverted B4 short interspersed nuclear elements (SINEs. Unexpectedly, the SINE-derived endo-siRNAs were found in a variety of sequencing data and virus-infected cells. Moreover, a murine miRNA was up-regulated more than 35 fold in infected than in mock-treated cells. The putative targets of the viral and the up-regulated murine miRNAs were potentially involved in processes of gene transcription and protein phosphorylation, and localized to membranes, suggesting their potential role in manipulating the host basal immune system during lytic infection. Our results extended the number of noncanonical miRNAs in mammals and shed new light on their potential functions of lytic infection of MHV68.

  2. Limited role of murine ATM in oncogene-induced senescence and p53-dependent tumor suppression.

    Directory of Open Access Journals (Sweden)

    Alejo Efeyan

    Full Text Available Recent studies in human fibroblasts have provided a new general paradigm of tumor suppression according to which oncogenic signaling produces DNA damage and this, in turn, results in ATM/p53-dependent cellular senescence. Here, we have tested this model in a variety of murine experimental systems. Overexpression of oncogenic Ras in murine fibroblasts efficiently induced senescence but this occurred in the absence of detectable DNA damage signaling, thus suggesting a fundamental difference between human and murine cells. Moreover, lung adenomas initiated by endogenous levels of oncogenic K-Ras presented abundant senescent cells, but undetectable DNA damage signaling. Accordingly, K-Ras-driven adenomas were also senescent in Atm-null mice, and the tumorigenic progression of these lesions was only modestly accelerated by Atm-deficiency. Finally, we have examined chemically-induced fibrosarcomas, which possess a persistently activated DNA damage response and are highly sensitive to the activity of p53. We found that the absence of Atm favored genomic instability in the resulting tumors, but did not affect the persistent DNA damage response and did not impair p53-dependent tumor suppression. All together, we conclude that oncogene-induced senescence in mice may occur in the absence of a detectable DNA damage response. Regarding murine Atm, our data suggest that it plays a minor role in oncogene-induced senescence or in p53-dependent tumor suppression, being its tumor suppressive activity probably limited to the maintenance of genomic stability.

  3. Trp53 activity is repressed in radio-adapted cultured murine limb bud cells

    International Nuclear Information System (INIS)

    Vares, Guillaume; Wang, Bing; Tanaka, Kaoru; Shang, Yi; Fujita, Kazuko; Hayata, Isamu; Nenoi, Mitsuru

    2011-01-01

    Understanding the effects of ionizing radiation (IR) at low dose in fetal models is of great importance, because the fetus is considered to be at the most radiosensitive stage of the development and prenatal radiation might influence subsequent development. We previously demonstrated the existence of an adaptive response (AR) in murine fetuses after pre-exposure to low doses of X-rays. Trp53-dependent apoptosis was suggested to be responsible for the teratogenic effects of IR; decreased apoptosis was observed in adapted animals. In this study, in order to investigate the role of Trp53 in AR, we developed a new model of irradiated micromass culture of fetal limb bud cells, which replicated proliferation, differentiation and response to IR in murine embryos. Murine fetuses were exposed to whole-body priming irradiation of 0.3 Gy or 0.5 Gy at embryonic day 11 (E11). Limb bud cells (collected from digital ray areas exhibiting radiation-induced apoptosis) were cultured and exposed to a challenging dose of 4 Gy at E12 equivalent. The levels of Trp53 protein and its phosphorylated form at Ser18 were investigated. Our results suggested that the induction of AR in mouse embryos was correlated with a repression of Trp53 activity. (author)

  4. Riboflavin-ultraviolet light pathogen reduction treatment does not impact the immunogenicity of murine red blood cells.

    Science.gov (United States)

    Tormey, Christopher A; Santhanakrishnan, Manjula; Smith, Nicole H; Liu, Jingchun; Marschner, Susanne; Goodrich, Raymond P; Hendrickson, Jeanne E

    2016-04-01

    Ultraviolet (UV) illumination/pathogen reduction effectively inactivates white blood cells (WBCs) in whole blood. Given that cotransfused WBCs may impact recipient immune responses, we hypothesized that pathogen reduction of whole blood may alter responses to RBC antigens. Transgenic mice expressing a model (HOD) antigen, authentic human (hGPA or KEL) antigens, or natural fluorescence (uGFP) on their RBCs were utilized as blood donors. Recipients were transfused with fresh whole blood to which riboflavin had been added or fresh whole blood treated by UV illumination/pathogen reduction treatment after the addition of riboflavin. Posttransfusion RBC recovery, survival, and alloimmunization were measured by flow cytometry. UV illumination/pathogen reduction treatment did not alter RBC antigen expression, and recipients of treated syngeneic RBCs had persistently negative direct antiglobulin tests. Greater than 75% of treated and untreated syngeneic RBCs were recovered 24 hours posttransfusion in all experiments, although alterations in the long-term posttransfusion survival of treated RBCs were observed. Treated and untreated KEL RBCs induced similar recipient alloimmune responses, with all recipients making anti-KEL glycoprotein immunoglobulins (p > 0.05). Alloimmune responses to treated HOD or hGPA RBCs were no different from untreated RBCs (p > 0.05). Pathogen inactivation treatment of fresh whole murine blood with riboflavin and UV illumination does not impact the rate or magnitude of RBC alloimmunization to three distinct RBC antigens. Further, UV illumination/pathogen reduction appears safe from an immunohematologic standpoint, with no immunogenic neoantigens detected on treated murine RBCs. Future studies with fresh and stored human RBCs are warranted to confirm these findings. © 2015 AABB.

  5. Protective antitumor activity induced by a fusion vaccine with murine ...

    African Journals Online (AJOL)

    Targeting angiogenesis is an effective strategy for anticancer therapy. The vascular endothelialcadherin (VE-cad) regulated angiogenesis is a potential target for anti-angiogenesis. Here, we develop a fusion vaccine plasmid DNA pSec-MBD2-VE-cad from VE-cad and murine beta defensin2 (MBD2) to induce immunity for ...

  6. Thrombopoietin inhibits murine mast cell differentiation

    Science.gov (United States)

    Martelli, Fabrizio; Ghinassi, Barbara; Lorenzini, Rodolfo; Vannucchi, Alessandro M; Rana, Rosa Alba; Nishikawa, Mitsuo; Partamian, Sandra; Migliaccio, Giovanni; Migliaccio, Anna Rita

    2009-01-01

    We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin, or addition of this growth factor to bone marrow-derived mast cell cultures, severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target anti-apoptotic gene Bcl2. PMID:18276801

  7. Human glioblastoma-associated microglia/monocytes express a distinct RNA profile compared to human control and murine samples.

    Science.gov (United States)

    Szulzewsky, Frank; Arora, Sonali; de Witte, Lot; Ulas, Thomas; Markovic, Darko; Schultze, Joachim L; Holland, Eric C; Synowitz, Michael; Wolf, Susanne A; Kettenmann, Helmut

    2016-08-01

    Glioblastoma (GBM) is the most aggressive brain tumor in adults. It is strongly infiltrated by microglia and peripheral monocytes that support tumor growth. In the present study we used RNA sequencing to compare the expression profile of CD11b(+) human glioblastoma-associated microglia/monocytes (hGAMs) to CD11b(+) microglia isolated from non-tumor samples. Hierarchical clustering and principal component analysis showed a clear separation of the two sample groups and we identified 334 significantly regulated genes in hGAMs. In comparison to human control microglia hGAMs upregulated genes associated with mitotic cell cycle, cell migration, cell adhesion, and extracellular matrix organization. We validated the expression of several genes associated with extracellular matrix organization in samples of human control microglia, hGAMs, and the hGAMs-depleted fraction via qPCR. The comparison to murine GAMs (mGAMs) showed that both cell populations share a significant fraction of upregulated transcripts compared with their respective controls. These genes were mostly related to mitotic cell cycle. However, in contrast to murine cells, human GAMs did not upregulate genes associated to immune activation. Comparison of human and murine GAMs expression data to several data sets of in vitro-activated human macrophages and murine microglia showed that, in contrast to mGAMs, hGAMs share a smaller overlap to these data sets in general and in particular to cells activated by proinflammatory stimulation with LPS + INFγ or TNFα. Our findings provide new insights into the biology of human glioblastoma-associated microglia/monocytes and give detailed information about the validity of murine experimental models. GLIA 2016 GLIA 2016;64:1416-1436. © 2016 Wiley Periodicals, Inc.

  8. Ultrastructural study on experimental infection of rotavirus in a murine heterologous model

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    Selma Majerowicz

    1994-09-01

    Full Text Available Viral replication, histopathological and ultrastructural changes were observed for a period of nine days in the small intestine of suckling mice infected with a simian rotavirus (SA11. Samples taken from duodenum, jejunun and ileum were prepared for light microscopy, transmission and scanning electron microscopy analysis. Histopathologic effect could be detected within 8 hr post-infection, when only a few altered cells were observed. Damage was extensive after 16 hr post-infection, showing swollen enterocytes and reduced and irregularly oriented microvilli at intestinal villi tips. Virus particles were detected at 16 and 48 hr post-infection, budding from the viroplasm into the rough endoplasmic reticulum cisternae in ileum enterocytes. Clear evidence of viral replication, observed by electron microscopy was not described before in heterologous murine models. Regeneration of the intestinal villi began at the third day post-infection. Despite some differences observed in clinical symptoms and microscopic analysis of homologous and heterologous rotavirus infections, we concluded that mechanisms of heterologous rotavirus infection in mice follow similar patterns to those observed in the homologous models.

  9. Localized conformational interrogation of antibody and antibody-drug conjugates by site-specific carboxyl group footprinting.

    Science.gov (United States)

    Pan, Lucy Yan; Salas-Solano, Oscar; Valliere-Douglass, John F

    Establishing and maintaining conformational integrity of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) during development and manufacturing is critical for ensuring their clinical efficacy. As presented here, we applied site-specific carboxyl group footprinting (CGF) for localized conformational interrogation of mAbs. The approach relies on covalent labeling that introduces glycine ethyl ester tags onto solvent-accessible side chains of protein carboxylates. Peptide mapping is used to monitor the labeling kinetics of carboxyl residues and the labeling kinetics reflects the conformation or solvent-accessibility of side chains. Our results for two case studies are shown here. The first study was aimed at defining the conformational changes of mAbs induced by deglycosylation. We found that two residues in C H 2 domain (D268 and E297) show significantly enhanced side chain accessibility upon deglycosylation. This site-specific result highlighted the advantage of monitoring the labeling kinetics at the amino acid level as opposed to the peptide level, which would result in averaging out of highly localized conformational differences. The second study was designed to assess conformational effects brought on by conjugation of mAbs with drug-linkers. All 59 monitored carboxyl residues displayed similar solvent-accessibility between the ADC and mAb under native conditions, which suggests the ADC and mAb share similar side chain conformation. The findings are well correlated and complementary with results from other assays. This work illustrated that site-specific CGF is capable of pinpointing local conformational changes in mAbs or ADCs that might arise during development and manufacturing. The methodology can be readily implemented within the industry to provide comprehensive conformational assessment of these molecules.

  10. Transcriptomic Analysis of Lung Tissue from Cigarette Smoke-Induced Emphysema Murine Models and Human Chronic Obstructive Pulmonary Disease Show Shared and Distinct Pathways.

    Science.gov (United States)

    Yun, Jeong H; Morrow, Jarrett; Owen, Caroline A; Qiu, Weiliang; Glass, Kimberly; Lao, Taotao; Jiang, Zhiqiang; Perrella, Mark A; Silverman, Edwin K; Zhou, Xiaobo; Hersh, Craig P

    2017-07-01

    Although cigarette smoke (CS) is the primary risk factor for chronic obstructive pulmonary disease (COPD), the underlying molecular mechanisms for the significant variability in developing COPD in response to CS are incompletely understood. We performed lung gene expression profiling of two different wild-type murine strains (C57BL/6 and NZW/LacJ) and two genetic models with mutations in COPD genome-wide association study genes (HHIP and FAM13A) after 6 months of chronic CS exposure and compared the results to human COPD lung tissues. We identified gene expression patterns that correlate with severity of emphysema in murine and human lungs. Xenobiotic metabolism and nuclear erythroid 2-related factor 2-mediated oxidative stress response were commonly regulated molecular response patterns in C57BL/6, Hhip +/- , and Fam13a -/- murine strains exposed chronically to CS. The CS-resistant Fam13a -/- mouse and NZW/LacJ strain revealed gene expression response pattern differences. The Fam13a -/- strain diverged in gene expression compared with C57BL/6 control only after CS exposure. However, the NZW/LacJ strain had a unique baseline expression pattern, enriched for nuclear erythroid 2-related factor 2-mediated oxidative stress response and xenobiotic metabolism, and converged to a gene expression pattern similar to the more susceptible wild-type C57BL/6 after CS exposure. These results suggest that distinct molecular pathways may account for resistance to emphysema. Surprisingly, there were few genes commonly modulated in mice and humans. Our study suggests that gene expression responses to CS may be largely species and model dependent, yet shared pathways could provide biologically significant insights underlying individual susceptibility to CS.

  11. Rabbit antibodies for hormone receptors and HER2 evaluation in breast cancer Anticorpos de coelho para avaliação de receptores hormonais e HER2 em câncer de mama

    Directory of Open Access Journals (Sweden)

    Rafael Malagoli Rocha

    2009-01-01

    Full Text Available BACKGROUND: Novel rabbit monoclonal antibodies (RabMab for estrogen (ER, progesterone (PR receptors and HER2 evaluation by immunohistochemistry have recently been commercially released. We compared the RabMab anti-ER, anti-PR and anti-HER2 to mouse monoclonal antibodies (Mab using tissue microarrays (TMA of breast carcinomas. METHODS: Two TMA containing breast carcinomas were built. Sections were immunostained using anti-ER and anti-PR, Mab and RabMab. The sections stained for ER and PR were evaluated considering positive those tumors in which more than 1% of the tumor cell nuclei stained moderate or strong. For HER2, the immunostained sections were evaluated using the ASCO/CAP guidelines for HER2. Chromogenic in situ hybridization (CISH was used as the gold standard for HER2 evaluation. CISH was evaluated using the Zymed HER2 CISH interpretation guidelines. RESULTS: RabMab against ER have similar staining patterns compared to the 6F11 (Mab, but stronger than 1D5 (Mab from three different suppliers. The RabMab against PR provide stronger and sharper immunohistochemical signals compared to Mab. The detection of HER2 protein overexpression was more prevalent with the polyclonal antibodies and RabMab than with the Mab. These were more specific than the RabMab, which were more sensitive when compared to CISH. CONCLUSION: The novel RabMab against ER and PR showed higher intensity of staining than the Mab. The RabMab against HER2 is more sensitive than Mab, however, Mab presented more specificity than RabMab when compared to CISH for HER2 evaluation of breast carcinomas.OBJETIVOS: Novos anticorpos monoclonais de coelho (RabMab para a avaliação imuno-histoquímica de receptores de estrógeno (RE, progesterona (RP e HER2 foram lançados comercialmente. Comparamos os RabMab anti-RE, anti-RP e anti-HER2 com os anticorpos monoclonais de camundongo (Mab utilizando tissue microarrays (TMA de carcinomas de mama. MÉTODOS: Foram construídos dois TMAs de

  12. Proteolytically modified human beta 2-microglobulin augments the specific cytotoxic activity in murine mixed lymphocyte culture

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Claësson, M H

    1987-01-01

    the endogenous production of interleukin 2 in the MLC culture; monoclonal antibody which reacts with both the native beta 2-m and M-beta 2-m molecule blocks the augmentation of cytotoxic T lymphocyte production induced by M-beta 2-m; murine as well as human MLC responder cells can proteolytically modify native......A proteolytically modified form of beta 2-microglobulin (beta 2-m) present in the serum of patients suffering from autoimmune, immunodeficient diseases and cancer has been reported in the literature. In the present study we show that human beta 2-m as well as the proteolytically modified human form...... (M-beta 2-m) bind to murine lymphocytes expressing H-2 class I antigens; M-beta 2-m, when added at day 0 and 1 of culture in nanomolar concentrations to a one-way murine allogeneic mixed lymphocyte culture (MLC) augments the generation of specific cytotoxic T lymphocytes; M-beta 2-m increases...

  13. Murine protein H is comprised of 20 repeating units, 61 amino acids in length

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Tack, B F

    1986-01-01

    A cDNA library constructed from size-selected (greater than 28 S) poly(A)+ RNA isolated from the livers of C57B10. WR mice was screened by using a 249-base-pair (bp) cDNA fragment encoding 83 amino acid residues of human protein H as a probe. Of 120,000 transformants screened, 30 hybridized......, 448 bp of 3'-untranslated sequence, and a polyadenylylated tail of undetermined length. Murine pre-protein H was deduced to consist of an 18-amino acid signal peptide and 1216 residues of H-protein sequence. Murine H was composed of 20 repetitive units, each about 61 amino acid residues in length...

  14. Diagnosing hypoxia in murine models of rheumatoid arthritis from reflectance multispectral images

    Science.gov (United States)

    Glinton, Sophie; Naylor, Amy J.; Claridge, Ela

    2017-07-01

    Spectra computed from multispectral images of murine models of Rheumatoid Arthritis show a characteristic decrease in reflectance within the 600-800nm region which is indicative of the reduction in blood oxygenation and is consistent with hypoxia.

  15. Disruption of canonical TGFβ-signaling in murine coronary progenitor cells by low level arsenic

    Energy Technology Data Exchange (ETDEWEB)

    Allison, Patrick; Huang, Tianfang; Broka, Derrick; Parker, Patti [Department of Pharmacology and Toxicology College of Pharmacy, Southwest Environmental Health Sciences Center, Steele Children' s Research Center and Bio5 Institute, University of Arizona, Tucson, AZ 85721 (United States); Barnett, Joey V. [Department of Pharmacology, Vanderbilt Medical University, Nashville, TN (United States); Camenisch, Todd D., E-mail: camenisch@pharmacy.arizona.edu [Department of Pharmacology and Toxicology College of Pharmacy, Southwest Environmental Health Sciences Center, Steele Children' s Research Center and Bio5 Institute, University of Arizona, Tucson, AZ 85721 (United States)

    2013-10-01

    Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGFβ family of ligands and receptors is essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronary smooth muscle cells. In this in vitro study, 18 hour exposure to 1.34 μM arsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGFβ2, TGFβ receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGFβ2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34 μM arsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGFβ2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGFβ2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity. - Highlights: • Arsenic blocks TGFβ2 induced expression of EMT genes. • Arsenic blocks TGFβ2 triggered Smad2/3 phosphorylation and nuclear translocation. • Arsenic blocks epicardial cell differentiation into cardiac mesenchyme.

  16. Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

    Science.gov (United States)

    Pissuwan, Dakrong; Valenzuela, Stella M.; Killingsworth, Murray C.; Xu, Xiaoda; Cortie, Michael B.

    2007-12-01

    Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (˜1×105 to 1×1010 W/m2). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5×102 W/m2 being sufficient, provided that a total fluence of ˜30 J/cm2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

  17. Computational identification of epitopes in the glycoproteins of novel bunyavirus (SFTS virus) recognized by a human monoclonal antibody (MAb 4-5)

    Science.gov (United States)

    Zhang, Wenshuai; Zeng, Xiaoyan; Zhang, Li; Peng, Haiyan; Jiao, Yongjun; Zeng, Jun; Treutlein, Herbert R.

    2013-06-01

    In this work, we have developed a new approach to predict the epitopes of antigens that are recognized by a specific antibody. Our method is based on the "multiple copy simultaneous search" (MCSS) approach which identifies optimal locations of small chemical functional groups on the surfaces of the antibody, and identifying sequence patterns of peptides that can bind to the surface of the antibody. The identified sequence patterns are then used to search the amino-acid sequence of the antigen protein. The approach was validated by reproducing the binding epitope of HIV gp120 envelop glycoprotein for the human neutralizing antibody as revealed in the available crystal structure. Our method was then applied to predict the epitopes of two glycoproteins of a newly discovered bunyavirus recognized by an antibody named MAb 4-5. These predicted epitopes can be verified by experimental methods. We also discuss the involvement of different amino acids in the antigen-antibody recognition based on the distributions of MCSS minima of different functional groups.

  18. Evolving GIS technologies in nature conservation and the spatial planning strategy of Tara NP (Serbia as a potential UNECSO MAB reserve

    Directory of Open Access Journals (Sweden)

    Radović Dejan

    2008-01-01

    Full Text Available Mt. Tara NP was nominated in 2004 within the UNESCO - ROSTE programme, for Man and the Biosphere (MAB Reserve status in Serbia as transboundary 'Peace Park' status between Serbia and Bosnia & Herzegovina. Mt. Tara is one of the most important centres of Balkans and European ecosystems and species diversity. They represent a unique example of well preserved forests in south eastern Europe with numerous endemic and relict species of flora and fauna. In this floristic diversity of Mt Tara of the greatest interest is the Serbian (Pančić's spruce Picea omorika. Mt. Tara NP is characterized by specific geomorphologic, hydrologic, geologic, soil and climatic features. The geographical information system (GIS that we have created has proved an excellent tool for the spatial planning strategy in assessment and conservation of all natural characteristics of Mt. Tara NP, and is helpful to Park management for sustainable use of landscape resources. GIS of Mt. Tara NP includes data on natural, artificial and management themes.

  19. Efficacy and immunological actions of FAHF-2 in a murine model of multiple food allergies.

    Science.gov (United States)

    Srivastava, Kamal D; Bardina, Ludmilla; Sampson, Hugh A; Li, Xiu-Min

    2012-05-01

    Food Allergy Herbal Formula-2 (FAHF-2) prevents anaphylaxis in a murine model of peanut allergy. Multiple food allergies (MFA) are common and associated with a higher risk of anaphylaxis. No well-characterized murine model of sensitization to multiple food allergens exists, and no satisfactory therapy for MFA is currently available. To determine the effect of FAHF-2 in a murine model of MFA. C3H/HeJ mice were orally sensitized to peanut, codfish, and egg concurrently. Oral FAHF-2 treatment commenced 1 day after completing sensitization and continued daily for 7 weeks. Mice were subsequently orally challenged with each allergen. Antibodies in sera from mice simultaneously sensitized with peanut, codfish, and egg recognized major allergens of all 3 foods, demonstrating sensitization to multiple unrelated food allergens (MFA mice). Sham-treated MFA mice exhibited anaphylactic symptoms accompanied by elevation of plasma histamine and hypothermia. In contrast, FAHF-2-treated MFA mice showed no anaphylactic symptoms, normal body temperature, and histamine levels after challenge with each allergen. Protection was accompanied by reduction in allergen-specific immunoglobulin E levels. Allergen-stimulated Th2 cytokine interleukin-4 and interleukin-13 production levels decreased, whereas the Th1 cytokine interferon-γ levels were elevated in cultured splenocytes and mesenteric lymph node cells in FAHF-2-treated mice. We established the first murine model of MFA. FAHF-2 prevents peanut, egg, and fish-induced anaphylactic reactions in this model, suggesting that FAHF-2 may have potential for treating human MFA. Copyright © 2012 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  20. Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation

    Science.gov (United States)

    Meng, Qing-Yuan; Akaike, Toshihiro

    2013-03-01

    Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells' self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

  1. Mechanism of infectivity of a murine leukemia virus in adult mice

    International Nuclear Information System (INIS)

    Levy, R.L.; Barrington, M.H.; Lerner, R.A.; Dixon, F.J.

    1976-01-01

    Infection of adult BALB/c mice with murine leukemia virus (MuLV) induces typical thymic lymphomas. Expression of virus was measured by using a radioimmunoassay for murine P-30, a virion core protein. Nineteen days after injection of MuLV-S into adult mice, there were 0.3μg P-30/ml of serum. X-irradiation permitted the early expression of high levels of viremia, when given before or after MuLV-S administration, and it also hastened the development of lymphomas. Seventeen to 21 days after injection of MuLV-S into x-irradiated (600 rads) adult mice, there were 2.7 μg of P-30/ml of serum. The virus produced by infected adult mice was infectious and oncogenic when given to newborn mice. Several lines of evidence are presented that suggest the mechanism by which x-irradiation permits early expession of virion proteins and lymphomas is not immunosuppression

  2. Identification of a murine cysteinyl leukotriene receptor by expression in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Mollerup, Jens; Jørgensen, Sune T.; Hougaard, Charlotte

    2001-01-01

    We report the identification of an EST encoding a murine cysteinyl leukotriene (mCysLT) receptor. LTD4, LTC4 and LTE4 but not LTB4 or various nucleotides activated Ca2+-evoked Cl- currents in mCysLT1 expressing Xenopus laevis oocytes. The response to LTD4 was blocked by MK-571, reduced by pretrea...... by pretreatment with pertussis toxin (PTX), and was partly dependent on extracellular Ca2+. The identified murine CysLT1 receptor differs from the hCysLT1 receptor with regard to PTX sensitivity, receptor-mediated Ca2+ influx, and antagonist sensitivity....

  3. Proteins of bovine viral diarrhea virus: characterization, biotype-specific differences, and immunological properties

    International Nuclear Information System (INIS)

    Donis, R.O.

    1987-01-01

    Virus-specific polypeptides in bovine viral diarrhea-mucosal disease (BVD) virus-infected bovine cells were studied by radiolabeling. A total of 12 polypeptides with apparent Mr of 165, 135, 118, 80, 75, 62, 56-58, 48, 37, 32, 25 and 19 kilodaltons (k) were identified in infected cells. Five glycoproteins were detected in infected cells. Two abundant species had apparent Mr of 48 k and 56-58 k while the minor species had masses of 118, 75 and 65 k. When cells were radiolabeled with L-[ 35 S]-methionine in the presence of tunicamycin the 56-58 k migrated with apparent masses of 54 k and 48-50 K in PAGE. Endoglycosidase F digestion of virus-induced polypeptides caused a 4-6 K reduction in the apparent molecular mass of the 56-58 k yielding a 52 k digested product. Tunicamycin caused a drastic reduction in the yield of infectious virus indicating that the carbohydrate moieties serve a vital role in the infection cycle of BVD virus. The noncytopathic biotype BVD (NCB-BVD) virus isolates can be consistently differentiated from cytopathic biotype BVD (CB-BVD) isolates on the basis of unique polypeptide profiles they induce in the infected cell: the most abundant polypeptide in CB-BVD infected cells is the 80 kD polypeptide while NCB-BVD lack this polypeptide and induce a predominant 118 k polypeptide. A panel of 25 murine monoclonal antibodies (Mabs) against the two major glycoproteins of BVD virus was produced. Based on their viral polypeptide specificity and on their ability to neutralize viral infectivity the Mabs in the panel were divided into 3 classes: Class 1 Mabs reacted with the 56-58 k glycoprotein and neutralized the virus, Class 2 Mabs recognized the 56-58 k glycoprotein but were not neutralizing and Class 3 Mabs reacted with the 48 k glycoprotein and did not neutralize the virus. These results identify the 56-58 k as one of the envelope glycoproteins of BVD virus

  4. Clinical and Epidemiological Characteristics of Scrub Typhus and Murine Typhus among Hospitalized Patients with Acute Undifferentiated Fever in Northern Vietnam

    Science.gov (United States)

    Hamaguchi, Sugihiro; Cuong, Ngo Chi; Tra, Doan Thu; Doan, Yen Hai; Shimizu, Kenta; Tuan, Nguyen Quang; Yoshida, Lay-Myint; Mai, Le Quynh; Duc-Anh, Dang; Ando, Shuji; Arikawa, Jiro; Parry, Christopher M.; Ariyoshi, Koya; Thuy, Pham Thanh

    2015-01-01

    A descriptive study on rickettsiosis was conducted at the largest referral hospital in Hanoi, Vietnam, to identify epidemiological and clinical characteristics of specific rickettsiosis. Between March 2001 and February 2003, we enrolled 579 patients with acute undifferentiated fever (AUF), excluding patients with malaria, dengue fever, and typhoid fever, and serologically tested for Orientia tsutsugamushi and Rickettsia typhi. Of the patients, 237 (40.9%) and 193 (33.3%) had scrub and murine typhus, respectively, and 149 (25.7%) had neither of them (non–scrub and murine typhus [non-ST/MT]). The proportion of murine typhus was highest among patients living in Hanoi whereas that of scrub typhus was highest in national or regional border areas. The presence of an eschar, dyspnea, hypotension, and lymphadenopathy was significantly associated with a diagnosis of scrub typhus (OR = 46.56, 10.90, 9.01, and 7.92, respectively). Patients with murine typhus were less likely to have these findings but more likely to have myalgia, rash, and relative bradycardia (OR = 1.60, 1.56, and 1.45, respectively). Scrub typhus and murine typhus were shown to be common causes of AUF in northern Vietnam although the occurrence of spotted fever group rickettsiae was not determined. Clinical and epidemiological information may help local clinicians make clinical diagnosis of specific rickettsioses in a resource-limited setting. PMID:25778504

  5. Atomic structure of the murine norovirus protruding domain and sCD300lf receptor complex.

    Science.gov (United States)

    Kilic, Turgay; Koromyslova, Anna; Malak, Virginie; Hansman, Grant S

    2018-03-21

    Human noroviruses are the leading cause of acute gastroenteritis in human. Noroviruses also infect animals such as cow, mice, cat, and dog. How noroviruses bind and enter host cells is still incompletely understood. Recently, the type I transmembrane protein CD300lf was recently identified as the murine norovirus receptor, yet it is unclear how the virus capsid and receptor interact at the molecular level. In this study, we determined the X-ray crystal structure of the soluble CD300lf (sCD300lf) and murine norovirus capsid-protruding domain complex at 2.05 Å resolution. We found that the sCD300lf binding site is located on the topside of the protruding domain and involves a network of hydrophilic and hydrophobic interactions. The sCD300lf locked nicely into a complementary cavity on the protruding domain that is additionally coordinated with a positive surface charge on the sCD300lf and a negative surface charge on the protruding domain. Five of six protruding domain residues interacting with sCD300lf were maintained between different murine norovirus strains, suggesting that the sCD300lf was capable of binding to a highly conserved pocket. Moreover, a sequence alignment with other CD300 paralogs showed that the sCD300lf interacting residues were partially conserved in CD300ld, but variable in other CD300 family members, consistent with previously reported infection selectivity. Overall, these data provide insights into how a norovirus engages a protein receptor and will be important for a better understanding of selective recognition and norovirus attachment and entry mechanisms. IMPORTANCE Noroviruses exhibit exquisite host-range specificity due to species-specific interactions between the norovirus capsid protein and host molecules. Given this strict host-range restriction it has been unclear how the viruses are maintained within a species between relatively sporadic epidemics. While much data demonstrates that noroviruses can interact with carbohydrates

  6. Megakaryocytes compensate for Kit insufficiency in murine arthritis.

    Science.gov (United States)

    Cunin, Pierre; Penke, Loka R; Thon, Jonathan N; Monach, Paul A; Jones, Tatiana; Chang, Margaret H; Chen, Mary M; Melki, Imene; Lacroix, Steve; Iwakura, Yoichiro; Ware, Jerry; Gurish, Michael F; Italiano, Joseph E; Boilard, Eric; Nigrovic, Peter A

    2017-05-01

    The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell-deficient murine lines: KitWsh/Wsh, which develops robust arthritis, and KitW/Wv, which does not. Reciprocal bone marrow transplantation between KitW/Wv and KitWsh/Wsh mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In KitW/Wv mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/megakaryocyte markers NF-E2 and glycoprotein VI. In KitW/Wv mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in KitW/Wv mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1-dependent manner. Transfer of WT but not IL-1-deficient megakaryocytes restored arthritis susceptibility to KitW/Wv mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1-driven systemic inflammatory disease.

  7. Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine model of Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Dhawan Gunjan

    2012-07-01

    Full Text Available Abstract Background Microglial activation is an important histologic characteristic of the pathology of Alzheimer’s disease (AD. One hypothesis is that amyloid beta (Aβ peptide serves as a specific stimulus for tyrosine kinase-based microglial activation leading to pro-inflammatory changes that contribute to disease. Therefore, inhibiting Aβ stimulation of microglia may prove to be an important therapeutic strategy for AD. Methods Primary murine microglia cultures and the murine microglia cell line, BV2, were used for stimulation with fibrillar Aβ1-42. The non-receptor tyrosine kinase inhibitor, dasatinib, was used to treat the cells to determine whether Src family kinase activity was required for the Aβ stimulated signaling response and subsequent increase in TNFα secretion using Western blot analysis and enzyme-linked immunosorbent assay (ELISA, respectively. A histologic longitudinal analysis was performed using an AD transgenic mouse model, APP/PS1, to determine an age at which microglial protein tyrosine kinase levels increased in order to administer dasatinib via mini osmotic pump diffusion. Effects of dasatinib administration on microglial and astroglial activation, protein phosphotyrosine levels, active Src kinase levels, Aβ plaque deposition, and spatial working memory were assessed via immunohistochemistry, Western blot, and T maze analysis. Results Aβ fibrils stimulated primary murine microglia via a tyrosine kinase pathway involving Src kinase that was attenuated by dasatinib. Dasatinib administration to APP/PS1 mice decreased protein phosphotyrosine, active Src, reactive microglia, and TNFα levels in the hippocampus and temporal cortex. The drug had no effect on GFAP levels, Aβ plaque load, or the related tyrosine kinase, Lyn. These anti-inflammatory changes correlated with improved performance on the T maze test in dasatinib infused animals compared to control animals. Conclusions These data suggest that amyloid

  8. Murine model of long term obstructive jaundice

    Science.gov (United States)

    Aoki, Hiroaki; Aoki, Masayo; Yang, Jing; Katsuta, Eriko; Mukhopadhyay, Partha; Ramanathan, Rajesh; Woelfel, Ingrid A.; Wang, Xuan; Spiegel, Sarah; Zhou, Huiping; Takabe, Kazuaki

    2016-01-01

    Background With the recent emergence of conjugated bile acids as signaling molecules in cancer, a murine model of obstructive jaundice by cholestasis with long-term survival is in need. Here, we investigated the characteristics of 3 murine models of obstructive jaundice. Methods C57BL/6J mice were used for total ligation of the common bile duct (tCL), partial common bile duct ligation (pCL), and ligation of left and median hepatic bile duct with gallbladder removal (LMHL) models. Survival was assessed by Kaplan-Meier method. Fibrotic change was determined by Masson-Trichrome staining and Collagen expression. Results 70% (7/10) of tCL mice died by Day 7, whereas majority 67% (10/15) of pCL mice survived with loss of jaundice. 19% (3/16) of LMHL mice died; however, jaundice continued beyond Day 14, with survival of more than a month. Compensatory enlargement of the right lobe was observed in both pCL and LMHL models. The pCL model demonstrated acute inflammation due to obstructive jaundice 3 days after ligation but jaundice rapidly decreased by Day 7. The LHML group developed portal hypertension as well as severe fibrosis by Day 14 in addition to prolonged jaundice. Conclusion The standard tCL model is too unstable with high mortality for long-term studies. pCL may be an appropriate model for acute inflammation with obstructive jaundice but long term survivors are no longer jaundiced. The LHML model was identified to be the most feasible model to study the effect of long-term obstructive jaundice. PMID:27916350

  9. Inhibition of Helicobacter pylori CagA-Induced Pathogenesis by Methylantcinate B from Antrodia camphorata

    Directory of Open Access Journals (Sweden)

    Chun-Jung Lin

    2013-01-01

    Full Text Available The bacterial pathogen Helicobacter pylori (Hp is the leading risk factor for the development of gastric cancer. Hp virulence factor, cytotoxin-associated gene A (CagA interacted with cholesterol-enriched microdomains and leads to induction of inflammation in gastric epithelial cells (AGS. In this study, we identified a triterpenoid methylantcinate B (MAB from the medicinal mushroom Antrodia camphoratawhich inhibited the translocation and phosphorylation of CagA and caused a reduction in hummingbird phenotype in HP-infected AGS cells. Additionally, MAB suppressed the Hp-induced inflammatory response by attenuation of NF-κB activation, translocation of p65 NF-κB, and phosphorylation of IκB-α, indicating that MAB modulates CagA-mediated signaling pathway. Additionally, MAB also suppressed the IL-8 luciferase activity and its secretion in HP-infected AGS cells. On the other hand, molecular structure simulations revealed that MAB interacts with CagA similarly to that of cholesterol. Moreover, binding of cholesterol to the immobilized CagA was inhibited by increased levels of MAB. Our results demonstrate that MAB is the first natural triterpenoid which competes with cholesterol bound to CagA leading to attenuation of Hp-induced pathogenesis of epithelial cells. Thus, this study indicates that MAB may have a scope to develop as a therapeutic candidate against Hp CagA-induced inflammation.

  10. Generation and Application of Monoclonal Antibody Against Lycopene.

    Science.gov (United States)

    Tsibezov, Valeriy V; Bashmakov, Yuriy K; Pristenskiy, Dmitry V; Zigangirova, Naylia A; Kostina, Ludmila V; Chalyk, Natalya E; Kozlov, Alexey Y; Morgunova, Elena Y; Chernyshova, Marina P; Lozbiakova, Marina V; Kyle, Nigel H; Petyaev, Ivan M

    2017-04-01

    A monoclonal antibody (Mab) against lycopene was developed from hybridoma clones obtained from BALB/c mice immunized with trans-isomer of lycopene (t-lycopene, t-LC) conjugated with colloidal gold particles. An alternating immunization schedule which included injection of both formulations of immunogen (without and with Freund's adjuvant) was most effective in the elucidation of a measurable immune response to the t-Lycopene conjugate. Selected hybridoma clones were able to produce an Mab positive in competition assay. In particular, preincubation of 6B9 Mabs with t-LC abolished the ability of 6B9 Mabs to bind LC in the competition assay. Mabs produced by other clones (4F10, 4A3, and 3B12) worked similarly. Analysis of antigen specificity showed that 6B9 Mab raised against t-LC did not recognize other carotenoids such as lutein and carotene. Mab 6B9 was shown to recognize lycopene on a glass surface and in the settings of indirect immunofluorescence experiments performed in cultured hepatocytes and alveolar macrophages incubated with and without lycopene, as well as in sebum and corneocyte specimens from the skin of volunteers supplemented with nutraceutical formulation of lycopene. Newly generated Mabs against lycopene may provide a valuable tool for different analytical assays of lycopene content in various biological, agricultural, and food products.

  11. Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

    International Nuclear Information System (INIS)

    Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won; Bae, Jin Ung; Kim, Gyu Hee; Park, So Youn; Kim, Chi Dae

    2012-01-01

    Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways led to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways. -- Highlights: ► Homocysteine (Hcy) induced MMP-9 production in murine macrophages. ► Hcy induced MMP-9 production through ERK and Akt signaling pathways. ► ERK and Akt signaling pathways were activated by Hcy in murine macrophages. ► ERK and Akt pathways were additively act on Hcy-induced MMP-9 production. ► Hcy enhances MMP-9 production in macrophages via activation of ERK and Akt signaling pathways in an independent manner.

  12. Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won; Bae, Jin Ung; Kim, Gyu Hee; Park, So Youn; Kim, Chi Dae, E-mail: chidkim@pusan.ac.kr

    2012-04-01

    Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways led to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways. -- Highlights: ► Homocysteine (Hcy) induced MMP-9 production in murine macrophages. ► Hcy induced MMP-9 production through ERK and Akt signaling pathways. ► ERK and Akt signaling pathways were activated by Hcy in murine macrophages. ► ERK and Akt pathways were additively act on Hcy-induced MMP-9 production. ► Hcy enhances MMP-9 production in macrophages via activation of ERK and Akt signaling pathways in an independent manner.

  13. The murine retinoblastoma homolog maps to chromosome 14 near Es-10

    NARCIS (Netherlands)

    Stone, J.C.; Crosby, J.J.; Kozak, C.A.; Schievella, A.R.; Bernards, R.A.; Nadeau, J.H.

    1989-01-01

    Restriction fragment length variants have been exploited to map genetically Rb-1, the murine homolog of the human retinoblastoma gene. Rb-1 localized to mouse chromosome 14 on the basis of results from analysis of somatic cell hybrids. In an interspecific backcross involving Mus spretus, Rb-1 and

  14. Differential gene expression in the murine gastric fundus lacking interstitial cells of Cajal

    Directory of Open Access Journals (Sweden)

    Ward Sean M

    2003-06-01

    Full Text Available Abstract Background The muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. The absence of intramuscular interstitial cells in W/WV mutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells. Method The gene expression profile of the gastric fundus of wild type and W/WV mice was assayed by murine microarray analysis displaying a total of 8734 elements. Queried genes from the microarray analysis were confirmed by semi-quantitative reverse transcription-polymerase chain reaction. Results Twenty-one genes were differentially expressed in wild type and W/WV mice. Eleven transcripts had 2.0–2.5 fold higher mRNA expression in W/WV gastric fundus when compared to wild type tissues. Ten transcripts had 2.1–3.9 fold lower expression in W/WV mutants in comparison with wild type animals. None of these genes have ever been implicated in any bowel motility function. Conclusions These data provides evidence that several important genes have significantly changed in the murine fundus of W/WV mutants that lack intramuscular interstitial cells of Cajal and have reduced enteric motor neurotransmission.

  15. Efficacy of posaconazole in murine experimental sporotrichosis.

    Science.gov (United States)

    Fernández-Silva, Fabiola; Capilla, Javier; Mayayo, Emilio; Guarro, Josep

    2012-05-01

    We developed a murine model of systemic sporotrichosis by using three strains of each of the two commonest species causing sporotrichosis, i.e., Sporothrix schenckii sensu stricto and Sporothrix brasiliensis, in order to evaluate the efficacy of posaconazole (PSC). The drug was administered at a dose of 2.5 or 5 mg/kg of body weight twice a day by gavage, and one group was treated with amphotericin B (AMB) as a control treatment. Posaconazole, especially at 5 mg/kg, showed good efficacy against all the strains tested, regardless of their MICs, as measured by prolonged survival, tissue burden reduction, and histopathology.

  16. Irradiation Design for an Experimental Murine Model

    International Nuclear Information System (INIS)

    Ballesteros-Zebadua, P.; Moreno-Jimenez, S.; Suarez-Campos, J. E.; Celis, M. A.; Larraga-Gutierrez, J. M.; Garcia-Garduno, O. A.; Rubio-Osornio, M. C.; Custodio-Ramirez, V.; Paz, C.

    2010-01-01

    In radiotherapy and stereotactic radiosurgery, small animal experimental models are frequently used, since there are still a lot of unsolved questions about the biological and biochemical effects of ionizing radiation. This work presents a method for small-animal brain radiotherapy compatible with a dedicated 6MV Linac. This rodent model is focused on the research of the inflammatory effects produced by ionizing radiation in the brain. In this work comparisons between Pencil Beam and Monte Carlo techniques, were used in order to evaluate accuracy of the calculated dose using a commercial planning system. Challenges in this murine model are discussed.

  17. Bone marrow stromal elements in murine leukemia; Decreased CSF-producing fibroblasts and normal IL-1 expression by macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Ishay, Z [Laboratory of Experimental Hematology, Department of Anatomy and Embryology, Hebrew University-Hadassah Medical School (Israel); Barak, V [Laboratory of Immunology, Department of Oncology, Hadassah University Hospital (Israel); Shoshan, S [Faculty of Dental Medicine, Connective Tissue Research Laboratory, Hebrew University, Jerusalem (Israel); Prindull, G [Department of Pediatrics, University of Gottingen, Gottingen (Germany, F.R.)

    1990-01-01

    A study of bone marrow stromal elements in murine acute myeloid leukemia (AML) was carried out. Our previous studies had indicated marrow stromal deficiency in murine AML. In the current investigation, separate stromal cells were cultured and the results obtained have shown that, while marrow stromal macrophages are normal in leukemia and express adequate amounts of IL-1, the fibroblasts are markedly reduced. However, if sufficient fibroblasts are pooled in vitro, they produce adequate amounts of CSF. Test of TNF{alpha} in leukemic cells CM, as possible cause of marrow stromal inhibition in leukemia, had not disclosed this cytokine. Further, it was observed that total body lethal irradiation of leukemic mice aggravates the stromal deficiency, confirming results of our previous investigations. It is concluded that bone marrow stromal deficiency in murine AML is due to decreased fibroblasts and, implicity, reduced CSF production. (author).

  18. Ribonucleases 6 and 7 have antimicrobial function in the human and murine urinary tract

    Science.gov (United States)

    Becknell, Brian; Eichler, Tad; Beceiro, Susana; Li, Birong; Easterling, Robert; Carpenter, Ashley R.; James, Cindy; McHugh, Kirk M.; Hains, David S.; Partida-Sanchez, Santiago; Spencer, John David

    2014-01-01

    Recent evidence suggests antimicrobial peptides protect the urinary tract from infection. Ribonuclease 7 (RNase 7), a member of the RNase A superfamily, is a potent epithelial-derived protein that maintains human urinary tract sterility. RNase 7 expression is restricted to primates, limiting evaluation of its antimicrobial activity in vivo. Here we identified Ribonuclease 6 (RNase 6) as the RNase A Superfamily member present in humans and mice that is most conserved at the amino acid level relative to RNase 7. Like RNase 7, recombinant human and murine RNase 6 has potent antimicrobial activity against uropathogens. Quantitative real-time PCR and immunoblot analysis indicate that RNase 6 mRNA and protein are up-regulated in the human and murine urinary tract during infection. Immunostaining located RNase 6 to resident and infiltrating monocytes, macrophages, and neutrophils. Uropathogenic E. coli induces RNase 6 peptide expression in human CD14+ monocytes and murine bone marrow derived macrophages. Thus, RNase 6 is an inducible, myeloid-derived protein with markedly different expression from the epithelial-derived RNase 7 but with equally potent antimicrobial activity. Our studies suggest RNase 6 serves as an evolutionarily conserved antimicrobial peptide that participates in the maintenance of urinary tract sterility. PMID:25075772

  19. Baicalein induces cell death in murine T cell lymphoma via inhibition of thioredoxin system.

    Science.gov (United States)

    Patwardhan, Raghavendra S; Pal, Debojyoti; Checker, Rahul; Sharma, Deepak; Sandur, Santosh K

    2017-10-01

    We have earlier demonstrated the radioprotective potential of baicalein using murine splenic lymphocytes. Here, we have studied the effect of baicalein on murine T cell lymphoma EL4 cells and investigated the underlying mechanism of action. We observed that baicalein induced a dose dependent cell death in EL4 cells in vitro and significantly reduced the frequency of cancer stem cells. Previously, we have reported that murine and human T cell lymphoma cells have increased oxidative stress tolerance capacity due to active thioredoxin system. Hence, we monitored the effect of baicalein on thioredoxin system in EL4 cells. Docking studies revealed that baicalein could bind to the active site of thioredoxin reductase. Baicalein treatment led to significant reduction in the activity of thioredoxin reductase and nuclear levels of thioredoxin-1 thereby increasing ASK1 levels and caspase-3 activity. Interestingly, CRISPR-Cas9 based knock-out of ASK1 or over-expression of thioredoxin-1 abolished anti-tumor effects of baicalein in EL4 cells. Further, baicalein administration significantly reduced intra-peritoneal tumor burden of EL4 cells in C57BL/6 mice. Thus, our study describes anti-tumor effects of baicalein in EL4 cells via inhibition of thioredoxin system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Gastrointestinal expression and partial cDNA cloning of murine Muc2

    NARCIS (Netherlands)

    van Klinken, B. J.; Einerhand, A. W.; Duits, L. A.; Makkink, M. K.; Tytgat, K. M.; Renes, I. B.; Verburg, M.; Büller, H. A.; Dekker, J.

    1999-01-01

    To help us investigate the role of mucin in the protection of the colonic epithelium in the mouse, we aimed to identify the murine colonic mucin (MCM) and its encoding gene. We isolated MCM, raised an anti-MCM antiserum, and studied the biosynthesis of MCM in the gastrointestinal tract. Isolated MCM

  1. Erythropoietin treatment alleviates ultrastructural myelin changes induced by murine cerebral malaria

    DEFF Research Database (Denmark)

    Hempel, Casper; Hyttel, Poul; Staalsø, Trine

    2012-01-01

    the effects of EPO treatment in this context. METHODS: The study consisted of two groups of Plasmodium berghei-infected mice and two groups of uninfected controls that were either treated with EPO or placebo (n = 4 mice/group). In the terminal phase of murine CM the brains were removed and processed...

  2. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

    Science.gov (United States)

    Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G

    2011-09-01

    Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  3. Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes

    Directory of Open Access Journals (Sweden)

    Korson Mark

    2007-04-01

    Full Text Available Abstract Background Methylmalonic acidemia (MMA, a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT. Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition. Methods To assess the effectiveness of gene delivery to restore the defective metabolism in this disorder, adenoviral correction experiments were performed using murine Mut embryonic fibroblasts and primary human methylmalonyl-CoA mutase deficient hepatocytes derived from a patient who harbored two early truncating mutations, E224X and R228X, in the MUT gene. Enzymatic and expression studies were used to assess the extent of functional correction. Results Primary hepatocytes, isolated from the native liver after removal subsequent to a combined liver-kidney transplantation procedure, or Mut murine fibroblasts were infected with a second generation recombinant adenoviral vector that expressed the murine methylmalonyl-CoA mutase as well as eGFP from distinct promoters. After transduction, [1-14C] propionate macromolecular incorporation studies and Western analysis demonstrated complete correction of the enzymatic defect in both cell types. Viral reconstitution of enzymatic expression in the human methylmalonyl-CoA mutase deficient hepatocytes exceeded that seen in fibroblasts or control hepatocytes. Conclusion These experiments provide proof of principle for viral correction in methylmalonic acidemia and suggest that hepatocyte-directed gene delivery will be an effective therapeutic treatment strategy in both murine models and in human patients. Primary hepatocytes from a liver that was unsuitable for transplantation provided an important resource for these studies.

  4. Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6–specific IgA mAb

    Science.gov (United States)

    Feng, Ningguo; Lawton, Jeffrey A.; Gilbert, Joana; Kuklin, Nelly; Vo, Phuoc; Prasad, B.V. Venkataram; Greenberg, Harry B.

    2002-01-01

    Rotaviruses are the leading cause of severe diarrheal disease in young children. Intestinal mucosal IgA responses play a critical role in protective immunity against rotavirus reinfection. Rotaviruses consist of three concentric capsid layers surrounding a genome of 11 segments of double-stranded RNA. The outer layer proteins, VP4 and VP7, which are responsible for viral attachment and entry, are targets for protective neutralizing antibodies. However, IgA mAb’s directed against the intermediate capsid protein VP6, which do not neutralize the virus, have also been shown to protect mice from rotavirus infection and clear chronic infection in SCID mice. We investigated whether the anti-VP6 IgA (7D9) mAb could inhibit rotavirus replication inside epithelial cells and found that 7D9 acted at an early stage of infection to neutralize rotavirus following antibody lipofection. Using electron cryomicroscopy, we determined the three-dimensional structure of the virus-antibody complex. The attachment of 7D9 IgA to VP6 introduces a conformational change in the VP6 trimer, rendering the particle transcriptionally incompetent and preventing the elongation of initiated transcripts. Based on these observations, we suggest that anti-VP6 IgA antibodies confers protection in vivo by inhibiting viral transcription at the start of the intracellular phase of the viral replication cycle. PMID:11994409

  5. Study of the epitope structure of purified Dac G I and Lol p I, the major allergens of Dactylis glomerata and Lolium perenne pollens, using monoclonal antibodies.

    Science.gov (United States)

    Mourad, W; Mécheri, S; Peltre, G; David, B; Hébert, J

    1988-11-15

    The use of mAb allowed us to further analyze the cross-reactivity between purified Dac g I and Lol p I, the major allergens of Dactylis glomerata (cocksfoot) and Lolium perenne (Rye grass), respectively. It was first shown, using IEF, followed by immunoprinting, that serum IgE antibodies from most grass-sensitive patients recognize both Dac g I and Lol p I. Second, three different anti-Lol p I mAb, 290A-167, 348A-6, and 539A-6, and one anti-Dac g I mAb, P3B2 were all shown to react with Dac g I and Lol p I, indicating that the two molecules share common epitopes. Epitope specificity of the mAb was determined by competitive binding inhibition of a given labeled mAb to solid phase fixed Dac g I or Lol p I by the mAb. The results indicated that the four mAb are directed against four different and non-overlapping epitopes present on both allergens. Using double-binding RIA, our data strongly suggest that the common epitopes are not repetitive on both molecules. In addition to their similar physicochemical characteristics, such as isolectric points and m.w., Dac g I and Lol p I share four identical epitopes. Binding inhibition of human IgE to Lol p I and Dac g I by the mAb was also assessed. The results indicated that each mAb was able to inhibit such reactions to variable degree but no additive inhibition was observed when two mAb of different specificities were used in combination, suggesting that the human IgE binding site is partially shared by each epitope recognized by the four mAb.

  6. Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media.

    Science.gov (United States)

    Hennings, Justin M; Zimmer, Randall L; Nabli, Henda; Davis, J Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L

    2016-03-01

    Validate single versus sequential culture media for murine embryo development. Prospective laboratory experiment. Assisted Reproduction Laboratory. Murine embryos. Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = culture. Human embryo studies are needed. © The Author(s) 2015.

  7. Hyper-radiation sensitivity of murine scid mutation and mapping of the human homologue HYRC1 gene

    International Nuclear Information System (INIS)

    Komatsu, Kenshi; Ohta, Tohru; Niikawa, Norio; Okumura, Yutaka; Kubota, Nobuo.

    1994-01-01

    The murine severe combined immunodeficient mutation (scid) is characterized by a lack of both B and T cells, due to a defect in lymphoid variable-(diversity)-joining(V(D)J) rearrangement. Scid cells are highly sensitive to both radiation-induced killing and chromosomal aberrations. Present experiments also demonstrated the high sensitivity of scid cells to killing, because of a deficient repair of double strand breaks(DSB). Scid cells can repair only 60% of radiation-induced DSB for 3 hours, while normal cells repair 85% of the DSB. Significantly reduced Do and n values were obtained from survival curves of scid cells and were similar to ataxia-telangiectasia(AT) cells (a unique human disease conferring whole body radiosensitivity). However, the kinetics of DNA synthesis after irradiation were different between the two cell types. In contrast with the radioresistant DNA synthesis of AT cells, DNA synthesis of scid cells was markedly inhibited after irradiation. The existence of different mutations was also supported by evidence of complementation in somatic cell hybrids between scid cells and AT cells. Using these hybrid cells, fragments of human chromosome 8 were introduced into scid cells HPRT mutant via X-irradiation and somatic cell fusion. The resulting hybrid clones contained human DNA fragment(s) which complemented the hyper-radiosensitivity of the scid cells. Alu-PCR products from these hybrids were used for chromosome painting using the technique of chromosome in situ suppression hybridization, allowing assignment of the human HYRC1 (hyper-radiosensitivity of murine scid mutation, complementing 1) gene, a candidate for a V(D)J recombinant gene, to human chromosome 8q11. (author)

  8. A fluorescence model of the murine lung for optical detection of pathogenic bacteria

    Science.gov (United States)

    Durkee, Madeleine S.; Cirillo, Jeffrey D.; Maitland, Kristen C.

    2017-07-01

    We present a computer model of intravital excitation and external fluorescence detection in the murine lungs validated with a three-dimensional lung tissue phantom. The model is applied to optical detection of pulmonary tuberculosis infection.

  9. Characterization of the Pathogenicity of Streptococcus intermedius TYG1620 Isolated from a Human Brain Abscess Based on the Complete Genome Sequence with Transcriptome Analysis and Transposon Mutagenesis in a Murine Subcutaneous Abscess Model.

    Science.gov (United States)

    Hasegawa, Noriko; Sekizuka, Tsuyoshi; Sugi, Yutaka; Kawakami, Nobuhiro; Ogasawara, Yumiko; Kato, Kengo; Yamashita, Akifumi; Takeuchi, Fumihiko; Kuroda, Makoto

    2017-02-01

    Streptococcus intermedius is known to cause periodontitis and pyogenic infections in the brain and liver. Here we report the complete genome sequence of strain TYG1620 (genome size, 2,006,877 bp; GC content, 37.6%; 2,020 predicted open reading frames [ORFs]) isolated from a brain abscess in an infant. Comparative analysis of S. intermedius genome sequences suggested that TYG1620 carries a notable type VII secretion system (T7SS), two long repeat regions, and 19 ORFs for cell wall-anchored proteins (CWAPs). To elucidate the genes responsible for the pathogenicity of TYG1620, transcriptome analysis was performed in a murine subcutaneous abscess model. The results suggest that the levels of expression of small hypothetical proteins similar to phenol-soluble modulin β1 (PSMβ1), a staphylococcal virulence factor, significantly increased in the abscess model. In addition, an experiment in a murine subcutaneous abscess model with random transposon (Tn) mutant attenuation suggested that Tn mutants with mutations in 212 ORFs in the Tn mutant library were attenuated in the murine abscess model (629 ORFs were disrupted in total); the 212 ORFs are putatively essential for abscess formation. Transcriptome analysis identified 37 ORFs, including paralogs of the T7SS and a putative glucan-binding CWAP in long repeat regions, to be upregulated and attenuated in vivo This study provides a comprehensive characterization of S. intermedius pathogenicity based on the complete genome sequence and a murine subcutaneous abscess model with transcriptome and Tn mutagenesis, leading to the identification of pivotal targets for vaccines or antimicrobial agents for the control of S. intermedius infections. Copyright © 2017 American Society for Microbiology.

  10. Non-contact respiration monitoring for in-vivo murine micro computed tomography: characterization and imaging applications

    International Nuclear Information System (INIS)

    Burk, Laurel M; Lee, Yueh Z; Wait, J Matthew; Lu Jianping; Zhou, Otto Z

    2012-01-01

    A cone beam micro-CT has previously been utilized along with a pressure-tracking respiration sensor to acquire prospectively gated images of both wild-type mice and various adult murine disease models. While the pressure applied to the abdomen of the subject by this sensor is small and is generally without physiological effect, certain disease models of interest, as well as very young animals, are prone to atelectasis with added pressure, or they generate too weak a respiration signal with this method to achieve optimal prospective gating. In this work we present a new fibre-optic displacement sensor which monitors respiratory motion of a subject without requiring physical contact. The sensor outputs an analogue signal which can be used for prospective respiration gating in micro-CT imaging. The device was characterized and compared against a pneumatic air chamber pressure sensor for the imaging of adult wild-type mice. The resulting images were found to be of similar quality with respect to physiological motion blur; the quality of the respiration signal trace obtained using the non-contact sensor was comparable to that of the pressure sensor and was superior for gating purposes due to its better signal-to-noise ratio. The non-contact sensor was then used to acquire in-vivo micro-CT images of a murine model for congenital diaphragmatic hernia and of 11-day-old mouse pups. In both cases, quality CT images were successfully acquired using this new respiration sensor. Despite the presence of beam hardening artefacts arising from the presence of a fibre-optic cable in the imaging field, we believe this new technique for respiration monitoring and gating presents an opportunity for in-vivo imaging of disease models which were previously considered too delicate for established animal handling methods. (paper)

  11. Developmental constraints revealed by co-variation within and among molar rows in two murine rodents.

    Science.gov (United States)

    Renaud, Sabrina; Pantalacci, Sophie; Quéré, Jean-Pierre; Laudet, Vincent; Auffray, Jean-Christophe

    2009-01-01

    Morphological integration corresponds to interdependency between characters that can arise from several causes. Proximal causes of integration include that different phenotypic features may share common genetic sets and/or interact during their development. Ultimate causes may be the prolonged effect of selection favoring integration of functionally interacting characters, achieved by the molding of these proximal causes. Strong and direct interactions among successive teeth of a molar row are predicted by genetic and developmental evidences. Functional constraints related to occlusion, however, should have selected more strongly for a morphological integration of occluding teeth and a corresponding evolution of the underlying developmental and genetic pathways. To investigate how these predictions match the patterns of phenotypic integration, we studied the co-variation among the six molars of the murine molar row, focusing on two populations of house mice (Mus musculus domesticus) and wood mice (Apodemus sylvaticus). The size and shape of the three upper and lower molars were quantified and compared. Our results evidenced similar patterns in both species, size being more integrated than shape among all the teeth, and both size and shape co-varying strongly between adjacent teeth, but also between occluding teeth. Strong co-variation within each molar row is in agreement with developmental models showing a cascade influence of the first molar on the subsequent molars. In contrast, the strong co-variation between molars of the occluding tooth rows confirms that functional constraints molded patterns of integration and probably the underlying developmental pathways despite the low level of direct developmental interactions occurring among molar rows. These patterns of co-variation are furthermore conserved between the house mouse and the wood mouse that diverged >10 Ma, suggesting that they may constitute long-running constraints to the diversification of the murine

  12. Functional imaging of murine hearts using accelerated self-gated UTE cine MRI

    NARCIS (Netherlands)

    Motaal, Abdallah G.; Noorman, Nils; de Graaf, Wolter L.; Hoerr, Verena; Florack, Luc M. J.; Nicolay, Klaas; Strijkers, Gustav J.

    2015-01-01

    We introduce a fast protocol for ultra-short echo time (UTE) Cine magnetic resonance imaging (MRI) of the beating murine heart. The sequence involves a self-gated UTE with golden-angle radial acquisition and compressed sensing reconstruction. The self-gated acquisition is performed asynchronously

  13. Long-term treatment of rheumatoid arthritis with adalimumab

    Directory of Open Access Journals (Sweden)

    Murdaca G

    2013-05-01

    Full Text Available Giuseppe Murdaca, Francesca Spanò, Francesco PuppoDepartment of Internal Medicine, Clinical Immunology Unit, University of Genoa, Genoa, ItalyAbstract: Rheumatoid arthritis (RA is a chronic inflammatory disease that is associated with joint damage and progressive disability, an increased risk of morbidity related to comorbid conditions and substantial socioeconomic costs. Tumor necrosis factor-alpha (TNF-α is a proinflammatory cytokine known to have a central role in the initial host response to infection and in the pathogenesis of various immune-mediated diseases, such as RA, ankylosing spondylitis, psoriasis and/or psoriatic arthritis, Crohn’s disease, and systemic lupus erythematosus. Five TNF-α inhibitors are available for the clinical use: infliximab; adalimumab; etanercept; golimumab; and certolizumab pegol. Infliximab is a chimeric human/murine IgG1 monoclonal antibody (mAb; adalimumab, and golimumab are human mAbs; certolizumab pegol is composed of the fragment antigen-binding anti-binding domain of a humanized anti-TNF-α mAb, combined with polyethylene glycol to increase its half-life in the body; etanercept is a fusion protein that acts as a “decoy receptor” for TNF-α. In this paper, we will briefly review the current data on efficacy and safety of adalimumab in patients with RA, its potential beneficial effects upon comorbid conditions, such as endothelial dysfunction and accelerated atherosclerosis in RA, and the immunogenicity.Keywords: adalimumab, efficacy, safety, rheumatoid arthritis, VEGF, immunogenicity, infections

  14. Scanning electron microscopy of the neuropathology of murine cerebral malaria

    Directory of Open Access Journals (Sweden)

    Brenneis Christian

    2006-11-01

    Full Text Available Abstract Background The mechanisms leading to death and functional impairments due to cerebral malaria (CM are yet not fully understood. Most of the knowledge about the pathomechanisms of CM originates from studies in animal models. Though extensive histopathological studies of the murine brain during CM are existing, alterations have not been visualized by scanning electron microscopy (SEM so far. The present study investigates the neuropathological features of murine CM by applying SEM. Methods C57BL/6J mice were infected with Plasmodium berghei ANKA blood stages. When typical symptoms of CM developed perfused brains were processed for SEM or light microscopy, respectively. Results Ultrastructural hallmarks were disruption of vessel walls, parenchymal haemorrhage, leukocyte sequestration to the endothelium, and diapedesis of macrophages and lymphocytes into the Virchow-Robin space. Villous appearance of observed lymphocytes were indicative of activated state. Cerebral oedema was evidenced by enlargement of perivascular spaces. Conclusion The results of the present study corroborate the current understanding of CM pathophysiology, further support the prominent role of the local immune system in the neuropathology of CM and might expose new perspectives for further interventional studies.

  15. Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

    Energy Technology Data Exchange (ETDEWEB)

    Pissuwan, Dakrong [University of Technology Sydney, Institute for Nanoscale Technology (Australia); Valenzuela, Stella M. [University of Technology Sydney, Department of Medical and Molecular Biosciences (Australia)], E-mail: stella.valenzuela@uts.edu.au; Killingsworth, Murray C. [Sydney South West Pathology Service (Australia)], E-mail: murray.killingsworth@swsahs.nsw.gov.au; Xu, Xiaoda; Cortie, Michael B. [University of Technology Sydney, Institute for Nanoscale Technology (Australia)], E-mail: michael.cortie@uts.edu.au

    2007-12-15

    Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation ({approx}1x10{sup 5} to 1x10{sup 10} W/m{sup 2}). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5x10{sup 2} W/m{sup 2} being sufficient, provided that a total fluence of {approx}30 J/cm{sup 2} is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm{sup 2} resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

  16. Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

    International Nuclear Information System (INIS)

    Pissuwan, Dakrong; Valenzuela, Stella M.; Killingsworth, Murray C.; Xu, Xiaoda; Cortie, Michael B.

    2007-01-01

    Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (∼1x10 5 to 1x10 10 W/m 2 ). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5x10 2 W/m 2 being sufficient, provided that a total fluence of ∼30 J/cm 2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm 2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells

  17. Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus

    Directory of Open Access Journals (Sweden)

    Qian Yajuan

    2011-05-01

    Full Text Available Abstract Background Cucumber green mottle mosaic virus (CGMMV, a member of the genus Tobamovirus, can be transmitted by seeds and infects many cucurbit species, causing serious yield losses in cucumber and watermelon plants. In this paper, five serological methods including antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA, triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA, Dot-immunobinding assay (DBIA, direct tissue blot immunoassay (DTBIA and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR were described for detection and diagnosis of CGMMV. Results Using the purified CGMMV particles as immunogens, six murine monoclonal antibodies (MAbs were produced. Five serological methods were established using the MAb 4H1 and detection sensitivity was compared using purified preparations and infected-plant tissue extracts. The detection sensitivity of ACP-ELISA was 0.16 ng of purified CGMMV, whereas TAS-ELISA was more sensitive than ACP-ELISA with a minimum detection of 0.04 ng of purified CGMMV. The sensitivities of TAS-ELISA and DBIA were similar for detecting CGMMV in infected-plant tissue extracts, and were four times higher than ACP-ELISA. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus. The detection sensitivity of IC-RT-PCR for CGMMV-infected plant tissues was about 400 times higher than that of TAS-ELISA and DBIA. Conclusions The established ACP-ELISA, TAS-ELISA, DBIA and DTBIA are suitable for routine CGMMV detection of large-scale samples in the field survey, while IC-RT-PCR is more sensitive and suitable for acquiring information about the viral genome.

  18. Selective host range restriction of goat cells for recombinant murine leukemia virus and feline leukemia virus type A.

    OpenAIRE

    Fischinger, P J; Thiel, H J; Blevins, C S; Dunlop, N M

    1981-01-01

    We isolated a strain of normal goat fibroblasts which was uniquely selective in that it allowed the replication of xenotropic murine leukemia virus but not polytropic recombinant murine leukemia virus. In addition, feline leukemia virus type A replication was severely diminished in these goat cells, whereas feline leukemia virus type B and feline endogenous RD114-CCC viruses replicated efficiently. No other known cells exhibit this pattern of virus growth restriction. These goat cells allow t...

  19. Murine model of long-term obstructive jaundice.

    Science.gov (United States)

    Aoki, Hiroaki; Aoki, Masayo; Yang, Jing; Katsuta, Eriko; Mukhopadhyay, Partha; Ramanathan, Rajesh; Woelfel, Ingrid A; Wang, Xuan; Spiegel, Sarah; Zhou, Huiping; Takabe, Kazuaki

    2016-11-01

    With the recent emergence of conjugated bile acids as signaling molecules in cancer, a murine model of obstructive jaundice by cholestasis with long-term survival is in need. Here, we investigated the characteristics of three murine models of obstructive jaundice. C57BL/6J mice were used for total ligation of the common bile duct (tCL), partial common bile duct ligation (pCL), and ligation of left and median hepatic bile duct with gallbladder removal (LMHL) models. Survival was assessed by Kaplan-Meier method. Fibrotic change was determined by Masson-Trichrome staining and Collagen expression. Overall, 70% (7 of 10) of tCL mice died by day 7, whereas majority 67% (10 of 15) of pCL mice survived with loss of jaundice. A total of 19% (3 of 16) of LMHL mice died; however, jaundice continued beyond day 14, with survival of more than a month. Compensatory enlargement of the right lobe was observed in both pCL and LMHL models. The pCL model demonstrated acute inflammation due to obstructive jaundice 3 d after ligation but jaundice rapidly decreased by day 7. The LHML group developed portal hypertension and severe fibrosis by day 14 in addition to prolonged jaundice. The standard tCL model is too unstable with high mortality for long-term studies. pCL may be an appropriate model for acute inflammation with obstructive jaundice, but long-term survivors are no longer jaundiced. The LHML model was identified to be the most feasible model to study the effect of long-term obstructive jaundice. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Formation and maturation of the murine meniscus.

    Science.gov (United States)

    Gamer, Laura W; Xiang, Lin; Rosen, Vicki

    2017-08-01

    Meniscal injuries are commonplace, but current surgical repair procedures do not prevent degenerative joint changes that occur after meniscal injury and often lead to osteoarthritis. Successful tissue regeneration in adults often recapitulates events that occur during embryogenesis, suggesting that understanding the regulatory pathways controlling these early processes may provide clues for developing strategies for tissue repair. While the mouse is now widely used to study joint diseases, detailed knowledge of the basic biology of murine meniscus is not readily available. Here, we examine meniscal morphogenesis in mice from embryonic day 13.5 (E13.5) to 6 months of age using histology, in situ hybridization, and immunohistochemistry. We find that the meniscus is a morphologically distinct structure at E16 when it begins to regionalize. At birth, the meniscus has a distinguishable inner, avascular, round chondrocyte cell region, an outer, vascularized, fibroblast cell region, and a surface superficial zone. Maturation begins at 2 weeks of age when the meniscus expresses type I collagen, type II collagen, type X collagen, and MMP-13 in specific patterns. By 4 weeks of age, small areas of ossification are detected in the anterior meniscal horn, a common feature seen in rodents. Maturation appears complete at 8 weeks of age, when the meniscus resembles the adult structure complete with ossifying tissue that contains bone marrow like areas. Our results provide, the first systematic study of mouse meniscal development and will be a valuable tool for analyzing murine models of knee joint formation and disease. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1683-1689, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  1. Effect of fucoidan on B16 murine melanoma cell melanin formation ...

    African Journals Online (AJOL)

    Background:Fucoidan is a complex sulfated polysaccharide extracted from brown seaweed and has a wide variety of biological activities. It not only inhibits cancer cell growth but also inhibits tyrosinase in vitro. Therefore, it is of interest to investigate the effect of fucoidan on B16 murine melanoma cells as the findings may ...

  2. A study of the use of radioimmunoscintigraphy (RIA) with the monoclonal antibody MAb-170, and fluorine-18 flurodeoxyglucose positron emission tomography (18FDG-PET) for the preoperative imaging of complex ovarian masses and their ability to identify ovarian cancer

    Science.gov (United States)

    Lieberman, Gidon

    . This method preserves antigen integrity at the cost of poor tissue architecture in comparison to paraffin embedded sections. This chapter employed two novel antigen retrieval techniques to provide better identification of antibody distribution using of formalin-fixed paraffin-embedded tissue sections. The antibody localised to tumour cell cytoplasm with relative sparing of the nucleus and cell membrane. Chapter four describes the evaluation of [18]FDG-DHC imaging in twenty patients with suspected ovarian cancer in a similar fashion to chapter 2. Twelve patients had pelvic malignancies, seven patients had benign pathologies and one patient had a borderline malignancy. All malignant pelvic masses were identified with DHC, and accurately estimated disease spread. Two of the benign pelvic masses localised [18]FDG. The overall for sensitivity and specificity for +FDG-DHC imaging was 100% and 78%. In conclusion. MAb-170 is unlikely to be incorporated into clinical practice due to a poor sensitivity and unsuitability for repeat use. Increasingly humanised and more specific antibodies against ovarian cancer histological types may be more suitable in the future. [18]FDG imaging with DHC was specific, acceptable to patients and has a distinct future in the diagnostic imaging and follow up of primary and recurrent ovarian cancer.

  3. Effect of small dose of radiation on induction of apoptosis in murine tumors

    International Nuclear Information System (INIS)

    Seong, Jin Sil; Pyo, Hong Ryull; Chung, Eun Ji; Kim, Sung Hee; Suh, Chang Ok

    1999-01-01

    To investigate the presence of adaptive response by low dose radiation in murine tumors in relation to radiation induced apoptosis as well as related mechanism. Syngeneic murine tumors, OCa-1 and HCa-l, were given 0.05 Gy pretreatment followed by therapeutic dose of 25 Gy radiation. Induction of apoptosis was analyzed for each treatment group. Regulating molecules of apoptosis. p53, Bcl-2, Sax, Bel-X, were also analyzed by Western blotting. In 0.05 Gy pretreatment group of OCa-l, 25 Gy-induced apoptosis per 1000 cells was 229, which was estimated at 30% lower level than the expected (p<0.05). In contrast, this reduction in radiation induced apoptosis was not seen in HCa-1. In the expression of apoptosis regulating molecules, p53 increased in both tumors in response to radiation. Bcl-2 and Bax did not show significant change in both tumors however, the expression of Bcl-2 surpassed that of Bax in 0.05 Gy pretreatment group of OCa-1. Bcl-X was not expressed in OCa-1. In HCa-l, ScI-X showed increased expression even with 0.05 Gy. Adaptive response by low dose radiation is shown in one murine tumor, OCa-I, in relation to radiation induced apoptosis. Apoptosis regulating molecules including Bcl-2/Bax and Bcl-X, appear to related. This study shows an evidence that adaptive response is present, but not a generalized phenomenon in vivo

  4. A novel method for murine intrahepatic islet transplantation via cecal vein.

    Science.gov (United States)

    Byun, Nari; Kim, Hyun-Je; Min, Byoung-Hoon; Shin, Jun-Seop; Yoon, Il-Hee; Kim, Jong-Min; Kim, Yong-Hee; Park, Chung-Gyu

    2015-12-01

    Islet transplantation is one of the most beneficial treatment modality to treat type 1 diabetic patients with frequent hypoglycemic unawareness. In clinical setting, human islets are infused via portal vein and are settled in the end-portal venules in the liver. However, mouse islets are transplanted into kidney subcapsule or liver through direct portal vein. These conventional transplantation methods have several drawbacks such as different physiological environments around the transplanted islets in kidney subcapsule from the liver and high mortality rate in direct portal vein approach. In this study, we introduced murine intrahepatic islet transplantation method via cecal vein to have the same surgical operation route in humans as well as guaranteeing low mortality rate after islet transplantation. With this protocol, consistent normoglycemia can be obtained in diabetic mice, while keeping operation-related mortality extremely low. This approach with easier accessibility and low mortality will make murine intrahepatic islet transplantation a useful model for studying immunological mechanisms such as strong innate and adaptive immune responses that occur in human islet transplantation. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Establishment of a new murine elastase-induced aneurysm model combined with transplantation.

    Directory of Open Access Journals (Sweden)

    Zuzanna Rowinska

    Full Text Available The aim of our study was to develop a reproducible murine model of elastase-induced aneurysm formation combined with aortic transplantation.Adult male mice (n = 6-9 per group underwent infrarenal, orthotopic transplantation of the aorta treated with elastase or left untreated. Subsequently, both groups of mice were monitored by ultrasound until 7 weeks after grafting.Mice receiving an elastase-pretreated aorta developed aneurysms and exhibited a significantly increased diastolic vessel diameter compared to control grafted mice at 7 week after surgery (1.11 ± 0.10 mm vs. 0.75 ± 0.03 mm; p ≤ 0,001. Histopathological examination revealed disruption of medial elastin, an increase in collagen content and smooth muscle cells, and neointima formation in aneurysm grafts.We developed a reproducible murine model of elastase-induced aneurysm combined with aortic transplantation. This model may be suitable to investigate aneurysm-specific inflammatory processes and for use in gene-targeted animals.

  6. Predominant antitumor effects by fully human anti-TRAIL-receptor2 (DR5) monoclonal antibodies in human glioma cells in vitro and in vivo

    Science.gov (United States)

    Nagane, Motoo; Shimizu, Saki; Mori, Eiji; Kataoka, Shiro; Shiokawa, Yoshiaki

    2010-01-01

    Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIPL, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIPL expression. Downregulation of c-FLIPL expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIPL conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIPL and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas. PMID:20511188

  7. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) stimulates murine macrophages infected with Citrobacter rodentium.

    Science.gov (United States)

    Hugo, Ayelén A; Rolny, Ivanna S; Romanin, David; Pérez, Pablo F

    2017-03-01

    Citrobacter rodentium is a specific murine enteropathogen which causes diarrheal disease characterized by colonic hyperplasia and intestinal inflammation. Recruitment of neutrophils and macrophages constitute a key step to control the infection. Since modulation of the activity of professional phagocytic cells could contribute to improve host´s defences against C. rodentium, we investigated the effect of Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) on the interaction between murine macrophages (RAW 264.7) and C. rodentium. Phagocytosis, surface molecules and inducible nitric oxide synthase (iNOs) expression were determined by flow cytometry. Reactive oxygen species (ROS) were assessed by fluorescence microscopy. The presence of lactobacilli increased phagocytosis of C. rodentium whereas C. rodentium had no effect on lactobacilli internalization. Survival of internalized C. rodentium diminished when strain CIDCA 133 was present. CD-86, MHCII, iNOs expression and nitrite production were increased when C. rodentium and lactobacilli were present even though strain CIDCA 133 alone had no effect. Strain CIDCA 133 led to a strong induction of ROS activity which was not modified by C. rodentium. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) is able to increase the activation of murine macrophages infected with C. rodentium. The sole presence of lactobacilli is enough to modify some stimulation markers (e.g. ROS induction) whereas other markers require the presence of both bacteria; thus, indicating a synergistic effect.

  8. Interleukin-12 promotes activation of effector cells that induce a severe destructive granulomatous form of murine experimental autoimmune thyroiditis.

    OpenAIRE

    Braley-Mullen, H.; Sharp, G. C.; Tang, H.; Chen, K.; Kyriakos, M.; Bickel, J. T.

    1998-01-01

    Granulomatous inflammatory lesions are a major histopathological feature of a wide spectrum of human infectious and autoimmune diseases. Experimental autoimmune thyroiditis (EAT) with granulomatous histopathological features can be induced by mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTg and anti-interleukin-2 receptor (anti-IL-2R), anti-IL-2, or anti-interferon-gamma (anti-IFN-gamma) monoclonal antibody (MAb). These studies suggested that IFN-gamma-producing T...

  9. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    International Nuclear Information System (INIS)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

    2016-01-01

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8"+ CAR-T cells had antigen-specific cytotoxic activity. • CD4"+ CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  10. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    Energy Technology Data Exchange (ETDEWEB)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp; Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  11. Activation of DNA damage repair pathways by murine polyomavirus

    Energy Technology Data Exchange (ETDEWEB)

    Heiser, Katie; Nicholas, Catherine; Garcea, Robert L., E-mail: Robert.Garcea@Colorado.edu

    2016-10-15

    Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. -- Highlights: •Murine polyomavirus activates and recruits DNA damage repair (DDR) proteins to replication centers. •Large T-antigen mediates recruitment of DDR proteins to viral replication centers. •Inhibition or knockout of CHK1, CHK2, DNA-PK or H2AX do not affect viral titers. •Inhibition of ATR activity reduces viral titers, but not viral DNA accumulation.

  12. Cytogenetic study of murine rodents inhabiting in uranium-mining regions of Akmolinskaya oblast

    International Nuclear Information System (INIS)

    Kazymbet, P.; Altaeva, N.; Bakhtin, M.; Zhapbasov, R.

    2010-01-01

    Republic of Kazakhstan is ranked as the world's leading uranium ore reserves. About 25% of the world's proven uranium ore reserves occur here. Strategy of study concerning ecology effects conditioned by ionizing radiation includes as one main element an analysis of genetic processes in natural populations and ecosystems. Therefore analysis of cytogenetic effects of murine rodents inhabiting in influence zones of uranium-mining regions is one of the most important elements of radio-bio-ecological monitoring and are not completed so far. In habitat of murine rodents in influence zone of Stepnogorsk Mining-Chemical Complex tailing it is shown that gamma radiation equivalent dose rate and beta-particle flux density exceed from 6 to 15 times check measurements. In soil, plant, and water samples the activity of radionuclides like 238 U, 226 Ra, 232 Th and 210 Pb exceeds the testing level from 2 to 52 times. Dose of ionizing radiation absorbed by murine rodents inhabited in radioactive contaminated areas exceeds from 10 to 19 times the one absorbed by control animals. Big Jerboa (Allactaga major Kern) inhabited nearby of Stepnogorsk Mining-Chemical Complex tailing has rate of occurrence of cells with hypo diploidy, and hyper diploidy in hematopoietic tissue which correspondingly 1,85 and 3,5 times exceeds the control level; and this factor of Jerboa (Allactaga saltator Eversman) is correspondingly 1,7 and 4,1 times higher than control level. Rate of occurrence of cells with polyploidy in Big Jerboa (Allactaga major Kern) from radioactively contaminated areas is 2,7 times higher than in control animal; and this factor of Jerboa (Allactaga saltator Eversman) by 6,4 times exceeds control level. Levels of chromosomal rearrangements of Big Jerboa (Allactaga major Kern) from trail and control areas are 3,39±0,60% and 0,60±0,19% correspondently; and these factors of Jerboa (Allactaga saltator Eversman) are 4,63±0,91% and 1,22±0,37%, correspondently which confirms existence

  13. Radioprotection by murine and human tumor-necrosis factor; Dose-dependent effects on hematopoiesis in the mouse

    Energy Technology Data Exchange (ETDEWEB)

    Sloerdal, L; Muench, M O; Warren, D J; Moore, M A.S. [James Ewing Laboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center, New York (USA)

    1989-01-01

    Tumor-necrosis factor (TNF) has been shown to confer significant radioprotection in murine models. Herein, we demonstrate a dose-dependent enhancement of hematological recovery when single doses of either murine or human recombinant TNF are administered prior to irradiation. In addition to its action upon leukocytes and erythocytes, TNF also alleviates radiation-induced thrombocytopenia in the mouse. These effects on circulating blood constituents are further reflected in increased numbers of both primitive (CFU-S) and more differentiated (CFU-GM, CFU-Mega) hematopoietic progenitors in TNF-treated animals. This suggests that TNF exerts it radioprotective effects on a pool of primitive multi-potential hematopoietic cells. (author).

  14. Collagen-Induced Arthritis: A model for Murine Autoimmune Arthritis

    OpenAIRE

    Pietrosimone, K. M.; Jin, M.; Poston, B.; Liu, P.

    2015-01-01

    Collagen-induced arthritis (CIA) is a common autoimmune animal model used to study rheumatoid arthritis (RA). The development of CIA involves infiltration of macrophages and neutrophils into the joint, as well as T and B cell responses to type II collagen. In murine CIA, genetically susceptible mice (DBA/1J) are immunized with a type II bovine collagen emulsion in complete Freund’s adjuvant (CFA), and receive a boost of type II bovine collagen in incomplete Freund’s adjuvant (IFA) 21 days aft...

  15. Newly Identified TLR9 Stimulant, M6-395 Is a Potent Polyclonal Activator for Murine B Cells.

    Science.gov (United States)

    Park, Mi-Hee; Jung, Yu-Jin; Kim, Pyeung-Hyeun

    2012-02-01

    Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-β1-induced germ line transcript α (GLTα) and IL-4-induced GLTγ1 as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching.

  16. Role of P2X7 on steroid synthesis in murine luteal cells

    Directory of Open Access Journals (Sweden)

    Chunping Zhang

    2016-03-01

    Full Text Available The extracellular adenosine triphosphate (ATP regulates different cellular functions through activating purinergic receptors as a signalling molecule or neurotransmitter. P2X7 is highly expressed in murine small luteal cells. In this study, murine luteal cells were cultured in vitro and treated with P2X7 agonists – ATP and 2′(3′-O-(4-benzoyl-benzoyl-adenosine 50-triphosphate (BzATP and with P2X7 antagonist – brilliant blue G (BBG. We found that ATP and BzATP increased the production of progesterone and had no influence on the production of estradiol. BBG reversed the effect of BzATP and ATP. Further studies demonstrated that ATP and BzATP promoted the expression of CYP11A. These results revealed that P2X7 receptor activation is involved in the steroid synthesis in corpus luteum.

  17. Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

    Science.gov (United States)

    Bergasa-Caceres, Fernando; Rabitz, Herschel A.

    2014-01-01

    A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.

  18. microRNA-222 modulates liver fibrosis in a murine model of biliary atresia

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Wen-jun; Dong, Rui; Chen, Gong, E-mail: chengongzlp@hotmail.com; Zheng, Shan

    2014-03-28

    Highlights: • The RRV infected group showed cholestasis, retardation and extrahepatic biliary atresia. • miR-222 was highly expressed, and PPP2R2A was inhibited in the murine biliary atresia model. • miR-222 profoundly modulated the process of fibrosis in the murine biliary atresia model. • miR-222 might represent a potential target for improving biliary atresia prognosis. - Abstract: microRNA-222 (miR-222) has been shown to initiate the activation of hepatic stellate cells, which plays an important role in the pathogenesis of liver fibrosis. The aim of our study was to evaluate the role of miR-22 in a mouse model of biliary atresia (BA) induced by Rhesus Rotavirus (RRV) infection. New-born Balb/c mice were randomized into control and RRV infected groups. The extrahepatic bile ducts were evaluated. The experimental group was divided into BA group and negative group based on histology. The expression of miR-222, protein phosphatase 2 regulatory subunit B alpha (PPP2R2A), proliferating cell nuclear antigen (PCNA) and phospho-Akt were detected. We found that the experimental group showed signs of cholestasis, retardation and extrahepatic biliary atresia. No abnormalities were found in the control group. In the BA group, miR-222, PCNA and Akt were highly expressed, and PPP2R2A expression was significantly inhibited. Our findings suggest that miR-222 profoundly modulated the process of fibrosis in the murine BA model, which might represent a potential target for improving BA prognosis.

  19. microRNA-222 modulates liver fibrosis in a murine model of biliary atresia

    International Nuclear Information System (INIS)

    Shen, Wen-jun; Dong, Rui; Chen, Gong; Zheng, Shan

    2014-01-01

    Highlights: • The RRV infected group showed cholestasis, retardation and extrahepatic biliary atresia. • miR-222 was highly expressed, and PPP2R2A was inhibited in the murine biliary atresia model. • miR-222 profoundly modulated the process of fibrosis in the murine biliary atresia model. • miR-222 might represent a potential target for improving biliary atresia prognosis. - Abstract: microRNA-222 (miR-222) has been shown to initiate the activation of hepatic stellate cells, which plays an important role in the pathogenesis of liver fibrosis. The aim of our study was to evaluate the role of miR-22 in a mouse model of biliary atresia (BA) induced by Rhesus Rotavirus (RRV) infection. New-born Balb/c mice were randomized into control and RRV infected groups. The extrahepatic bile ducts were evaluated. The experimental group was divided into BA group and negative group based on histology. The expression of miR-222, protein phosphatase 2 regulatory subunit B alpha (PPP2R2A), proliferating cell nuclear antigen (PCNA) and phospho-Akt were detected. We found that the experimental group showed signs of cholestasis, retardation and extrahepatic biliary atresia. No abnormalities were found in the control group. In the BA group, miR-222, PCNA and Akt were highly expressed, and PPP2R2A expression was significantly inhibited. Our findings suggest that miR-222 profoundly modulated the process of fibrosis in the murine BA model, which might represent a potential target for improving BA prognosis

  20. Murine homeobox-containing gene, Msx-1: analysis of genomic organization, promoter structure, and potential autoregulatory cis-acting elements.

    Science.gov (United States)

    Kuzuoka, M; Takahashi, T; Guron, C; Raghow, R

    1994-05-01

    Detailed molecular organization of the coding and upstream regulatory regions of the murine homeodomain-containing gene, Msx-1, is reported. The protein-encoding portion of the gene is contained in two exons, 590 and 1214 bp in length, separated by a 2107-bp intron; the homeodomain is located in the second exon. The two-exon organization of the murine Msx-1 gene resembles a number of other homeodomain-containing genes. The 5'-(GTAAGT) and 3'-(CCCTAG) splicing junctions and the mRNA polyadenylation signal (UAUAA) of the murine Msx-1 gene are also characteristic of other vertebrate genes. By nuclease protection and primer extension assays, the start of transcription of the Msx-1 gene was located 256 bp upstream of the first AUG. Computer analysis of the promoter proximal 1280-bp sequence revealed a number of potentially important cis-regulatory sequences; these include the recognition elements for Ap-1, Ap-2, Ap-3, Sp-1, a possible binding site for RAR:RXR, and a number of TCF-1 consensus motifs. Importantly, a perfect reverse complement of (C/G)TTAATTG, which was recently shown to be an optimal binding sequence for the homeodomain of Msx-1 protein (K.M. Catron, N. Iler, and C. Abate (1993) Mol. Cell. Biol. 13:2354-2365), was also located in the murine Msx-1 promoter. Binding of bacterially expressed Msx-1 homeodomain polypeptide to Msx-1-specific oligonucleotide was experimentally demonstrated, raising a distinct possibility of autoregulation of this developmentally regulated gene.

  1. Neurological Disorders in a Murine Model of Chronic Renal Failure

    Directory of Open Access Journals (Sweden)

    Jean-Marc Chillon

    2014-01-01

    Full Text Available Cardiovascular disease is highly prevalent in patients with chronic renal failure (CRF. However, data on the impact of CRF on the cerebral circulatory system are scarce—despite the fact that stroke is the third most common cause of cardiovascular death in people with CRF. In the present study, we examined the impact of CRF on behavior (anxiety, recognition and ischemic stroke severity in a well-defined murine model of CRF. We did not observe any significant increases between CRF mice and non-CRF mice in terms of anxiety. In contrast, CRF mice showed lower levels of anxiety in some tests. Recognition was not impaired (vs. controls after 6 weeks of CRF but was impaired after 10 weeks of CRF. Chronic renal failure enhances the severity of ischemic stroke, as evaluated by the infarct volume size in CRF mice after 34 weeks of CRF. Furthermore, neurological test results in non-CRF mice tended to improve in the days following ischemic stroke, whereas the results in CRF mice tended to worsen. In conclusion, we showed that a murine model of CRF is suitable for evaluating uremic toxicity and the associated neurological disorders. Our data confirm the role of uremic toxicity in the genesis of neurological abnormalities (other than anxiety.

  2. Hyaluronan preserves the proliferation and differentiation potentials of long-term cultured murine adipose-derived stromal cells

    International Nuclear Information System (INIS)

    Chen, P.-Y.; Huang, Lynn L.H.; Hsieh, H.-J.

    2007-01-01

    For long-term culture, murine adipose-derived stromal cells (mADSCs) at latter passages demonstrated a marked decline in proliferative activity, exhibited senescent morphology and reduced differentiation potentials, particularly osteogenesis. To extend the lifespan of mADSCs, two culture conditions containing hyaluronan (HA) was compared in our study, one as a culture medium supplement (SHA), and the other where HA was pre-coated on culture surface (CHA). mADSCs cultivated with SHA exhibited a prolonged lifespan, reduced cellular senescence, and enhanced osteogenic potential compared to regular culture condition (control). Upon CHA treatment, mADSCs tended to form cell aggregates with gradual growth profiles, while their differentiation activities remained similar to SHA groups. After transferring mADSCs from CHA to control surface, they were shown to have an extended lifespan and an increase of osteogenic potential. Our results suggested that HA can be useful for preserving the proliferation and differentiation potentials of long-term cultured mADSCs

  3. Major differences between human atopic dermatitis and murine models, as determined by using global transcriptomic profiling

    DEFF Research Database (Denmark)

    Ewald, David A.; Noda, Shinji; Oliva, Margeaux

    2017-01-01

    , and a comparison of these models with the human AD transcriptomic fingerprint is lacking. Objective We sought to evaluate the transcriptomic profiles of 6 common murine models and determine how they relate to human AD skin. Methods Transcriptomic profiling was performed by using microarrays and quantitative RT......-PCR on biopsy specimens from NC/Nga, flaky tail, Flg-mutated, ovalbumin-challenged, oxazolone-challenged, and IL-23–injected mice. Gene expression data of patients with AD, psoriasis, and contact dermatitis were obtained from previous patient cohorts. Criteria of a fold change of 2 or greater and a false...... discovery rate of 0.05 or less were used for gene arrays. Results IL-23–injected, NC/Nga, and oxazolone-challenged mice show the largest homology with our human meta-analysis–derived AD transcriptome (37%, 18%, 17%, respectively). Similar to human AD, robust TH1, TH2, and also TH17 activation are seen in IL...

  4. Corn silk induced cyclooxygenase-2 in murine macrophages.

    Science.gov (United States)

    Kim, Kyung A; Shin, Hyun-Hee; Choi, Sang Kyu; Choi, Hye-Seon

    2005-10-01

    Stimulation of murine macrophages with corn silk induced cyclooxygenase (COX)-2 with secretion of PGE2. Expression of COX-2 was inhibited by pyrolidine dithiocarbamate (PDTC), and increased DNA binding by nuclear factor kappa B (NF-kappaB), indicating that COX-2 induction proceeds also via the NF-kappaB signaling pathway. A specific inhibitor of COX-2 decreased the expression level of inducible nitric oxide synthase (iNOS) stimulated by corn silk. PGE2 elevated the expression level of iNOS, probably via EP2 and EP4 receptors on the surface of the macrophages.

  5. Approaching the basis set limit for DFT calculations using an environment-adapted minimal basis with perturbation theory: Formulation, proof of concept, and a pilot implementation

    International Nuclear Information System (INIS)

    Mao, Yuezhi; Horn, Paul R.; Mardirossian, Narbe; Head-Gordon, Teresa; Skylaris, Chris-Kriton; Head-Gordon, Martin

    2016-01-01

    Recently developed density functionals have good accuracy for both thermochemistry (TC) and non-covalent interactions (NC) if very large atomic orbital basis sets are used. To approach the basis set limit with potentially lower computational cost, a new self-consistent field (SCF) scheme is presented that employs minimal adaptive basis (MAB) functions. The MAB functions are optimized on each atomic site by minimizing a surrogate function. High accuracy is obtained by applying a perturbative correction (PC) to the MAB calculation, similar to dual basis approaches. Compared to exact SCF results, using this MAB-SCF (PC) approach with the same large target basis set produces <0.15 kcal/mol root-mean-square deviations for most of the tested TC datasets, and <0.1 kcal/mol for most of the NC datasets. The performance of density functionals near the basis set limit can be even better reproduced. With further improvement to its implementation, MAB-SCF (PC) is a promising lower-cost substitute for conventional large-basis calculations as a method to approach the basis set limit of modern density functionals.

  6. Receptor for advanced glycation end-products and World Trade Center particulate induced lung function loss: A case-cohort study and murine model of acute particulate exposure.

    Science.gov (United States)

    Caraher, Erin J; Kwon, Sophia; Haider, Syed H; Crowley, George; Lee, Audrey; Ebrahim, Minah; Zhang, Liqun; Chen, Lung-Chi; Gordon, Terry; Liu, Mengling; Prezant, David J; Schmidt, Ann Marie; Nolan, Anna

    2017-01-01

    World Trade Center-particulate matter(WTC-PM) exposure and metabolic-risk are associated with WTC-Lung Injury(WTC-LI). The receptor for advanced glycation end-products (RAGE) is most highly expressed in the lung, mediates metabolic risk, and single-nucleotide polymorphisms at the AGER-locus predict forced expiratory volume(FEV). Our objectives were to test the hypotheses that RAGE is a biomarker of WTC-LI in the FDNY-cohort and that loss of RAGE in a murine model would protect against acute PM-induced lung disease. We know from previous work that early intense exposure at the time of the WTC collapse was most predictive of WTC-LI therefore we utilized a murine model of intense acute PM-exposure to determine if loss of RAGE is protective and to identify signaling/cytokine intermediates. This study builds on a continuing effort to identify serum biomarkers that predict the development of WTC-LI. A case-cohort design was used to analyze a focused cohort of male never-smokers with normal pre-9/11 lung function. Odds of developing WTC-LI increased by 1.2, 1.8 and 1.0 in firefighters with soluble RAGE (sRAGE)≥97pg/mL, CRP≥2.4mg/L, and MMP-9≤397ng/mL, respectively, assessed in a multivariate logistic regression model (ROCAUC of 0.72). Wild type(WT) and RAGE-deficient(Ager-/-) mice were exposed to PM or PBS-control by oropharyngeal aspiration. Lung function, airway hyperreactivity, bronchoalveolar lavage, histology, transcription factors and plasma/BAL cytokines were quantified. WT-PM mice had decreased FEV and compliance, and increased airway resistance and methacholine reactivity after 24-hours. Decreased IFN-γ and increased LPA were observed in WT-PM mice; similar findings have been reported for firefighters who eventually develop WTC-LI. In the murine model, lack of RAGE was protective from loss of lung function and airway hyperreactivity and was associated with modulation of MAP kinases. We conclude that in a multivariate adjusted model increased sRAGE is

  7. Receptor for advanced glycation end-products and World Trade Center particulate induced lung function loss: A case-cohort study and murine model of acute particulate exposure.

    Directory of Open Access Journals (Sweden)

    Erin J Caraher

    Full Text Available World Trade Center-particulate matter(WTC-PM exposure and metabolic-risk are associated with WTC-Lung Injury(WTC-LI. The receptor for advanced glycation end-products (RAGE is most highly expressed in the lung, mediates metabolic risk, and single-nucleotide polymorphisms at the AGER-locus predict forced expiratory volume(FEV. Our objectives were to test the hypotheses that RAGE is a biomarker of WTC-LI in the FDNY-cohort and that loss of RAGE in a murine model would protect against acute PM-induced lung disease. We know from previous work that early intense exposure at the time of the WTC collapse was most predictive of WTC-LI therefore we utilized a murine model of intense acute PM-exposure to determine if loss of RAGE is protective and to identify signaling/cytokine intermediates. This study builds on a continuing effort to identify serum biomarkers that predict the development of WTC-LI. A case-cohort design was used to analyze a focused cohort of male never-smokers with normal pre-9/11 lung function. Odds of developing WTC-LI increased by 1.2, 1.8 and 1.0 in firefighters with soluble RAGE (sRAGE≥97pg/mL, CRP≥2.4mg/L, and MMP-9≤397ng/mL, respectively, assessed in a multivariate logistic regression model (ROCAUC of 0.72. Wild type(WT and RAGE-deficient(Ager-/- mice were exposed to PM or PBS-control by oropharyngeal aspiration. Lung function, airway hyperreactivity, bronchoalveolar lavage, histology, transcription factors and plasma/BAL cytokines were quantified. WT-PM mice had decreased FEV and compliance, and increased airway resistance and methacholine reactivity after 24-hours. Decreased IFN-γ and increased LPA were observed in WT-PM mice; similar findings have been reported for firefighters who eventually develop WTC-LI. In the murine model, lack of RAGE was protective from loss of lung function and airway hyperreactivity and was associated with modulation of MAP kinases. We conclude that in a multivariate adjusted model increased s

  8. Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

    DEFF Research Database (Denmark)

    Rasch, Morten G; Lund, Ida K; Illemann, Martin

    2010-01-01

    MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full......-length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits......Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine...

  9. Pathogenesis of Candida albicans infections in the alternative chorio-allantoic membrane chicken embryo model resembles systemic murine infections.

    Directory of Open Access Journals (Sweden)

    Ilse D Jacobsen

    Full Text Available Alternative models of microbial infections are increasingly used to screen virulence determinants of pathogens. In this study, we investigated the pathogenesis of Candida albicans and C. glabrata infections in chicken embryos infected via the chorio-allantoic membrane (CAM and analyzed the virulence of deletion mutants. The developing immune system of the host significantly influenced susceptibility: With increasing age, embryos became more resistant and mounted a more balanced immune response, characterized by lower induction of proinflammatory cytokines and increased transcription of regulatory cytokines, suggesting that immunopathology contributes to pathogenesis. While many aspects of the chicken embryo response resembled murine infections, we also observed significant differences: In contrast to systemic infections in mice, IL-10 had a beneficial effect in chicken embryos. IL-22 and IL-17A were only upregulated after the peak mortality in the chicken embryo model occurred; thus, the role of the Th17 response in this model remains unclear. Abscess formation occurs frequently in murine models, whereas the avian response was dominated by granuloma formation. Pathogenicity of the majority of 15 tested C. albicans deletion strains was comparable to the virulence in mouse models and reduced virulence was associated with significantly lower transcription of proinflammatory cytokines. However, fungal burden did not correlate with virulence and for few mutants like bcr1Δ and tec1Δ different outcomes in survival compared to murine infections were observed. C. albicans strains locked in the yeast stage disseminated significantly more often from the CAM into the embryo, supporting the hypothesis that the yeast morphology is responsible for dissemination in systemic infections. These data suggest that the pathogenesis of C. albicans infections in the chicken embryo model resembles systemic murine infections but also differs in some aspects. Despite

  10. Lactobacillus reuteri 100-23 modulates urea hydrolysis in the murine stomach.

    Science.gov (United States)

    Wilson, Charlotte M; Loach, Diane; Lawley, Blair; Bell, Tracey; Sims, Ian M; O'Toole, Paul W; Zomer, Aldert; Tannock, Gerald W

    2014-10-01

    Comparisons of in vivo (mouse stomach) and in vitro (laboratory culture) transcriptomes of Lactobacillus reuteri strain 100-23 were made by microarray analysis. These comparisons revealed the upregulation of genes associated with acid tolerance, including urease production, in the mouse stomach. Inactivation of the ureC gene reduced the acid tolerance of strain 100-23 in vitro, and the mutant was outcompeted by the wild type in the gut of ex-Lactobacillus-free mice. Urine analysis showed that stable isotope-labeled urea, administered by gavage, was metabolized to a greater extent in Lactobacillus-free mice than animals colonized by strain 100-23. This surprising observation was associated with higher levels of urease activity and fecal-type bacteria in the stomach digesta of Lactobacillus-free mice. Despite the modulation of urea hydrolysis in the stomach, recycling of urea nitrogen in the murine host was not affected since the essential amino acid isoleucine, labeled with a stable isotope, was detected in the livers of both Lactobacillus-free and 100-23-colonized animals. Therefore, our experiments reveal a new and unexpected impact of Lactobacillus colonization on urea hydrolysis in the murine gut. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. Shark Variable New Antigen Receptor (VNAR Single Domain Antibody Fragments: Stability and Diagnostic Applications

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    Stewart Nuttall

    2013-01-01

    Full Text Available The single variable new antigen receptor domain antibody fragments (VNARs derived from shark immunoglobulin new antigen receptor antibodies (IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1 from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.

  12. Poly-thymidine oligonucleotides mediate activation of murine glial cells primarily through TLR7, not TLR8.

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    Min Du

    Full Text Available The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS.

  13. Acanthus ilicifolius plant extract prevents DNA alterations in a transplantable Ehrlich ascites carcinoma-bearing murine model.

    Science.gov (United States)

    Chakraborty, Tridib; Bhuniya, Dipak; Chatterjee, Mary; Rahaman, Mosiur; Singha, Dipak; Chatterjee, Baidya Nath; Datta, Subrata; Rana, Ajay; Samanta, Kartick; Srivastawa, Sunil; Maitra, Sankar K; Chatterjee, Malay

    2007-12-28

    To investigate the chemopreventive efficacy of the Indian medicinal plant Acanthus ilicifolius L Acanthaceae in a transplantable Ehrlich ascites carcinoma (EAC)-bearing murine model. Male Swiss albino mice were divided into four groups: Group A was the untreated normal control; Group B was the EAC control mice group that received serial, intraperitoneal (ip) inoculations of rapidly proliferating 2 x 10(5) viable EAC cells in 0.2 mL of sterile phosphate buffered saline; Group C was the plant extract-treated group that received the aqueous leaf extract (ALE) of the plant at a dose of 2.5 mg/kg body weight by single ip injections, once daily for 10, 20 and 30 consecutive days following tumour inoculation (ALE control); and Group D was the EAC + ALE-treatment group. The chemopreventive potential of the ALE was evaluated in a murine model by studying various biological parameters and genotoxic markers, such as tumour cell count, mean survival of the animals, haematological indices, hepatocellular histology, immunohistochemical expression of liver metallothionein (MT) protein, sister-chromatid exchanges (SCEs), and DNA alterations. Treatment of the EAC-bearing mice with the ALE significantly (P decrement (P single-strand breaks (SSBs) by 38.53% (3.14 +/- 0.31 vs 1.93 +/- 0.23, P < 0.01) in EAC-bearing murine liver. Our data indicate that, ALE is beneficial in restoring haematological and hepatic histological profiles and in lengthening the survival of the animals against the proliferation of ascites tumour in vivo. Finally, the chemopreventive efficacy of the ALE is manifested in limiting MT expression and in preventing DNA alterations in murine liver. The promising results of this study suggest further investigation into the chemopreventive mechanisms of the medicinal plant A. ilicifolius in vivo and in vitro.

  14. Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

    Science.gov (United States)

    Geis, Franziska K; Galla, Melanie; Hoffmann, Dirk; Kuehle, Johannes; Zychlinski, Daniela; Maetzig, Tobias; Schott, Juliane W; Schwarzer, Adrian; Goffinet, Christine; Goff, Stephen P; Schambach, Axel

    2017-05-31

    Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC. In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation. We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.

  15. Development of an ex vivo BrdU labeling procedure for the murine LLNA

    Science.gov (United States)

    The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [3H]m...

  16. SEP-induced activity and its thermographic cortical representation in a murine model.

    Science.gov (United States)

    Hoffmann, Klaus-Peter; Ruff, Roman; Kirsch, Matthias

    2013-06-01

    This article is a methodical report on the generation of reproducible changes in brain activity in a murine model. Somatosensory evoked potentials (SEP) are used to generate synchronized cortical activity. After electrical stimulation of mice forelimbs, the potentials were recorded with a flexible thin-film polyimide electrode structure directly from the cortex. Every registration included a simultaneous recording from both hemispheres that repeated four times to reproduce and compare the results. The SEPs in the murine model were shown to generate a very stable signal. The latency of the second positive wave (P2 wave) ranged between 16 and 19 ms, and the N1-P2 amplitude ranged between 39 and 48 µV. In addition, the temperature distribution of the cortex was acquired using infrared thermography. Surface cortical temperature changed during electrical stimulation without a clear hemispheric correlation. These initial results could be a step toward a better understanding of the different synchronized cortical activities and basic methods of evaluation of various mathematical algorithms to detect them.

  17. Decay-accelerating factor 1 deficiency exacerbates Trypanosoma cruzi-induced murine chronic myositis.

    Science.gov (United States)

    Solana, María E; Ferrer, María F; Novoa, María Mercedes; Song, Wen-Chao; Gómez, Ricardo M

    2012-10-01

    Murine infection with Trypanosoma cruzi (Tc) has been used to study the role of T-cells in the pathogenesis of human inflammatory idiopathic myositis. Absence of decay-accelerating factor 1 (Daf1) has been shown to enhance murine T-cell responses and autoimmunity. To determine whether Daf1 deficiency can exacerbate Tc-induced myositis, C57BL/6 DAF(+/+) and DAF(-/-) mice were inoculated with 5 × 10(4) trypomastigotes, and their morbidity, parasitemia, parasite burden, histopathology, and T-cell expansion were studied in the acute and chronic stages. DAF(-/-) mice had lower parasitemia and parasite burden but higher morbidity, muscle histopathology, and increased number of CD44(+) (activated/memory phenotype) splenic CD4(+) and CD8(+) T-cells. An enhanced CD8(+) T-cell immune-specific response may explain the lower parasitemia and parasite burden levels and the increase in histopathological lesions. We propose that Tc-inoculated DAF(-/-) mice are a useful model to study T-cell mediated immunity in skeletal muscle tissues. Copyright © 2012 Wiley Periodicals, Inc.

  18. Dendritic cell-based vaccination in cancer: therapeutic implications emerging from murine models

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    Soledad eMac Keon

    2015-05-01

    Full Text Available Dendritic cells (DCs play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel T there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts towards an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment.

  19. Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis

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    Want MY

    2017-03-01

    Full Text Available Muzamil Y Want,1 Mohammad Islammudin,1 Garima Chouhan,1 Hani A Ozbak,2 Hassan A Hemeg,2 Asoke P Chattopadhyay,3 Farhat Afrin2 1Parasite Immunology Laboratory, Department of Biotechnology, Jamia Hamdard, Hamdard University, New Delhi, India; 2Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Taibah University, Medina, Saudi Arabia; 3Department of Chemistry, University of Kalyani, Kalyani, India Abstract: Visceral leishmaniasis (VL is a fatal, vector-borne disease caused by the intracellular protozoa of the genus Leishmania. Most of the therapeutics for VL are toxic, expensive, or ineffective. Sesquiterpenes are a new class of drugs with proven antimicrobial and antiviral activities. Artemisinin is a sesquiterpene lactone with potent antileishmanial activity, but with limited access to infected cells, being a highly lipophilic molecule. Association of artemisinin with liposome is a desirable strategy to circumvent the problem of poor accessibility, thereby improving its efficacy, as demonstrated in a murine model of experimental VL. Nanoliposomal artemisinin (NLA was prepared by thin-film hydration method and optimized using Box–Behnken design with a mean particle diameter of 83±16 nm, polydispersity index of 0.2±0.03, zeta potential of -27.4±5.7 mV, and drug loading of 33.2%±2.1%. Morphological study of these nanoliposomes by microscopy showed a smooth and spherical surface. The mechanism of release of artemisinin from the liposomes followed the Higuchi model in vitro. NLA was free from concomitant signs of toxicity, both ex vivo in murine macrophages and in vivo in healthy BALB/c mice. NLA significantly denigrated the intracellular infection of Leishmania donovani amastigotes and the number of infected macrophages ex vivo with an IC50 of 6.0±1.4 µg/mL and 5.1±0.9 µg/mL, respectively. Following treatment in a murine model of VL, NLA demonstrated superior efficacy compared to artemisinin with a

  20. Activation of farnesoid X receptor downregulates monocyte chemoattractant protein-1 in murine macrophage

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    Li, Liangpeng; Zhang, Qian; Peng, Jiahe; Jiang, Chanjui; Zhang, Yan; Shen, Lili; Dong, Jinyu; Wang, Yongchao; Jiang, Yu, E-mail: yujiang0207@163.com

    2015-11-27

    Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily, which plays important roles in bile acids/lipid homeostasis and inflammation. Monocyte chemoattractant protein-1 (MCP-1) contributes to macrophage infiltration into body tissues during inflammation. Here we investigated whether FXR can regulate MCP-1 expression in murine macrophage. FXR activation down regulate MCP-1 mRNA and protein levels in ANA-1 and Raw264.7 cells. Luciferase reporter assay, Gel shift and Chromatin immunoprecipitation assays have revealed that the activated FXR bind to the FXR element located in −738 bp ∼  −723 bp in MCP-1 promoter. These results suggested that FXR may serve as a novel target for regulating MCP-1 levels for the inflammation related diseases therapies. - Highlights: • FXR is expressed in murine macrophage cell line. • FXR down regulates MCP-1 expression. • FXR binds to the DR4 in MCP-1 promoter.

  1. Endogenous murine tau promotes neurofibrillary tangles in 3xTg-AD mice without affecting cognition.

    Science.gov (United States)

    Baglietto-Vargas, David; Kitazawa, Masashi; Le, Elaine J; Estrada-Hernandez, Tatiana; Rodriguez-Ortiz, Carlos J; Medeiros, Rodrigo; Green, Kim N; LaFerla, Frank M

    2014-02-01

    Recent studies on tauopathy animal models suggest that the concomitant expression of the endogenous murine tau delays the pathological accumulation of human tau, and interferes with the disease progression. To elucidate the role of endogenous murine tau in a model with both plaques and tangles, we developed a novel transgenic mouse model by crossing 3xTg-AD with mtauKO mice (referred to as 3xTg-AD/mtauKO mice). Therefore, this new model allows us to determine the pathological consequences of the murine tau. Here, we show that 3xTg-AD/mtauKO mice have lower tau loads in both soluble and insoluble fractions, and lower tau hyperphosphorylation level in the soluble fraction relative to 3xTg-AD mice. In the 3xTg-AD model endogenous mouse tau is hyperphosphorylated and significantly co-aggregates with human tau. Despite the deletion of the endogenous tau gene in 3xTg-AD/mtauKO mice, cognitive dysfunction was equivalent to 3xTg-AD mice, as there was no additional impairment on a spatial memory task, and thus despite increased tau phosphorylation, accumulation and NFTs in 3xTg-AD mice no further effects on cognition are seen. These findings provide better understanding about the role of endogenous tau to Alzheimer's disease (AD) pathology and for developing new AD models. © 2013.

  2. β-Arrestin2 mediates progression of murine primary myelofibrosis.

    Science.gov (United States)

    Rein, Lindsay Am; Wisler, James W; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Minyong; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia Kl; Premont, Richard T; Lefkowitz, Robert J

    2017-12-21

    Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2-/- cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.

  3. H4 histamine receptors mediate cell cycle arrest in growth factor-induced murine and human hematopoietic progenitor cells.

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    Anne-France Petit-Bertron

    Full Text Available The most recently characterized H4 histamine receptor (H4R is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs.

  4. Characterization of Periplasmic Protein BP26 Epitopes of Brucella melitensis Reacting with Murine Monoclonal and Sheep Antibodies

    Science.gov (United States)

    Wu, Jingbo; Zhang, Hui; Wang, Yuanzhi; Qiao, Jun; Chen, Chuangfu; Gao, Goege F.; Allain, Jean-Pierre; Li, Chengyao

    2012-01-01

    More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues 93DRDLQTGGI101 (position 93 to 101) or residues 104QPIYVYPD111, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65–70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90. PMID:22457830

  5. Antioxidative effects in vivo and colonization of Lactobacillus plantarum MA2 in the murine intestinal tract.

    Science.gov (United States)

    Tang, Wei; Xing, Zhuqing; Hu, Wei; Li, Chao; Wang, Jinju; Wang, Yanping

    2016-08-01

    Lactobacillus plantarum MA2 was isolated from traditional Chinese Tibet kefir grains, which possess several excellent properties and functions. We previously demonstrated the antioxidant activities of this bacterium in vitro. However, the maintenance and survival of L. plantarum MA2 inside the murine intestinal tract, where it exerts its probiotic properties, and whether its effects are elicited directly on the host remain unknown. Therefore, this study investigated the mechanisms of L. plantarum MA2 in aging mice following D-galactose administration. The levels of malondialdehyde decreased significantly in the L. plantarum MA2 groups after oral ingestion compared to the D-galactose model group, and total antioxidant capacity and glutathione peroxidase and superoxide dismutase activities increased significantly in the serum and liver. We combined fluorescein isothiocyanate labeling and green fluorescent protein expression to dynamically monitor the colonization and distribution of L. plantarum MA2 in the murine intestinal tract. The results indicated that L. plantarum MA2 was detected in the ileum, colon, and feces after single and continuous oral administration at day 21 and was maintained at 10(4)-10(5) CFU/g. These results suggest that L. plantarum MA2 colonizes and survives in the murine intestinal tract to exert its antioxidative effects.

  6. Ribosomopathy-like properties of murine and human cancers.

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    Sucheta Kulkarni

    Full Text Available Ribosomopathies comprise a heterogeneous group of hematologic and developmental disorders, often characterized by bone marrow failure, skeletal and other developmental abnormalities and cancer predisposition. They are associated with mutations and/or haplo-insufficiencies of ribosomal proteins (RPs and inefficient ribosomal RNA (rRNA processing. The resulting ribosomal stress induces the canonical p19ARF/Mdm2/p53 tumor suppressor pathway leading to proliferative arrest and/or apoptosis. It has been proposed that this pathway is then inactivated during subsequent neoplastic evolution. We show here that two murine models of hepatoblastoma (HB and hepatocellular carcinoma (HCC unexpectedly possess features that mimic the ribosomopathies. These include loss of the normal stoichiometry of RP transcripts and proteins and the accumulation of unprocessed rRNA precursors. Silencing of p19ARF, cytoplasmic sequestration of p53, binding to and inactivation of Mdm2 by free RPs, and up-regulation of the pro-survival protein Bcl-2 may further cooperate to drive tumor growth and survival. Consistent with this notion, re-instatement of constitutive p19ARF expression in the HB model completely suppressed tumorigenesis. In >2000 cases of human HCC, colorectal, breast, and prostate cancer, RP transcript deregulation was a frequent finding. In HCC and breast cancer, the severity of this dysregulation was associated with inferior survival. In HCC, the presence of RP gene mutations, some of which were identical to those previously reported in ribosomopathies, were similarly negatively correlated with long-term survival. Taken together, our results indicate that many if not all cancers possess ribosomopathy-like features that may affect their biological behaviors.

  7. Murine cell glycolipids customization by modular expression of glycosyltransferases.

    Science.gov (United States)

    Cid, Emili; Yamamoto, Miyako; Buschbeck, Marcus; Yamamoto, Fumiichiro

    2013-01-01

    Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.

  8. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    International Nuclear Information System (INIS)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

    2011-01-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  9. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

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    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

    2011-07-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  10. Innate immunity drives the initiation of a murine model of primary biliary cirrhosis.

    Directory of Open Access Journals (Sweden)

    Chao-Hsuan Chang

    Full Text Available Invariant natural killer T (iNKT cells play complex roles in bridging innate and adaptive immunity by engaging with glycolipid antigens presented by CD1d. Our earlier work suggested that iNKT cells were involved in the initiation of the original loss of tolerance in primary biliary cirrhosis (PBC. To address this issue in more detail and, in particular, to focus on whether iNKT cells activated by a Th2-biasing agonist (2s,3s,4r-1-O-(α-D-galactopyranosyl-N-tetracosanoyl-2-amino-1,3,4-nonanetriol (OCH, can influence the development of PBC in a xenobiotic-induced PBC murine model. Groups of mice were treated with either OCH or, as a control, α-galactosylceramide (α-GalCer and thence serially followed for cytokine production, markers of T cell activation, liver histopathology and anti-mitochondrial antibody responses. Further, additional groups of CD1d deleted mice were similarly studied. Our data indicate that administration of OCH has a dramatic influence with exacerbation of portal inflammation and hepatic fibrosis similar to mice treated with α-GalCer. Further, iNKT cell deficient CD1d knockout mice have decreased inflammatory portal cell infiltrates and reduced anti-mitochondrial antibody responses. We submit that activation of iNKT cells can occur via overlapping and/or promiscuous pathways and highlight the critical role of innate immunity in the natural history of autoimmune cholangitis. These data have implications for humans with PBC and emphasize that therapeutic strategies must focus not only on suppressing adaptive responses, but also innate immunity.

  11. Gene expression in IFN-g-activated murine macrophages

    Directory of Open Access Journals (Sweden)

    Pereira C.A.

    2004-01-01

    Full Text Available Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated or BALB/c (297 and 58 genes, respectively, up- and down-regulated mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

  12. Natural IgM antibodies that bind neoepitopes exposed as a result of spinal cord injury , drive secondary injury by activating complement.

    Science.gov (United States)

    Narang, Aarti; Qiao, Fei; Atkinson, Carl; Zhu, Hong; Yang, Xiaofeng; Kulik, Liudmila; Holers, V Michael; Tomlinson, Stephen

    2017-06-19

    Natural IgM antibodies (Abs) function as innate immune sensors of injury via recognition of neoepitopes expressed on damaged cells, although how this recognition systems function following spinal cord injury (SCI) exposes various neoepitopes and their precise nature remains largely unknown. Here, we investigated the role of two natural IgM monoclonal Abs (mAbs), B4 and C2, that recognize post-ischemic neoepitopes following ischemia and reperfusion in other tissues. Identification of post-SCI expressed neoepitopes was examined using previously characterized monoclonal Abs (B4 and C2 mAbs). The role of post-SCI neoepitopes and their recognition by natural IgM Abs in propagating secondary injury was examined in Ab-deficient Rag1-/- or wild type C57BL/6 mice using Ab reconstitution experiments and neoepitope-targeted therapeutic studies, respectively. Administration of B4 or C2 mAb following murine SCI increased lesion size and worsened functional outcome in otherwise protected Ab-deficient Rag1-/- mice. Injury correlated with colocalized deposition of IgM and C3d in injured spinal cords from both mAb reconstituted Rag1-/- mice and untreated wild-type mice. Depletion of peritoneal B1 B cells, a source of natural Abs, reduced circulating levels of IgM with B4 (annexin-IV) and C2 (subset of phospholipids) reactivity, reduced IgM and complement deposition in the spinal cord, and protected against SCI. We therefore investigated whether the B4 neoepitope represents a therapeutic target for complement inhibition. B4-Crry, a fusion protein consisting of a single-chain Ab derived from B4 mAb, linked to the complement inhibitor Crry, significantly protected against SCI. B4-Crry exhibited a dual function in that it inhibited both the binding of pathogenic IgM and blocked complement activation in the spinal cord. This study identifies important neoepitopes expressed withi