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Sample records for factor receptor antibody

  1. Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

    NARCIS (Netherlands)

    Spaargaren, M.; Defize, L. H.; Boonstra, J.; de Laat, S. W.

    1991-01-01

    The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized

  2. Acetylcholine receptor antibody

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003576.htm Acetylcholine receptor antibody To use the sharing features on this page, please enable JavaScript. Acetylcholine receptor antibody is a protein found in the blood of ...

  3. [Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

    Science.gov (United States)

    Han, Dong-gang; Duan, Xiao-yi; Guo, You-min; Zhou, Qi; Wang, Quan-ying; Yang, Guang-xiao

    2010-01-01

    To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

  4. Antibody-induced activation of the epidermal growth factor receptor tyrosine kinase requires the presence of detergent

    NARCIS (Netherlands)

    Spaargaren, M.; Defize, L. H.; de Laat, S. W.; Boonstra, J.

    1990-01-01

    Activation of the epidermal growth factor receptor (EGF-R) tyrosine kinase was investigated in membrane preparations as well as intact A431 cells, using anti-EGF-R antibodies directed against extra- and intracellular receptor domains. In vitro assay conditions were mimicked on whole cells by a mild

  5. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    Directory of Open Access Journals (Sweden)

    Yongjun Yin

    2016-05-01

    Full Text Available Activating mutations in fibroblast growth factor receptor 3 (FGFR3 have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9, a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11 with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3.

  6. Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

    Energy Technology Data Exchange (ETDEWEB)

    Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa; Cavallaro, Ugo [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Marco, Ario de, E-mail: ario.demarco@ung.si [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Dept. Environmental Sciences, University of Nova Gorica (UNG), Vipavska 13, P.O. Box 301-SI-5000, Rozna Dolina, Nova Gorica (Slovenia)

    2011-05-20

    Highlights: {yields} Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. {yields} These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. {yields} The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.

  7. Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

    International Nuclear Information System (INIS)

    Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa; Cavallaro, Ugo; Marco, Ario de

    2011-01-01

    Highlights: → Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. → These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. → The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.

  8. 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.

  9. 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

  10. Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity

    DEFF Research Database (Denmark)

    Engelmann, H; Holtmann, H; Brakebusch, C

    1990-01-01

    Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ....... These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies...

  11. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition

    DEFF Research Database (Denmark)

    Green, M R; Couchman, J R

    1985-01-01

    , the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing......Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map...... whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar...

  12. Antibody guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor receptor and placental alkaline phosphatase

    International Nuclear Information System (INIS)

    Kalofonos, H.P.; Pawlikowska, T.R.; Hemingway, A.

    1989-01-01

    Twenty-seven patients with brain glioma were scanned using 123 I-labeled monoclonal antibodies against epidermal growth factor receptor (EGFR1) or placental alkaline phosphatase (H17E2). Successful localization was achieved in 18 out of 27 patients. Eleven out of 27 patients were also studied using a nonspecific control antibody (11.4.1) of the same immunoglobulin subclass and observable tumor localization was also achieved in five patients. The specificity of targeting was assessed by comparing images obtained with specific and nonspecific antibodies and by examining tumor and normal tissue biopsies after dual antibody administration. Ten patients with recurrent grade III or IV glioma who showed good localization of radiolabeled antibody were treated with 40-140 mCi of 131 I-labeled antibody delivered to the tumor area intravenously (n = 5) or by infusion into the internal carotid artery (n = 5). Six patients showed clinical improvement lasting from 6 mo to 3 yr. One patient continues in remission (3 yr after therapy), but the other five who responded initially relapsed 6-9 mo after therapy and died. No major toxicity was attributable to antibody-guided irradiation. Targeted irradiation by monoclonal antibody may be clinically useful and should be explored further in the treatment of brain gliomas resistant to conventional forms of treatment

  13. Antibody guided irradiation of brain glioma by arterial infusion of radioactive monoclonal antibody against epidermal growth factor receptor and blood group A antigen

    Energy Technology Data Exchange (ETDEWEB)

    Epenetos, A.A.; Courtenay-Luck, N.; Pickering, D.; Hooker, G.; Lavender, J.P.; McKenzie, C.G. (Hammersmith Hospital, London (UK)); Durbin, H. (Imperial Cancer Research Fund, London (UK). Labs.)

    1985-05-18

    In a patient with recurrent grade IV glioma of the brain resistant to conventional treatment an antibody guided isotopic scan showed uptake by the tumour of a monoclonal antibody (9A) that was developed against epidermal growth factor receptor but cross reacted with blood group A antigen. As a therapeutic attempt antibody labelled with 1665 MBq (45.0 mCi) iodine-131 was delivered to the tumour area by infusion into the internal carotid artery. Computed tomography showed regression of the tumour after treatment, and an appreciable and sustained clinical improvement was noted without any toxicity. Delivery of irradiation guided by monoclonal antibody delivered by arterial infusion of the tumour area may be of clinical value in the treatment of brain gliomas resistant to conventional forms of treatment.

  14. Epitope mapping of the alpha-chain of the insulin-like growth factor I receptor using antipeptide antibodies.

    Science.gov (United States)

    Delafontaine, P; Ku, L; Ververis, J J; Cohen, C; Runge, M S; Alexander, R W

    1994-12-01

    Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells (VSMC). The IGF I receptor (IGF IR) is a heterotetramer composed of two cross-linked extracellular alpha-chains and two membrane-spanning beta-chains that contain a tyrosine-kinase domain. It has a high degree of sequence similarity to the insulin receptor (IR), and the putative ligand-specific binding site has been localized to a cysteine-rich region (CRR) of the alpha-chain. To obtain insights into antigenic determinants of the IGF IR, we raised a panel of site-specific polyclonal antibodies against short peptide sequences N-terminal to and within the CRR. Several antibodies raised against linear epitopes within the CRR bound to solubilized and native rat and human IGF IR by ELISA, did not cross-react with IR, but unexpectedly failed to inhibit 125I-IGF I binding. A polyclonal antibody directed against a 48-amino acid synthetic peptide, corresponding to a region of the CRR postulated to be essential for ligand binding, failed to react with either solubilized, reduced or intact IGF IR. Three antibodies specific for the N-terminus of the alpha-chain reacted with solubilized and native IGF IR. One of these, RAB 6, directed against amino acids 38-44 of the IGF IR, inhibited 125I-IGF I binding to rat aortic smooth muscle cells (RASM) and to IGF IR/3T3 cells (overexpressing human IGF IR) by up to 45%. Immunohistochemical analysis revealed strong IGF IR staining in the medial smooth muscle cell layer of rat aorta. These findings are consistent with a model wherein conformational epitopes within the CRR and linear epitopes within the N-terminus of the alpha-chain contribute to the IGF I binding pocket. These antibodies should provide a valuable tool to study structure-function relationships and in vivo regulation of the IGF IR.

  15. Development and Characterization of a Camelid Single Domain Antibody-Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2.

    Science.gov (United States)

    Tian, Baomin; Wong, Wah Yau; Uger, Marni D; Wisniewski, Pawel; Chao, Heman

    2017-01-01

    Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor, which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFRs). In this study, we describe the development and characterization of V21-DOS47, an immunoconjugate that targets VEGFR2. V21-DOS47 is composed of a camelid single domain anti-VEGFR2 antibody (V21) and the enzyme urease. The conjugate specifically binds to VEGFR2 and urease converts endogenous urea into ammonia, which is toxic to tumor cells. Previously, we developed a similar antibody-urease conjugate, L-DOS47, which is currently in clinical trials for non-small cell lung cancer. Although V21-DOS47 was designed from parameters learned from the generation of L-DOS47, additional optimization was required to produce V21-DOS47. In this study, we describe the expression and purification of two versions of the V21 antibody: V21H1 and V21H4. Each was conjugated to urease using a different chemical cross-linker. The conjugates were characterized by a panel of analytical techniques, including SDS-PAGE, size exclusion chromatography, Western blotting, and LC-MS E peptide mapping. Binding characteristics were determined by ELISA and flow cytometry assays. To improve the stability of the conjugates at physiologic pH, the pIs of the V21 antibodies were adjusted by adding several amino acid residues to the C-terminus. For V21H4, a terminal cysteine was also added for use in the conjugation chemistry. The modified V21 antibodies were expressed in the E. coli BL21 (DE3) pT7 system. V21H1 was conjugated to urease using the heterobifunctional cross-linker succinimidyl-[( N -maleimidopropionamido)-diethyleneglycol] ester (SM(PEG) 2 ), which targets lysine resides in the antibody. V21H4 was conjugated to urease using the homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol

  16. 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).

  17. Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

    Science.gov (United States)

    Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

    2013-02-01

    We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

  18. Epidermal growth factor receptor antibody plus recombinant human endostatin in treatment of hepatic metastases after remnant gastric cancer resection

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    We report a 55-year-old male who developed advanced hepatic metastasis and peritoneal carcinomatosis after resection of remnant gastric cancer resection 3 mo ago. The patient only received epidermal growth factor (EGF) receptor antibody (Cetuximab) plus recombinant human endostatin (Endostar).Anti-tumor activity was assessed by 18F-fluorodeoxyglucose (18F-FDG)positron emission tomography/computer tomography (PET/CT) at baseline and then every 4 wk. The case illustrates that 18FDG-PET/CT could make an early prediction of the response to Cetuximab plus Endostar in such clinical situations. 18FDG-PET/CT is a useful molecular imaging modality to evaluate the biological response advanced hepatic metastasis and peritoneal carcinomatosis to Cetuximab plus Endostar in patients after remnant gastric cancer resection.

  19. A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library.

    Science.gov (United States)

    Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu

    2016-11-04

    When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of V H and V L genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  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. Chimeric Monoclonal Antibody Cetuximab Targeting Epidermal Growth Factor-Receptor in Advanced Non-Melanoma Skin Cancer.

    Science.gov (United States)

    Wollina, Uwe; Tchernev, Georgi; Lotti, Torello

    2018-01-25

    Non-melanoma skin cancer (NMSC) is the most common malignancy in humans. Targeted therapy with monoclonal antibody cetuximab is an option in case of advanced tumor or metastasis. We present and update of the use of cetuximab in NMSC searching PUBMED 2011-2017. The monoclonal antibody cetuximab against epidermal growth factor receptor (EGFR) has been investigated for its use in NMSC during the years 2011 to 2017 by a PUBMED research using the following items: "Non-melanoma skin cancer AND cetuximab," "cutaneous squamous cell carcinoma AND cetuximab," and "basal cell carcinoma AND cetuximab", and "cetuximab AND skin toxicity". Available data were analyzed including case reports. Current evidence of cetuximab efficacy in NMSC was mainly obtained in cutaneous SCC and to a lesser extend in BCC. Response rates vary for neoadjuvant, adjuvant, mono- and combined therapy with cetuximab. Management of cutaneous toxicities is necessary. Guidelines are available. Cetuximab is an option for recurrent or advanced NMSC of the skin. It seems to be justified particularly in very high-risk tumors. There is a need for phase III trials.

  2. Fixation effect of SurePath preservative fluids using epidermal growth factor receptor mutation-specific antibodies for immunocytochemistry.

    Science.gov (United States)

    Kawahara, Akihiko; Taira, Tomoki; Abe, Hideyuki; Watari, Kosuke; Murakami, Yuichi; Fukumitsu, Chihiro; Takase, Yorihiko; Yamaguchi, Tomohiko; Azuma, Koichi; Akiba, Jun; Ono, Mayumi; Kage, Masayoshi

    2014-02-01

    Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids. Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers. PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased. These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future. © 2013 American Cancer Society.

  3. [Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv].

    Science.gov (United States)

    Sheng, Wei-Jin; Miao, Qing-Fang; Zhen, Yong-Su

    2009-06-01

    Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.

  4. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    Science.gov (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  5. Preclinical evaluation of radiolabelled nimotuzumab, a promising monoclonal antibody targeting the epidermal growth factor receptor

    Czech Academy of Sciences Publication Activity Database

    Bárta, P.; Lázníčková, A.; Lázníček, M.; Beckford, Denis R.; Beran, Miloš

    2013-01-01

    Roč. 56, č. 5 (2013), s. 280-288 ISSN 0362-4803 Grant - others:GA ČR(CZ) GAP304/10/1738 Institutional support: RVO:61389005 Keywords : radiopharmaceutical * 177Lu * 131I * nimotuzumab * EGFR * preclinical biodistribution Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 1.187, year: 2013

  6. Inhibition of the release of soluble tumor necrosis factor receptors in experimental endotoxemia by an anti-tumor necrosis factor-alpha antibody

    NARCIS (Netherlands)

    Jansen, J.; van der Poll, T.; Levi, M. [=Marcel M.; ten Cate, H.; Gallati, H.; ten Cate, J. W.; van Deventer, S. J.

    1995-01-01

    The role of tumor necrosis factor-alpha in the shedding of soluble tumor necrosis factor receptors in endotoxemia was investigated. The appearance of the soluble tumor necrosis factor receptors was assessed in four healthy volunteers following an intravenous injection of tumor necrosis factor-alpha

  7. Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

    Directory of Open Access Journals (Sweden)

    Ganesh Kolumam

    2015-07-01

    Full Text Available Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

  8. In vitro and in vivo imaging of prostate cancer angiogenesis using anti-vascular endothelial growth factor receptor 2 antibody-conjugated quantum dot

    International Nuclear Information System (INIS)

    Kwon, Haejin; Lee, Jiyeon; Song, Rita; Lee, Jung Han; Hwang, Sung Il; Lee, Hak Jong; Kim, Young Hwa

    2013-01-01

    Authors aimed to determine the targeting ability of vascular endothelial growth factor receptor 2 (VEGFR2)-conjugated quantum dots (QDs) in vitro, and apply it for a xenograft prostate cancer mouse model. Conjugation reaction of QDs was performed by using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and sulfo-(N-hydroxysulfosuccinimide) (Sulfo-NHS). The human umbilical vein cord endothelial cells (HUVECs) were incubated with QDs, conjugated with antiVGFR2, to see a specific binding in vitro. Fluorescent cell images were taken by a confocal microscope. The human prostate cancer cells (PC3) were injected to five nude mice on hind limbs to make the xenograft tumor model. QD-antiVEGFR2 antibody complex was injected into the tumor model and fluorescence measurements were performed at 1, 4, 9, 12, 15, and 24 hours after the injection. The specific interaction between HUVECs and QD-antiVEGFR2 antibody was clearly shown in vitro. The in vivo fluorescence image disclosed that there was an increased signal of tumor, 12 hours after the injection of QDs. By showing endothelial cells binding with QDs-antiVEGFR2 antibodyand an experimental application of the antibody for VEGFR2 imaging in the prostate cancer xenograft mouse model, we suggests that the antibody-conjugated QDs can be a potential imaging tool for angiogenesis of the cancer.

  9. Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system.

    Science.gov (United States)

    Hur, Byung-ung; Yoon, Jae-bong; Liu, Li-Kun; Cha, Sang-hoon

    2010-11-30

    Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6×10(17)) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5×10(9) human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4×10(8) and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K(D) of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts

    International Nuclear Information System (INIS)

    Flier, J.S.; Usher, P.; Moses, A.C.

    1986-01-01

    Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [ 3 H]thymidine into DNA in these cells, the identify of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody αIR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125 I-labeled IGF-I but not 125 I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. αIR-3 competitively inhibits IGF-I-mediated stimulation of [ 3 H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of αIR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of 3 H]thymidine incorporation is not inhibited by αIR-3. However, the incremental effects of higher concentrations (> 1 μg/ml) of insulin on [ 3 H]thymidine incorporation are inhibited by αIR-3. αIR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself

  11. EFFICACY EVALUATION OF A MONOCLONAL ANTIBODY AGAINST THE EPIDERMAL GROWTH FACTORS RECEPTOR IN THE MODEL OF SUBCUTANEOUS XENOGRAFT IN IMMUNODEFICIENT MICE

    Directory of Open Access Journals (Sweden)

    Ya. Yu. Ustyugov

    2015-01-01

    Full Text Available This article presents the results of the comparative antitumor efficacy study of two test articles of therapeutic humanized monoclonal antibodies against epidermal growth factor receptor (EGFR manufactured by Russian biopharmaceutical company CJSC “Biocad” and the commercial drug “Erbitux®” (Merck, Germany in subcutaneous xenografts model using human epidermoid carcinoma A431NS cell line. EGFR overexpression in epithelial tumor cells is a commonly known fact that determines use of this receptor as a target for therapeutic monoclonal antibodies. The basic mechanism of action of such drugs is blocking of epithelial cells proliferation through competitive binding to EGFR. Evaluation of tumor growth dynamics in immunodeficient (Nu/Nu mice was performed during in vivo experiment using two parameters: tumor growth index and tumor growth inhibition (TGI, %. The results received with used study design show that antitumor effects of the test articles manufactured by CJSC “Biocad” and the commercial comparator drug “Erbitux®” estimated by values of TGI and tumor growth index are comparable.

  12. Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

    Science.gov (United States)

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

    2017-10-01

    The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

  13. Antiproliferative and apoptotic effects of a specific anti-insulin-like growth factor I receptor single chain antibody on breast cancer cells.

    Science.gov (United States)

    Motallebnezhad, Morteza; Younesi, Vahid; Aghebati-Maleki, Leili; Nickho, Hamid; Safarzadeh, Elham; Ahmadi, Majid; Movassaghpour, Ali Akbar; Hosseini, Ahmad; Yousefi, Mehdi

    2016-11-01

    Insulin-like growth factor I receptor (IGF-IR) is expressed on breast cancer cells and involves in metastasis, survival, and proliferation. Currently, application of IGF-IR-targeting monoclonal antibodies (mAbs), alone or in combination with other drugs, is a promising strategy for breast cancer therapy. Single-chain fragment variable (scFv) antibodies have been introduced as appropriate tools for tumor-targeting purposes because of their advantages over whole antibodies. In the present study, we employed a naïve phage library and isolated scFvs against a specific epitope from extracellular domain of IGF-IR by panning process. The selected scFvs were further characterized using polyclonal and monoclonal phage ELISA, soluble monoclonal ELISA, and colony PCR and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies on breast cancer cell lines were also evaluated by MTT and Annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the IGF-IR peptide, and analyses of PCR product and sequencing confirmed the presence of full length V H and Vκ inserts. Treatment of MCF7 and SKBR3 cells with anti-IGF-IR scFv led to a significant growth inhibition. The results also showed that scFv treatment significantly augmented trastuzumab growth inhibitory effects on SKBR3 cells. The percentage of the apoptotic MCF7 and SKBR3 cells after 24-h treatment with scFv was 39 and 30.70 %, respectively. Twenty-four-hour treatment with scFv in combination with trastuzumab resulted in 44.75 % apoptosis of SKBR3 cells. Taken together, our results demonstrate that the targeting of IGF-IR by scFv can be an effective strategy in the treatment of breast cancer and provide further evidence for effectiveness of dual targeting of HER2 and IGF-IR in breast cancer therapy.

  14. 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.

  15. Production of two hemopoietic growth factors is differentially regulated in single T lymphocytes activated with an anti-T cell receptor antibody

    DEFF Research Database (Denmark)

    Kelso, A; Owens, T

    1988-01-01

    A method has been developed to measure the production by single activated T lymphocytes of two hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and multipotential CSF (multi-CSF or IL-3). When individual cells of the L3T4 (CD4)+ F23.1+ T cell clone E9.D4 were transferred by microma......A method has been developed to measure the production by single activated T lymphocytes of two hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and multipotential CSF (multi-CSF or IL-3). When individual cells of the L3T4 (CD4)+ F23.1+ T cell clone E9.D4 were transferred...... by micromanipulation into wells coated with the monoclonal anti-T cell receptor antibody F23.1, up to 90% of cells produced CSF as detected by CSF-dependent hemopoietic cell lines. Production occurred in the absence of proliferation and did not require the addition of accessory cells or IL-2. Both the frequency of CSF......-producing cells and the average production per positive cell depended on the density of the immobilized stimulating ligand, indicating that the response of each cell is not an all-or-none phenomenon but varies with the strength of stimulation. Individual cells of the clone varied over a 100-fold range...

  16. Increased anaerobic metabolism is a distinctive signature in a colorectal cancer cellular model of resistance to antiepidermal growth factor receptor antibody.

    Science.gov (United States)

    Monteleone, Francesca; Rosa, Roberta; Vitale, Monica; D'Ambrosio, Chiara; Succoio, Mariangela; Formisano, Luigi; Nappi, Lucia; Romano, Maria Fiammetta; Scaloni, Andrea; Tortora, Giampaolo; Bianco, Roberto; Zambrano, Nicola

    2013-03-01

    Cetuximab is a chimeric antibody approved for the treatment of metastatic colorectal cancer that selectively targets epidermal growth factor receptor (EGFR) signaling. Treatment efficacy with this drug is often impaired by acquired resistance and poor information has been accumulated on the mechanisms underlying such a phenomenon. By taking advantage of a syngenic cellular system of sensitivity and acquired resistance to anti-EGFR therapy in the colorectal carcinoma GEO cell line, we profiled protein expression differences between Cetuximab-sensitive and -resistant cells. Combined 2D DIGE and MS analyses revealed a main proteomic signature resulting from selective deregulation of various metabolic enzymes, including glucose-6-phosphate dehydrogenase, transketolase, lactate dehydrogenase B, and pyruvate dehydrogenase E1, which was also confirmed by Western blotting experiments. Lactate dehydrogenase B downregulation has been already related to an increased anaerobic utilization of glucose by tumor cells; accordingly, we verified that Cetuximab-resistant cells have a significantly higher production of lactate. Resistant cells also showed decreased nicotinamide adenine dinucleotide phosphate (NADPH) levels. Observed protein deregulations were not related to functional alterations of the hypoxia-inducible factor 1-associated pathways. Our data demonstrate that increased anaerobic metabolism is a prominent feature observed in the GEO syngenic model of acquired resistance to anti-EGFR therapy in colorectal cancer. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Location of Primary Tumor and Benefit From Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies in Patients With RAS and BRAF Wild-Type Metastatic Colorectal Cancer

    Science.gov (United States)

    Moretto, Roberto; Cremolini, Chiara; Rossini, Daniele; Pietrantonio, Filippo; Battaglin, Francesca; Mennitto, Alessia; Bergamo, Francesca; Loupakis, Fotios; Marmorino, Federica; Berenato, Rosa; Marsico, Valentina Angela; Caporale, Marta; Antoniotti, Carlotta; Masi, Gianluca; Salvatore, Lisa; Borelli, Beatrice; Fontanini, Gabriella; Lonardi, Sara; De Braud, Filippo

    2016-01-01

    Introduction. Right- and left-sided colorectal cancers (CRCs) differ in clinical and molecular characteristics. Some retrospective analyses suggested that patients with right-sided tumors derive less benefit from anti-epidermal growth factor receptor (EGFR) antibodies; however, molecular selection in those studies was not extensive. Patients and Methods. Patients with RAS and BRAF wild-type metastatic CRC (mCRC) who were treated with single-agent anti-EGFRs or with cetuximab-irinotecan (if refractory to previous irinotecan) were included in the study. Differences in outcome between patients with right- and left-sided tumors were investigated. Results. Of 75 patients, 14 and 61 had right- and left-sided tumors, respectively. None of the right-sided tumors responded according to RECIST, compared with 24 left-sided tumors (overall response rate: 0% vs. 41%; p = .0032), and only 2 patients with right-sided tumors (15%) versus 47 patients with left-sided tumors (80%) achieved disease control (p < .0001). The median duration of progression-free survival was 2.3 and 6.6 months in patients with right-sided and left-sided tumors, respectively (hazard ratio: 3.97; 95% confidence interval: 2.09–7.53; p < .0001). Conclusion. Patients with right-sided RAS and BRAF wild-type mCRC seemed to derive no benefit from single-agent anti-EGFRs. Implications for Practice: Right- and left-sided colorectal tumors have peculiar epidemiological and clinicopathological characteristics, distinct gene expression profiles and genetic alterations, and different prognoses. This study assessed the potential predictive impact of primary tumor site with regard to anti-epidermal growth factor receptor (EGFR) monoclonal antibody treatment in patients with RAS and BRAF wild-type metastatic colorectal cancer. The results demonstrated the lack of activity of anti-EGFRs in RAS and BRAF wild-type, right-sided tumors, thus suggesting a potential role for primary tumor location in driving treatment choices

  18. Pharmacokinetics, biodistribution and dosimetry of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats

    Energy Technology Data Exchange (ETDEWEB)

    Escobar, Normando Iznaga; Morales, Alejo Morales; Duconge, Jorge; Torres, Idania Caballero; Fernandez, Eduardo; Gomez, Jose A

    1998-01-01

    The pharmacokinetics, biodistribution and dosimetry of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t{sub (1(2{alpha}}{sub ))}) of 0.250 h and a mean elimination (t{sub (1(2{beta}}{sub ))}) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with {sup 99m}Tc-labeled humanized MAb R3 conjugate in patients should be supported.

  19. Pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats

    International Nuclear Information System (INIS)

    Escobar, Normando Iznaga; Morales, Alejo Morales; Duconge, Jorge; Torres, Idania Caballero; Fernandez, Eduardo; Gomez, Jose A.

    1998-01-01

    The pharmacokinetics, biodistribution and dosimetry of 99m Tc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t (1(2α)) ) of 0.250 h and a mean elimination (t (1(2β)) ) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with 99m Tc-labeled humanized MAb R3 conjugate in patients should be supported

  20. Establishment of EMab-134, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Detecting Squamous Cell Carcinoma Cells of the Oral Cavity.

    Science.gov (United States)

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

    2017-12-01

    Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, activates downstream signaling cascades in many tumors. In this study, we established novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We immunized mice with a combination of the extracellular domain of EGFR and EGFR-overexpressing LN229 glioblastoma cells (LN229/EGFR) and performed the first screening using enzyme-linked immunosorbent assay. Next, we selected mAbs using flow cytometry. Among 156 established clones, two mAbs, EMab-51 (IgG 1 , kappa) and EMab-134 (IgG 1 , kappa), reacted with EGFR in Western blot analysis; EMab-134 showed a much higher sensitivity compared with EMab-51. We compared the binding affinities of EMab-51 and EMab-134 using flow cytometry; the calculated K D values for EMab-51 and EMab-134 against SAS cells/HSC-2 cells were 9.2 × 10 -9 M/9.9 × 10 -9 M and 2.6 × 10 -9 M/8.3 × 10 -9 M, respectively, indicating that EMab-134 has a higher affinity to EGFR-expressing cells. Immunohistochemical analysis of EMab-51 and EMab-134 showed sensitive and specific reactions against oral cancer cells; EMab-134 demonstrated a much higher sensitivity (36/38 cases; 94.7%) to oral squamous cell carcinomas compared with EMab-51 (6/38 cases; 15.8%). This novel anti-EGFR mAb, EMab-134, could be advantageous for detecting EGFR in the pathological analysis of EGFR-expressing cancers.

  1. IMPACT STUDY: MK-0646 (DALOTUZUMAB, INSULIN GROWTH FACTOR 1 RECEPTOR (IGF-1R ANTIBODY COMBINED WITH PEMETREXED AND CISPLATIN IN STAGE IV METASTATIC NON-SQUAMOUS LUNG CANCER

    Directory of Open Access Journals (Sweden)

    Chao H Huang

    2016-01-01

    Full Text Available Background: Insulin-like growth factor 1 receptor (IGF-1R regulates cell growth, proliferation and apoptosis. Adenocarcinoma and never-smokers have a higher expression of IGF-1R, which is associated with worse overall survival. Dalotuzumab-MK0646 (D is a humanized monoclonal antibody that targets IGF-1R. Pemetrexed (P has higher activity in non-squamous lung cancer (NSQL. We initiated a randomized phase II trial to test the combination of P and Cisplatin (C +/- D in NSQL.Methods: Eligibility criteria: untreated NSQL stage IIIB or IV, ECOG 0 or 1, measurable disease, adequate renal, hepatic and hematologic function, and no other intercurrent illness. P at 500mg/m2 and C at 75mg/m2 IV were given every 3 weeks. D was given at 10mg/kg IV weekly on days 1, 8 and 15 of every 3-week cycle in the experimental group. The patients had a radiographic assessment after every 2 cycles and were treated for a maximum of 6 cycles if there was a response or stable disease. The primary objective of the study was to compare the clinical response rates of PC vs PC+D. Results: From 1/2009 to 2/2011, the study accrued 26 subjects: 16 male and 10 female, with a median age of 59; 14 were treated with PC and 12 were treated with PC+D. We observed 2 partial responses (PR, 7 stable disease (SD, 3 progressive disease (PD, and 2 were not evaluable (NE in the PC arm. In comparison, for the PC+D arm there were: 3 PR, 4 SD, 4 PD and 1 NE. The hematologic toxicity was similar in both groups. There higher incidence of hyperglycemia the experimental group; 4 cases with grade 3 and 1 case with grade 4. Conclusion: PC+D had a similar response rate compared to PC, with a higher rate of hyperglycemia. Identification of responders using predictive markers would be key to continuing the study of D in NSQL.

  2. A chimeric receptor of the insulin-like growth factor receptor type 1 (IGFR1) and a single chain antibody specific to myelin oligodendrocyte glycoprotein activates the IGF1R signalling cascade in CG4 oligodendrocyte progenitors.

    Science.gov (United States)

    Annenkov, Alexander; Rigby, Anne; Amor, Sandra; Zhou, Dun; Yousaf, Nasim; Hemmer, Bernhard; Chernajovsky, Yuti

    2011-08-01

    In order to generate neural stem cells with increased ability to survive after transplantation in brain parenchyma we developed a chimeric receptor (ChR) that binds to myelin oligodendrocyte glycoprotein (MOG) via its ectodomain and activates the insulin-like growth factor receptor type 1 ‎‎(IGF1R) signalling cascade. Activation of this pro-survival pathway in response to ligand broadly available in the brain might increase neuroregenerative potential of transplanted precursors. The ChR was produced by fusing a MOG-specific single ‎chain antibody with the extracellular boundary of the IGF1R transmembrane segment. The ChR is expressed on the cellular surface, predominantly as a monomer, and is not N-glycosylated. To show MOG-dependent functionality of the ChR, neuroblastoma cells B104 expressing this ChR were stimulated with monolayers of cells expressing recombinant MOG. The ChR undergoes MOG-dependent tyrosine phosphorylation and homodimerisation. It promotes insulin and IGF-independent growth of the oligodendrocyte progenitor cell line CG4. The proposed mode of the ChR activation is by MOG-induced dimerisation which promotes kinase domain transphosphorylation, by-passing the requirement of conformation changes known to be important for IGF1R activation. Another ChR, which contains a segment of the β-chain ectodomain, was produced in an attempt to recapitulate some of these conformational changes, but proved non-functional. 2011 Elsevier B.V. All rights reserved.

  3. Development of antibodies against the rat brain somatostatin receptor.

    Science.gov (United States)

    Theveniau, M; Rens-Domiano, S; Law, S F; Rougon, G; Reisine, T

    1992-05-15

    Somatostatin (SRIF) is a neurotransmitter in the brain involved in the regulation of motor activity and cognition. It induces its physiological actions by interacting with receptors. We have developed antibodies against the receptor to investigate its structural properties. Rabbit polyclonal antibodies were generated against the rat brain SRIF receptor. These antibodies (F4) were able to immunoprecipitate solubilized SRIF receptors from rat brain and the cell line AtT-20. The specificity of the interaction of these antibodies with SRIF receptors was further demonstrated by immunoblotting. F4 detected SRIF receptors of 60 kDa from rat brain and adrenal cortex and the cell lines AtT-20, GH3, and NG-108, which express high densities of SRIF receptors. They did not detect immunoreactive material from rat liver or COS-1, HEPG, or CRL cells, which do not express functional SRIF receptors. In rat brain, 60-kDa immunoreactivity was detected by F4 in the hippocampus, cerebral cortex, and striatum, which have high densities of SRIF receptors. However, F4 did not interact with proteins from cerebellum and brain stem, which express few SRIF receptors. Immunoreactive material cannot be detected in rat pancreas or pituitary, which have been reported to express a 90-kDa SRIF receptor subtype. The selective detection of 60-kDa SRIF receptors by F4 indicates that the 60- and 90-kDa SRIF receptor subtypes are immunologically distinct. The availability of antibodies that selectively detect native and denatured brain SRIF receptors provides us with a feasible approach to clone the brain SRIF receptor gene(s).

  4. Neutron capture therapy of epidermal growth factor receptor (EGFR)vIII positive gliomas using boronated monoclonal antibody L8A4

    International Nuclear Information System (INIS)

    Yang, Weilian; Barth, Rolf F.; Wu, Gong

    2006-01-01

    The purpose of the present study was to evaluate the EGFRvIII specific monoclonal antibody, L8A4 as a boron delivery agent for NCT of the receptor (+) rat glioma, F98 npEGFRvIII . A heavily boronated polyamidoamine (PAMAM) dendrimer (BD) was linked to L8A4 by means of heterobifunctional reagents. Wild type (F98 WT ) receptor(-) or EGFRvIII human gene transfected receptor(+) F98 npEGFRvIII glioma cells were implanted into the brains of Fischer rats. Biodistribution studies were initiated 14 d later. Animals received 125 I-labeled BD-L8A4 by either convection enhanced delivery (CED) or intratumoral(i.t.) injection and were euthanized 6, 12, 24 or 48 h later. At 6 h following CED, equivalent amounts of the bioconjugate were detected in receptor(+) and (-) tumors, but by 24 h the amounts retained by receptor(+) gliomas were 60.1% following CED and 43.7% following i.t. injection, compared to 14.6% ID/g by receptor(-) tumors. Tumor boron concentrations were 32.7 and 44.5 μg/g, respectively, for BD-L8A4 alone or in combination with i.v. BPA. BNCT was carried out at the MITR-II Reactor 24 h after CED of BD-L8A4 (∼40 μg 10 B/∼750 μg protein) and 2.5 h after i.v. injection of BPA (500 mg/kg). Rats that received BD-L8A4 alone or in combination with BPA had mean survival times of 70.4 and 85d, respectively, with 20% and 10% long term survivors, respectively, compared to 40.1 d for i.v. BPA and 30.3 and 26.3 d for irradiated and untreated controls, respectively. These data convincingly demonstrate the therapeutic efficacy of molecular targeting of EGFRvIII and should provide a platform for the future development of combinations of high and low molecular weight delivery agents for BNCT of brain tumors. (author)

  5. Agonistic effects of a monoclonal antibody specific for the interleukin-2 receptor

    International Nuclear Information System (INIS)

    Eardley, D.D.; Makrides, V.

    1986-01-01

    Interleukin-2 (IL-2) mediated immune responses can be blocked by monoclonal antibodies to the IL-2 receptor. The monoclonal antibody, M720, is defined as specific for the IL-2 receptor because it blocks 35 S-IL-2 binding to Con A blasts, reacts with lymphoblasts but not resting splenocytes, and inhibits IL-2 induced proliferation to mitogen, antigen, or allogeneic stimuli. Under appropriate culture conditions, the IL-2 receptor-specific antibody can act like IL-2 in that it will induce proliferation in T cells in the absence of additional antigen or mitogen. This agonistic effect is dependent on time, dose of antibody, and requires fetal calf serum (FCS) in the media. Because the FCS is not mitogenic by itself, the authors propose that the FCS components act as incomplete mitogen to induce appearance of IL-2 receptors but lack a factor which would push the majority of the cells into the S phase of the cell cycle. This factor is usually IL-2, but in the authors experiments, the IL-2 receptor-specific antibody can provide the same stimulus. These data indicate that factors like FCS can induce IL-2 receptors, but without additional IL-2 or receptor triggering, the cells will not proceed through the synthetic and proliferative phases of cell growth

  6. Agonistic Human Monoclonal Antibodies against Death Receptor 4 (DR4) | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute is seeking parties interested in licensing human monoclonal antibodies (mAbs) that bind to death receptor 4 ("DR4"). The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been recognized as promising targets for cancer treatment.

  7. A chimeric receptor of the insulin-like growth factor receptor type 1 (IGFR1) and a single chain antibody specific to myelin oligodendrocyte glycoprotein activates the IGF1R signalling cascade in CG4 oligodendrocyte progenitors

    NARCIS (Netherlands)

    Annenkov, A.; Rigby, A.; Amor, S.; Zhou, D.M.; Yousaf, N.; Hemmer, B.; Chernajovsky, Y.

    2011-01-01

    In order to generate neural stem cells with increased ability to survive after transplantation in brain parenchyma we developed a chimeric receptor (ChR) that binds to myelin oligodendrocyte glycoprotein (MOG) via its ectodomain and activates the insulin-like growth factor receptor type 1 (IGF1R)

  8. Measurement of anti- acetylcholine receptor auto-antibodies in ...

    African Journals Online (AJOL)

    auto-antibodies in myasthenia gravis. K. J. Steenkamp, W. Duim, M. s. Myer,. S. C. K. Malfeld, R. Anderson. Two different acetylcholine receptor (AChR) preparations derived from ... the detection of AChR auto-antibodies in serum specimens from 20 ... 4°C. Thereafter, 1 ml of washing solution (phosphate- buffered saline ...

  9. R1507, an Anti-Insulin-Like Growth Factor-1 Receptor (IGF-1R) Antibody, and EWS/FLI-1 siRNA in Ewing's Sarcoma: Convergence at the IGF/IGFR/Akt Axis

    Science.gov (United States)

    Rodon, Jordi; Sun, Michael; Kuenkele, Klaus-Peter; Parsons, Henrique A.; Trent, Jonathan C.; Kurzrock, Razelle

    2011-01-01

    A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si) RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71) was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays). TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney) cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt. PMID:22022506

  10. R1507, an anti-insulin-like growth factor-1 receptor (IGF-1R antibody, and EWS/FLI-1 siRNA in Ewing's sarcoma: convergence at the IGF/IGFR/Akt axis.

    Directory of Open Access Journals (Sweden)

    Helen J Huang

    Full Text Available A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71 was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays. TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt.

  11. Anti-idiotypic antibody: A new strategy for the development of a growth hormone receptor antagonist.

    Science.gov (United States)

    Lan, Hainan; Zheng, Xin; Khan, Muhammad Akram; Li, Steven

    2015-11-01

    In general, traditional growth hormone receptor antagonist can be divided into two major classes: growth hormone (GH) analogues and anti-growth hormone receptor (GHR) antibodies. Herein, we tried to explore a new class of growth hormone receptor (GHR) antagonist that may have potential advantages over the traditional antagonists. For this, we developed a monoclonal anti-idiotypic antibody growth hormone, termed CG-86. A series of experiments were conducted to characterize and evaluate this antibody, and the results from a competitive receptor-binding assay, Enzyme Linked Immunosorbent Assays (ELISA) and epitope mapping demonstrate that CG-86 behaved as a typical Ab2β. Next, we examined its antagonistic activity using in vitro cell models, and the results showed that CG-86 could effectively inhibit growth hormone receptor-mediated signalling and effectively inhibit growth hormone-induced Ba/F3-GHR638 proliferation. In summary, these studies show that an anti-idiotypic antibody (CG-86) has promise as a novel growth hormone receptor antagonist. Furthermore, the current findings also suggest that anti-idiotypic antibody may represent a novel strategy to produce a new class of growth hormone receptor antagonist, and this strategy may be applied with other cytokines or growth factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Physiochemical and biochemical factors influencing the pharmacokinetics of antibody therapeutics.

    Science.gov (United States)

    Bumbaca, Daniela; Boswell, C Andrew; Fielder, Paul J; Khawli, Leslie A

    2012-09-01

    Monoclonal antibodies are increasingly being developed to treat multiple disease areas, including those related to oncology, immunology, neurology, and ophthalmology. There are multiple factors, such as charge, size, neonatal Fc receptor (FcRn) binding affinity, target affinity and biology, immunoglobulin G (IgG) subclass, degree and type of glycosylation, injection route, and injection site, that could affect the pharmacokinetics (PK) of these large macromolecular therapeutics, which in turn could have ramifications on their efficacy and safety. This minireview examines how characteristics of the antibodies could be altered to change their PK profiles. For example, it was observed that a net charge modification of at least a 1-unit shift in isoelectric point altered antibody clearance. Antibodies with enhanced affinity for FcRn at pH 6.0 display longer serum half-lives and slower clearances than wild type. Antibody fragments have different clearance rates and tissue distribution profiles than full length antibodies. Fc glycosylation is perceived to have a minimal effect on PK while that of terminal high mannose remains unclear. More investigation is warranted to determine if injection route and/or site impacts PK. Nonetheless, a better understanding of the effects of all these variations may allow for the better design of antibody therapeutics.

  13. Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

    Science.gov (United States)

    Laricchia-Robbio, L; Liedberg, B; Platou-Vikinge, T; Rovero, P; Beffy, P; Revoltella, R P

    1996-10-01

    An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct

  14. Measurement of antiacetylcholine receptor auto-antibodies in ...

    African Journals Online (AJOL)

    Two different acetylcholine receptor (AChR) preparations derived from amputated human muscle (AChRAMP) and from the human rhabdomyosarcoma cell line TE671 (AChRTE67,) were compared in radio-immunoprecipitation assays for the detection of AChR auto-antibodies in serum specimens from 20 patients with ...

  15. 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

  16. 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.

  17. Monoclonal Antibodies to the Thyrotropin Receptor

    Directory of Open Access Journals (Sweden)

    Takao Ando

    2005-01-01

    Full Text Available The thyrotropin receptor (TSHR is a seven transmembrane G-protein linked glycoprotein expressed on the thyroid cell surface and which, under the regulation of TSH, controls the production and secretion of thyroid hormone from the thyroid gland. This membrane protein is also a major target antigen in the autoimmune thyroid diseases. In Graves' disease, autoantibodies to the TSHR (TSHR-Abs stimulate the TSHR to produce thyroid hormone excessively. In autoimmune thyroid failure, some patients exhibit TSHR-Abs which block TSH action on the receptor. There have been many attempts to generate human stimulating TSHR-mAbs, but to date, only one pathologically relevant human stimulating TSHR-mAb has been isolated. Most mAbs to the TSHR have been derived from rodents immunized with TSHR antigen from bacteria or insect cells. These antigens lacked the native conformation of the TSHR and the resulting mAbs were exclusively blocking or neutral TSHR-mAbs. However, mAbs raised against intact native TSHR antigen have included stimulating mAbs. One such stimulating mAb has demonstrated a number of differences in its regulation of TSHR post-translational processing. These differences are likely to be reflective of TSHR-Abs seen in Graves' disease.

  18. Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier

    International Nuclear Information System (INIS)

    Friden, P.M.; Walus, L.R.; Musso, G.F.; Taylor, M.A.; Malfroy, B.; Starzyk, R.M.

    1991-01-01

    Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. The authors have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration, labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate ∼ 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26

  19. Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

    Directory of Open Access Journals (Sweden)

    William A. McEwan

    2016-11-01

    Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

  20. Adult celiac disease with acetylcholine receptor antibody positive myasthenia gravis

    Institute of Scientific and Technical Information of China (English)

    Hugh J Freeman; Helen R Gillett; Peter M Gillett; Joel Oger

    2009-01-01

    Celiac disease has been associated with some autoimmune disorders. A 40-year-old competitive strongman with celiac disease responded to a glutenfree diet, but developed profound and generalized motor weakness with acetylcholine receptor antibody positive myasthenia gravis, a disorder reported to occur in about 1 in 5000. This possible relationship between myasthenia gravis and celiac disease was further explored in serological studies. Frozen stored serum samples from 23 acetylcholine receptor antibody positive myasthenia gravis patients with no intestinal symptoms were used to screen for celiac disease. Both endomysial and tissue transglutaminase antibodies were examined. One of 23 (or, about 4.3%) was positive for both IgA-endomysial and IgA tissue transglutaminase antibodies. Endoscopic studies subsequently showed duodenal mucosal scalloping and biopsies confirmed the histopathological changes of celiac disease. Celiac disease and myasthenia gravis may occur together more often than is currently appreciated. The presence of motor weakness in celiac disease may be a clue to occult myasthenia gravis, even in the absence of intestinal symptoms.

  1. Antibodies to the α-subunit of insulin receptor from eggs of immunized hens

    International Nuclear Information System (INIS)

    Song, C.; Yu, J.; Bai, D.H.; Hester, P.Y.; Kim, K.

    1985-01-01

    Simple methods for the generation, purification, and assay of antibodies to the α-subunit of insulin receptor from eggs of immunized hen have been described. Chicken antibodies against the α-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the β-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen

  2. A randomized, phase II study of the anti-insulin-like growth factor receptor type 1 (IGF-1R) monoclonal antibody robatumumab (SCH 717454) in patients with advanced colorectal cancer

    International Nuclear Information System (INIS)

    Lin, Edward H; Lenz, Heinz-Josef; Saleh, Mansoor N; Mackenzie, Mary J; Knost, James A; Pathiraja, Kumudu; Langdon, Ronald B; Yao, Siu-Long; Lu, Brian D

    2014-01-01

    Overexpression of insulin-like growth factor receptor type 1 (IGF-1R) may promote tumor development and progression in some cancer patients. Our objective was to assess tumor uptake of fluorodeoxyglucose by positron-emission tomography in patients with chemotherapy-refractory colorectal cancer treated with an anti-insulin-like growth factor receptor type 1 (anti-IGF-1R) monoclonal antibody, robatumumab. This was a randomized, open-label study with two periods (P1 and P2). Patients were randomized 3:1 into treatment arms R/R and C/R that received, respectively, one cycle of 0.3 mg/kg robatumumab or one or more cycles of second-line chemotherapy in P1, followed in either case by 10 mg/kg robatumumab biweekly in P2. The primary measure of fluorodeoxyglucose uptake was maximum standardized uptake value (SUV max ). The primary endpoint was the proportion of patients in the R/R arm having a mean percent decrease from baseline in SUV max (DiSUV) greater than 20% 12–14 days postdose in P2. Secondary endpoints included Response Evaluation Criteria in Solid Tumors (RECIST)-defined tumor response and pharmacodynamic measures of target engagement. Among 41 patients who were evaluable for the primary endpoint, seven (17%, 95% CI 7%–32%) had DiSUV greater than 20%. Fifty robatumumab-treated patients were evaluable for RECIST-defined tumor response and six (12%) had stable disease lasting greater than or equal to 7 weeks in P2. Pharmacodynamic endpoints indicated target engagement after dosing with 10 mg/kg robatumumab, but not 0.3 mg/kg. The most frequently reported adverse events were fatigue/asthenia, nausea, anorexia, and gastrointestinal disturbances. In this study, few patients with chemotherapy-refractory colorectal cancer appeared to benefit from treatment with the IGF-1R antagonist robatumumab

  3. C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies.

    Science.gov (United States)

    Fuss, Alexander; Hope, Christopher M; Deayton, Susan; Bennett, Greg Donald; Holdsworth, Rhonda; Carroll, Robert P; Coates, P Toby H

    2015-07-01

    Acute antibody-mediated rejection can occur in absence of circulating donor-specific antibodies. Agonistic antibodies targeting the anti-angiotensin II type 1 receptor (anti-AT1 R) are emerging as important non-human leucocyte antigen (HLA) antibodies. Elevated levels of anti-angiotensin II receptor antibodies were first observed in kidney transplant recipients with malignant hypertension and allograft rejection. They have now been studied in three separate kidney transplant populations and associate to frequency of rejection, severity of rejection and graft failure. We report 11 cases of biopsy-proven, Complement 4 fragment d (C4d)-negative, acute rejection occurring without circulating donor-specific anti-HLA antibodies. In eight cases, anti-angiotensin receptor antibodies were retrospectively examined. The remaining three subjects were identified from our centre's newly instituted routine anti-angiotensin receptor antibody screening. All subjects fulfilled Banff 2013 criteria for antibody-mediated rejection and all responded to anti-rejection therapy, which included plasma exchange and angiotensin receptor blocker therapy. These cases support the routine assessment of anti-AT1 R antibodies in kidney transplant recipients to identify subjects at risk. Further studies will need to determine optimal assessment protocol and the effectiveness of pre-emptive treatment with angiotensin receptor blockers. © 2015 Asian Pacific Society of Nephrology.

  4. HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design.

    Science.gov (United States)

    de Goeij, Bart E C G; Peipp, Matthias; de Haij, Simone; van den Brink, Edward N; Kellner, Christian; Riedl, Thilo; de Jong, Rob; Vink, Tom; Strumane, Kristin; Bleeker, Wim K; Parren, Paul W H I

    2014-01-01

    The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (Kadcyla(TM)). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA') fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA', was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA'-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.

  5. Fibroblast growth factor receptors in breast cancer.

    Science.gov (United States)

    Wang, Shuwei; Ding, Zhongyang

    2017-05-01

    Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of breast cancer. Recent genetic studies have identified some subtypes of fibroblast growth factor receptors as strong genetic loci associated with breast cancer. In this article, we review the recent epidemiological findings and experiment results of fibroblast growth factor receptors in breast cancer. First, we summarized the structure and physiological function of fibroblast growth factor receptors in humans. Then, we discussed the common genetic variations in fibroblast growth factor receptors that affect breast cancer risk. In addition, we also introduced the potential roles of each fibroblast growth factor receptors isoform in breast cancer. Finally, we explored the potential therapeutics targeting fibroblast growth factor receptors for breast cancer. Based on the biological mechanisms of fibroblast growth factor receptors leading to the pathogenesis in breast cancer, targeting fibroblast growth factor receptors may provide new opportunities for breast cancer therapeutic strategies.

  6. The main immunogenic region of acetylcholine receptors does not provoke the formation of antibodies of a predominant idiotype.

    Science.gov (United States)

    Killen, J A; Hochschwender, S M; Lindstrom, J M

    1985-08-01

    Anti-idiotype antibodies were induced in rats by immunization with rat monoclonal antibodies to the main immunogenic region of acetylcholine receptors. These anti-idiotype antibodies showed very little crossreaction with other rat monoclonal antibodies which bind to the same region of the receptor. When the rats producing these anti-idiotype antibodies were immunized with receptor, they showed no net decrease in anti-receptor antibody production. These data indicate that, although more than half of the antibodies produced by rats immunized with receptor are directed at a small region, many anti-receptor idiotypes are involved in this response and anti-idiotype therapy is not beneficial.

  7. Antibodies Against Hypocretin Receptor 2 Are Rare in Narcolepsy.

    Science.gov (United States)

    Giannoccaro, Maria Pia; Waters, Patrick; Pizza, Fabio; Liguori, Rocco; Plazzi, Giuseppe; Vincent, Angela

    2017-02-01

    Recently, antibodies to the hypocretin receptor 2 (HCRTR2-Abs) were reported in a high proportion of narcolepsy patients who developed the disease following Pandemrix® vaccination. We tested a group of narcolepsy patients for the HCRTR2-Abs using a newly established cell-based assay. Sera from 50 narcolepsy type 1 (NT1) and 11 narcolepsy type 2 (NT2) patients, 22 patients with other sleep disorders, 15 healthy controls, and 93 disease controls were studied. Cerebrospinal fluid (CSFs) from three narcoleptic patients were subsequently included. Human embryonic kidney cells were transiently transfected with human HCRTR2, incubated with patients' sera for 1 hr at 1:20 dilution and then fixed. Binding of antibodies was detected by fluorescently labeled secondary antibodies to human immunoglobulin G (IgG) and the different IgG subclasses. A nonlinear visual scoring system was used from 0 to 4; samples scoring ≥1 were considered positive. Only 3 (5%) of 61 patients showed a score ≥1, one with IgG1- and two with IgG3-antibodies, but titers were low (1:40-1:100). CSFs from these patients were negative. The three positive patients included one NT1 case with associated psychotic features, one NT2 patient, and an NT1 patient with normal hypocretin CSF levels. Low levels of IgG1 or IgG3 antibodies against HCRTR2 were found in 3 of 61 patients with narcolepsy, although only 1 presented with full-blown NT1. HCRTR2-Abs are not common in narcolepsy unrelated to vaccination. © Sleep Research Society 2016. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

  8. Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

    International Nuclear Information System (INIS)

    Conroy, W.G.

    1988-01-01

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG 3 k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [ 3 H]naloxone. The antibody which did not inhibit the binding of [ 3 H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG 3 k antibody that blocked the binding of [ 3 H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form

  9. Dual Mechanism of Interleukin-3 Receptor Blockade by an Anti-Cancer Antibody

    Directory of Open Access Journals (Sweden)

    Sophie E. Broughton

    2014-07-01

    Full Text Available Interleukin-3 (IL-3 is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD, a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF, IL-5, and IL-13 receptors, adopting unique “open” and classical “closed” conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling. Significantly, whereas “open-like” IL3Rα mutants can simultaneously bind IL-3 and CSL362, CSL362 still prevents the assembly of a higher-order IL-3 receptor-signaling complex. The discovery of open forms of cytokine receptors provides the framework for development of potent antibodies that can achieve a “double hit” cytokine receptor blockade.

  10. The anti-epidermal growth factor receptor (EGFR) monoclonal antibody, C225, enhances radiation-induced apoptosis in primary glioma cell lines through mediation of MAPK/JNK/p38 signaling pathways

    International Nuclear Information System (INIS)

    Chakravarti, A.; Noll, E.; Black, P.M.; Loeffler, J.S.

    2001-01-01

    Purpose: Increasing evidence suggests that signaling mediated by the epidermal growth factor receptor (EGFR) pathway contributes to radiation resistance. The anti-EGFR monoclonal antibody, C225, has been shown to enhance radiation response for several tumor types in preclinical models. Malignant gliomas are known to express, and quite frequently overexpress, EGFR. Our objectives in this study were to 1) Evaluate the efficacy of C225 as a radiation response modifier in EGFR-expressing glioma cell lines and to 2) Investigate the underlying molecular mechanisms mediating C225-induced enhancement of radiation response. Materials and Methods: Twelve EGFR-expressing glioma cells lines, established from patient tumors, were used for this study. Cells were incubated with C225, irradiated, and then evaluated for radiation response. Assays used to evaluate efficacy of C225-mediated radiosensitization included time-course apoptosis assays (Annexin V and TUNEL), viability assays (MTT), and clonogenic survival assays. The changes along MAPK (p44/p42)/JNK/p38-MAPK signal transduction pathways were then investigated using quantitative Western analysis with phospho-specific antibodies to determine the molecular mechanisms by which C225 mediates a given response. Results: C225 clearly enhanced radiation response for 7 of the 12 primary glioma cell lines studied. Enhancement of both immediate and delayed apoptotic responses was evident in these 7 responsive cell lines after C225 administration. The average apoptosis index at 6 hours post-RT+C225 for the 7 responsive lines was 9.5%, compared to 1.2% for the RT-only controls. A pattern of delayed apoptosis was evident in these 7 lines, with secondary apoptotic peaks (∼ 8.0%) occurring at 24 hours post-RT+C225. Time course viability measurements revealed a steady decrease in viable tumor cells in these responsive cell lines from 75% at 6 hours post-RT+C225 to 20% at 7 days. Clonogenic survival was also diminished in these 7 lines

  11. Type-I Insulin-Like Growth Factor Receptor (IGF1R)-Estrogen Receptor (ER) Crosstalk Contributes to Antiestrogen Therapy Resistance in Breast Cancer Cells

    Science.gov (United States)

    2013-02-01

    vitro have downregulated J GF1R making antibodies directed agai nst th is receptor ineffective. Inhlbition of IH may be necessary to manage ...monoclonal antibody to insulin-like growth factor receptor 1. J Clin Oncol 2009;27:580Q-7. 31. Drury s. Detre s. Leary A, Salter J, Reis-Filho J

  12. Valence of acetylcholine-receptor-antibody-titers in myasthenia gravis

    International Nuclear Information System (INIS)

    Zeitlhofer, J.; Maida, E.M.; Mamoli, B.; Mayr, N.

    1986-01-01

    In a retrospective study in 47 patients with myasthenia gravis acetylcholine-receptor-antibody-titers (AChR-AB) were correlated with the severity of the disease. In 18 patients the course of titers was studied and two groups of patients could be differentiated: patients with relative constant and patients with fluctuating titers. Age, age of begin of myasthenia and sex did not influence the titers. Also the duration of the disease and the severity of symptoms did not influence the level of AChR-AB-titers. In this retrospective study the influence of immunsuppressive therapy on the intra-individual course of AB-titers and their correlation with the clinical symptoms could not be judged. Measurement of AChR-AB is of value for the diagnosis of myasthenia gravis and important for judging the clinical course and the effect of therapy. (Author)

  13. Downregulation of transferrin receptor surface expression by intracellular antibody

    International Nuclear Information System (INIS)

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin

    2007-01-01

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 ± 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors

  14. Clinical Translation and Validation of a Predictive Biomarker for Patritumab, an Anti-human Epidermal Growth Factor Receptor 3 (HER3) Monoclonal Antibody, in Patients With Advanced Non-small Cell Lung Cancer.

    Science.gov (United States)

    Mendell, Jeanne; Freeman, Daniel J; Feng, Wenqin; Hettmann, Thore; Schneider, Matthias; Blum, Sabine; Ruhe, Jens; Bange, Johannes; Nakamaru, Kenji; Chen, Shuquan; Tsuchihashi, Zenta; von Pawel, Joachim; Copigneaux, Catherine; Beckman, Robert A

    2015-03-01

    During early clinical development, prospective identification of a predictive biomarker and validation of an assay method may not always be feasible. Dichotomizing a continuous biomarker measure to classify responders also leads to challenges. We present a case study of a prospective-retrospective approach for a continuous biomarker identified after patient enrollment but defined prospectively before the unblinding of data. An analysis of the strengths and weaknesses of this approach and the challenges encountered in its practical application are also provided. HERALD (NCT02134015) was a double-blind, phase 2 study in patients with non-small cell lung cancer (NSCLC) randomized to erlotinib with placebo or with high or low doses of patritumab, a monoclonal antibody targeted against human epidermal growth factor receptor 3 (HER3). While the primary objective was to assess safety and progression-free survival (PFS), a secondary objective was to determine a single predictive biomarker hypothesis to identify subjects most likely to benefit from the addition of patritumab. Although not identified as the primary biomarker in the study protocol, on the basis of preclinical results from 2 independent laboratories, expression levels of the HER3 ligand heregulin (HRG) were prospectively declared the predictive biomarker before data unblinding but after subject enrollment. An assay to measure HRG mRNA was developed and validated. Other biomarkers, such as epidermal growth factor receptor (EGFR) mutation status, were also evaluated in an exploratory fashion. The cutoff value for high vs. low HRG mRNA levels was set at the median delta threshold cycle. A maximum likelihood analysis was performed to evaluate the provisional cutoff. The relationship of HRG values to PFS hazard ratios (HRs) was assessed as a measure of internal validation. Additional NSCLC samples were analyzed to characterize HRG mRNA distribution. The subgroup of patients with high HRG mRNA levels ("HRG

  15. Anticardiolipin antibodies in proliferative diabetic retinopathy: An additional risk factor

    International Nuclear Information System (INIS)

    Shahin, Maha; ElDiasty, Amany M; Mabed, Mohamed

    2009-01-01

    To report the prevalence of anticardiolipin antibodies in patients with proliferative diabetic retinopathy (PDR) having high-risk criteria (HRC). Diabetic patients having PDR with HRC and diabetics free of retinopathy were compared for the presence of anticardiolipin antibodies. Among the 34 patients, 6 (17.7%) of diabetics having PDR with HRC were positive for anticardiolipin antibodies. There was no significant association of aCL antibodies with sex or type of diabetes. Using Pearson's correlation test, no significant associations of aCL antibodies with duration of diabetes or age of patients were found. All patients who were positive for anticardiolipin antibodies had PDR with HRC. The difference was statistically significant. Presence of anticardiolipin antibodies may represent an additional risk factor for PDR. (author)

  16. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    International Nuclear Information System (INIS)

    Berger, Christian; Madshus, Inger Helene; Stang, Espen

    2012-01-01

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: ► Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. ► Antibody combination causes internalization of EGFR by macropinocytosis. ► Antibody-induced internalization of EGFR is independent of EGFR kinase activity. ► Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  17. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene [Institute of Pathology, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Stang, Espen, E-mail: espsta@rr-research.no [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway)

    2012-12-10

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  18. Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies.

    Science.gov (United States)

    Dalmau, Josep; Gleichman, Amy J; Hughes, Ethan G; Rossi, Jeffrey E; Peng, Xiaoyu; Lai, Meizan; Dessain, Scott K; Rosenfeld, Myrna R; Balice-Gordon, Rita; Lynch, David R

    2008-12-01

    A severe form of encephalitis associated with antibodies against NR1-NR2 heteromers of the NMDA receptor was recently identified. We aimed to analyse the clinical and immunological features of patients with the disorder and examine the effects of antibodies against NMDA receptors in neuronal cultures. We describe the clinical characteristics of 100 patients with encephalitis and NR1-NR2 antibodies. HEK293 cells ectopically expressing single or assembled NR1-NR2 subunits were used to determine the epitope targeted by the antibodies. Antibody titres were measured with ELISA. The effect of antibodies on neuronal cultures was determined by quantitative analysis of NMDA-receptor clusters. Median age of patients was 23 years (range 5-76 years); 91 were women. All patients presented with psychiatric symptoms or memory problems; 76 had seizures, 88 unresponsiveness (decreased consciousness), 86 dyskinesias, 69 autonomic instability, and 66 hypoventilation. 58 (59%) of 98 patients for whom results of oncological assessments were available had tumours, most commonly ovarian teratoma. Patients who received early tumour treatment (usually with immunotherapy) had better outcome (p=0.004) and fewer neurological relapses (p=0.009) than the rest of the patients. 75 patients recovered or had mild deficits and 25 had severe deficits or died. Improvement was associated with a decrease of serum antibody titres. The main epitope targeted by the antibodies is in the extracellular N-terminal domain of the NR1 subunit. Patients' antibodies decreased the numbers of cell-surface NMDA receptors and NMDA-receptor clusters in postsynaptic dendrites, an effect that could be reversed by antibody removal. A well-defined set of clinical characteristics are associated with anti-NMDA-receptor encephalitis. The pathogenesis of the disorder seems to be mediated by antibodies.

  19. Anti-idiotypes against a monoclonal anti-haloperidol antibody bind to dopamine receptor

    International Nuclear Information System (INIS)

    Elazar, Z.; Kanety, H.; Schreiber, M.; Fuchs, S.

    1988-01-01

    Anti-idiotypic antibodies were raised in rabbits by immunization with a monoclonal anti-haloperidol antibody. Some of these anti-idiotypic antibodies bind in a concentration dependent manner to bovine striatal membranes. Following affinity purification, these antibodies inhibit haloperidol binding to striatal membranes and deplete [ 3 H]-spiperone binding sites from a solubilized preparation of striatal membranes. It is thus concluded that these anti-idiotypic antibodies are an internal image of haloperidol and as such can interact with D 2 -dopamine receptors

  20. Maintenance immunosuppression with intermittent intravenous IL-2 receptor antibody therapy in renal transplant recipients.

    LENUS (Irish Health Repository)

    Gabardi, Steven

    2011-09-01

    To report what we believe to be the first 2 cases of long-term (>24 months) intermittent intravenous interleukin-2 receptor antibody (IL-2RA) therapy for maintenance immunosuppression following renal transplantation.

  1. Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor

    Directory of Open Access Journals (Sweden)

    Ceran Ceyhan

    2012-10-01

    Full Text Available Abstract Background One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α. Methods Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation. Results We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells

  2. Thyroid-Stimulating Hormone Receptor Antibodies in Pregnancy: Clinical Relevance

    Science.gov (United States)

    Bucci, Ines; Giuliani, Cesidio; Napolitano, Giorgio

    2017-01-01

    Graves’ disease is the most common cause of thyrotoxicosis in women of childbearing age. Approximately 1% of pregnant women been treated before, or are being treated during pregnancy for Graves’ hyperthyroidism. In pregnancy, as in not pregnant state, thyroid-stimulating hormone (TSH) receptor (TSHR) antibodies (TRAbs) are the pathogenetic hallmark of Graves’ disease. TRAbs are heterogeneous for molecular and functional properties and are subdivided into activating (TSAbs), blocking (TBAbs), or neutral (N-TRAbs) depending on their effect on TSHR. The typical clinical features of Graves’ disease (goiter, hyperthyroidism, ophthalmopathy, dermopathy) occur when TSAbs predominate. Graves’ disease shows some peculiarities in pregnancy. The TRAbs disturb the maternal as well as the fetal thyroid function given their ability to cross the placental barrier. The pregnancy-related immunosuppression reduces the levels of TRAbs in most cases although they persist in women with active disease as well as in women who received definitive therapy (radioiodine or surgery) before pregnancy. Changes of functional properties from stimulating to blocking the TSHR could occur during gestation. Drug therapy is the treatment of choice for hyperthyroidism during gestation. Antithyroid drugs also cross the placenta and therefore decrease both the maternal and the fetal thyroid hormone production. The management of Graves’ disease in pregnancy should be aimed at maintaining euthyroidism in the mother as well as in the fetus. Maternal and fetal thyroid dysfunction (hyperthyroidism as well as hypothyroidism) are in fact associated with several morbidities. Monitoring of the maternal thyroid function, TRAbs measurement, and fetal surveillance are the mainstay for the management of Graves’ disease in pregnancy. This review summarizes the biochemical, immunological, and therapeutic aspects of Graves’ disease in pregnancy focusing on the role of the TRAbs in maternal and fetal

  3. Thyroid-Stimulating Hormone Receptor Antibodies in Pregnancy: Clinical Relevance

    Directory of Open Access Journals (Sweden)

    Ines Bucci

    2017-06-01

    Full Text Available Graves’ disease is the most common cause of thyrotoxicosis in women of childbearing age. Approximately 1% of pregnant women been treated before, or are being treated during pregnancy for Graves’ hyperthyroidism. In pregnancy, as in not pregnant state, thyroid-stimulating hormone (TSH receptor (TSHR antibodies (TRAbs are the pathogenetic hallmark of Graves’ disease. TRAbs are heterogeneous for molecular and functional properties and are subdivided into activating (TSAbs, blocking (TBAbs, or neutral (N-TRAbs depending on their effect on TSHR. The typical clinical features of Graves’ disease (goiter, hyperthyroidism, ophthalmopathy, dermopathy occur when TSAbs predominate. Graves’ disease shows some peculiarities in pregnancy. The TRAbs disturb the maternal as well as the fetal thyroid function given their ability to cross the placental barrier. The pregnancy-related immunosuppression reduces the levels of TRAbs in most cases although they persist in women with active disease as well as in women who received definitive therapy (radioiodine or surgery before pregnancy. Changes of functional properties from stimulating to blocking the TSHR could occur during gestation. Drug therapy is the treatment of choice for hyperthyroidism during gestation. Antithyroid drugs also cross the placenta and therefore decrease both the maternal and the fetal thyroid hormone production. The management of Graves’ disease in pregnancy should be aimed at maintaining euthyroidism in the mother as well as in the fetus. Maternal and fetal thyroid dysfunction (hyperthyroidism as well as hypothyroidism are in fact associated with several morbidities. Monitoring of the maternal thyroid function, TRAbs measurement, and fetal surveillance are the mainstay for the management of Graves’ disease in pregnancy. This review summarizes the biochemical, immunological, and therapeutic aspects of Graves’ disease in pregnancy focusing on the role of the TRAbs in maternal and

  4. Potentiated antibodies to mu-opiate receptors: effect on integrative activity of the brain.

    Science.gov (United States)

    Geiko, V V; Vorob'eva, T M; Berchenko, O G; Epstein, O I

    2003-01-01

    The effect of homeopathically potentiated antibodies to mu-receptors (10(-100) wt %) on integrative activity of rat brain was studied using the models of self-stimulation of the lateral hypothalamus and convulsions produced by electric current. Electric current was delivered through electrodes implanted into the ventromedial hypothalamus. Single treatment with potentiated antibodies to mu-receptors increased the rate of self-stimulation and decreased the threshold of convulsive seizures. Administration of these antibodies for 7 days led to further activation of the positive reinforcement system and decrease in seizure thresholds. Distilled water did not change the rate of self-stimulation and seizure threshold.

  5. Radiotherapy and receptor of epidermal growth factor

    International Nuclear Information System (INIS)

    Deberne, M.

    2009-01-01

    The expression level of the receptor of the epidermal growth factor is in correlation with the tumor cells radiosensitivity. An overexpression of the E.G.F.R. is often present in the bronchi cancer, epidermoid carcinomas of the O.R.L. sphere, esophagus, uterine cervix, and anal duct but also in the rectum cancers and glioblastomas. At the clinical level, the E.G.F.R. expression is in correlation with an unfavourable prognosis after radiotherapy in numerous tumoral localizations. In the rectum cancers it is an independent prognosis factor found in multifactorial analysis: increase of the rate of nodes and local recurrence when the E.G.F.R. is over expressed. In the uterine cervix cancers, the survival is is negatively affected in multifactorial analysis by the E.G.F.R. membranes expression level. At the therapy level, the development of anti E.G.F.R. targeted therapies (tyrosine kinase inhibitors and monoclonal antibodies) opens a new therapy field at radio-sensitivity potentiality. The irradiation makes an activation of the E.G.F.R. way that would be partially responsible of the post irradiation tumoral repopulation. This activation leads the phosphorylation of the PI3 kinase ways and M.A.P. kinase ones, then the Akt protein one that acts an apoptotic modulator part. It has been shown that blocking the E.G.F.R. way acts on three levels: accumulation of ells in phase G1, reduction of the cell repair and increasing of apoptosis. he inhibition of post irradiation action of the E.G.F.R. signal way is a factor explaining the ionizing radiation - anti E.G.F.R. synergy. The preclinical data suggest that the E.G.F.R. blocking by the monoclonal antibodies is more important than the use of tyrosine kinase inhibitors. A first positive randomized study with the cetuximab, published in 2006 in the epidermoid carcinomas of the O.R.L. sphere lead to its authorization on the market with the radiotherapy for this localization. The use of cetuximab in other indication with or in

  6. T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment.

    Directory of Open Access Journals (Sweden)

    Keith R Miller

    Full Text Available A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2, with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.

  7. Netrin-1 receptor antibodies in thymoma-associated neuromyotonia with myasthenia gravis.

    Science.gov (United States)

    Torres-Vega, Estefanía; Mancheño, Nuria; Cebrián-Silla, Arantxa; Herranz-Pérez, Vicente; Chumillas, María J; Moris, Germán; Joubert, Bastien; Honnorat, Jérôme; Sevilla, Teresa; Vílchez, Juan J; Dalmau, Josep; Graus, Francesc; García-Verdugo, José Manuel; Bataller, Luis

    2017-03-28

    To identify cell-surface antibodies in patients with neuromyotonia and to describe the main clinical implications. Sera of 3 patients with thymoma-associated neuromyotonia and myasthenia gravis were used to immunoprecipitate and characterize neuronal cell-surface antigens using reported techniques. The clinical significance of antibodies against precipitated proteins was assessed with sera of 98 patients (neuromyotonia 46, myasthenia gravis 52, thymoma 42; 33 of them with overlapping syndromes) and 219 controls (other neurologic diseases, cancer, and healthy volunteers). Immunoprecipitation studies identified 3 targets, including the Netrin-1 receptors DCC (deleted in colorectal carcinoma) and UNC5A (uncoordinated-5A) as well as Caspr2 (contactin-associated protein-like 2). Cell-based assays with these antigens showed that among the indicated patients, 9 had antibodies against Netrin-1 receptors (7 with additional Caspr2 antibodies) and 5 had isolated Caspr2 antibodies. Only one of the 219 controls had isolated Caspr2 antibodies with relapsing myelitis episodes. Among patients with neuromyotonia and/or myasthenia gravis, the presence of Netrin-1 receptor or Caspr2 antibodies predicted thymoma ( p myasthenia gravis, and neuromyotonia, often with Morvan syndrome ( p = 0.009). Expression of DCC, UNC5A, and Caspr2 proteins was demonstrated in paraffin-embedded thymoma samples (3) and normal thymus. Antibodies against Netrin-1 receptors (DCC and UNC5a) and Caspr2 often coexist and associate with thymoma in patients with neuromyotonia and myasthenia gravis. This study provides Class III evidence that antibodies against Netrin-1 receptors can identify patients with thymoma (sensitivity 21.4%, specificity 100%). © 2017 American Academy of Neurology.

  8. Chemokine Receptor-Specific Antibodies in Cancer Immunotherapy: Achievements and Challenges

    Science.gov (United States)

    Vela, Maria; Aris, Mariana; Llorente, Mercedes; Garcia-Sanz, Jose A.; Kremer, Leonor

    2015-01-01

    The 1990s brought a burst of information regarding the structure, expression pattern, and role in leukocyte migration and adhesion of chemokines and their receptors. At that time, the FDA approved the first therapeutic antibodies for cancer treatment. A few years later, it was reported that the chemokine receptors CXCR4 and CCR7 were involved on directing metastases to liver, lung, bone marrow, or lymph nodes, and the over-expression of CCR4, CCR6, and CCR9 by certain tumors. The possibility of inhibiting the interaction of chemokine receptors present on the surface of tumor cells with their ligands emerged as a new therapeutic approach. Therefore, many research groups and companies began to develop small molecule antagonists and specific antibodies, aiming to neutralize signaling from these receptors. Despite great expectations, so far, only one anti-chemokine receptor antibody has been approved for its clinical use, mogamulizumab, an anti-CCR4 antibody, granted in Japan to treat refractory adult T-cell leukemia and lymphoma. Here, we review the main achievements obtained with anti-chemokine receptor antibodies for cancer immunotherapy, including discovery and clinical studies, proposed mechanisms of action, and therapeutic applications. PMID:25688243

  9. Antibody Selection for Cancer Target Validation of FSH-Receptor in Immunohistochemical Settings

    Directory of Open Access Journals (Sweden)

    Nina Moeker

    2017-10-01

    Full Text Available Background: The follicle-stimulating hormone (FSH-receptor (FSHR has been reported to be an attractive target for antibody therapy in human cancer. However, divergent immunohistochemical (IHC findings have been reported for FSHR expression in tumor tissues, which could be due to the specificity of the antibodies used. Methods: Three frequently used antibodies (sc-7798, sc-13935, and FSHR323 were validated for their suitability in an immunohistochemical study for FSHR expression in different tissues. As quality control, two potential therapeutic anti-hFSHR Ylanthia® antibodies (Y010913, Y010916 were used. The specificity criteria for selection of antibodies were binding to native hFSHR of different sources, and no binding to non-related proteins. The ability of antibodies to stain the paraffin-embedded Flp-In Chinese hamster ovary (CHO/FSHR cells was tested after application of different epitope retrieval methods. Results: From the five tested anti-hFSHR antibodies, only Y010913, Y010916, and FSHR323 showed specific binding to native, cell-presented hFSHR. Since Ylanthia® antibodies were selected to specifically recognize native FSHR, as required for a potential therapeutic antibody candidate, FSHR323 was the only antibody to detect the receptor in IHC/histochemical settings on transfected cells, and at markedly lower, physiological concentrations (ex., in Sertoli cells of human testes. The pattern of FSH323 staining noticed for ovarian, prostatic, and renal adenocarcinomas indicated that FSHR was expressed mainly in the peripheral tumor blood vessels. Conclusion: Of all published IHC antibodies tested, only antibody FSHR323 proved suitable for target validation of hFSHR in an IHC setting for cancer. Our studies could not confirm the previously reported FSHR overexpression in ovarian and prostate cancer cells. Instead, specific overexpression in peripheral tumor blood vessels could be confirmed after thorough validation of the antibodies used.

  10. Ion channels in EEG: isolating channel dysfunction in NMDA receptor antibody encephalitis.

    Science.gov (United States)

    Symmonds, Mkael; Moran, Catherine H; Leite, M Isabel; Buckley, Camilla; Irani, Sarosh R; Stephan, Klaas Enno; Friston, Karl J; Moran, Rosalyn J

    2018-04-30

    Neurological and psychiatric practice frequently lack diagnostic probes that can assess mechanisms of neuronal communication non-invasively in humans. In N-methyl-d-aspartate (NMDA) receptor antibody encephalitis, functional molecular assays are particularly important given the presence of NMDA antibodies in healthy populations, the multifarious symptomology and the lack of radiological signs. Recent advances in biophysical modelling techniques suggest that inferring cellular-level properties of neural circuits from macroscopic measures of brain activity is possible. Here, we estimated receptor function from EEG in patients with NMDA receptor antibody encephalitis (n = 29) as well as from encephalopathic and neurological patient controls (n = 36). We show that the autoimmune patients exhibit distinct fronto-parietal network changes from which ion channel estimates can be obtained using a microcircuit model. Specifically, a dynamic causal model of EEG data applied to spontaneous brain responses identifies a selective deficit in signalling at NMDA receptors in patients with NMDA receptor antibody encephalitis but not at other ionotropic receptors. Moreover, though these changes are observed across brain regions, these effects predominate at the NMDA receptors of excitatory neurons rather than at inhibitory interneurons. Given that EEG is a ubiquitously available clinical method, our findings suggest a unique re-purposing of EEG data as an assay of brain network dysfunction at the molecular level.

  11. A monoclonal antibody TrkB receptor agonist as a potential therapeutic for Huntington's disease.

    Directory of Open Access Journals (Sweden)

    Daniel Todd

    Full Text Available Huntington's disease (HD is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for in vivo proof of concept studies in transgenic HD models.

  12. The Roles of the TSH Receptor Antibodies in Autoimmune Thyroid Diseases

    International Nuclear Information System (INIS)

    Koh, Chang Soon

    1986-01-01

    relapsed within 1 year after discontinuation of antithyroid drugs. The positive rate of TBII at the end of antithyroid drug treatment in relapse group (n=33) was significantly higher than those in remission group (n=26) (63.6% vs 23,1%; P 1 '2 5 I-bTSH to the TSH receptor were ranges of 0.1-2.6 mg/dl. One patient who had high titer of TBII in her serum delivered a hypothyroid baby due to transplacental transfer of maternal TBII. These findings suggested that 1) TSH receptor antibodies are closely related to a pathogenetic factor of Graves' hyperthyroidism and of some patients with primary nongoitrous myxedema, 2) measurement of TSH receptor antibodies is helpful m evaluating the clinical outcome of patients with Graves disease during antithyroid drug treatment and in predicting the neonatal transient hypothyroidism of baby delivered from primary myxedema patients. 3) there are 2 or more different types of TSH receptor antibodies in autoimmune thyroid diseases including one which stimulates thyroid by binding to the TSH receptor and another which blocks adenylate cyclase stimulation by TSH.

  13. Expression of epidermal growth factor receptors in human endometrial carcinoma

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Ottesen, B

    1993-01-01

    Little data exist on the expression of epidermal growth factor receptors (EGF-Rs) in human endometrial cancer. EGF-R status was studied in 65 patients with endometrial carcinomas and in 26 women with nonmalignant postmenopausal endometria, either inactive/atrophic endometrium or adenomatous...... hyperplasia. EGF-R was identified on frozen tissue sections by means of an indirect immunoperoxidase technique with a monoclonal antibody against the external domain of the EGF-R. Seventy-one percent of the carcinomas expressed positive EGF-R immunoreactivity. In general, staining was most prominent...

  14. Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

    Directory of Open Access Journals (Sweden)

    Miguel Aste-Amézaga

    2010-02-01

    Full Text Available Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD, and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR. The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50 values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL. In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell

  15. Subcellular localization of estradiol receptor in MCF7 cells studied with nanogold-labelled antibody fragments.

    Science.gov (United States)

    Kessels, M M; Qualmann, B; Thole, H H; Sierralta, W D

    1998-01-01

    Ultrastructural localization studies of estradiol receptor in hormone-deprived and hormone-stimulated MCF7 cells were done using F(ab') fragments of three different antibodies (#402, 13H2, HT277) covalently linked to nanogold. These ultra-small, non-charged immunoreagents, combined with a size-enlargement by silver enhancement, localized estradiol receptor in both nuclear and cytoplasmic areas of non-stimulated target cells; stimulation with the steroid induced a predominantly nuclear labelling. In the cytoplasm of resting cells, tagging was often observed at or in the proximity of stress fibers. In the nucleus a large proportion of receptor was found inside the nucleolus, specially with the reagent derived from antibody 13H2. We postulate that different accessibilities of receptor epitopes account for the different labelling densities observed at cytoskeletal elements and the nucleoli.

  16. Preparation and Characterization of an Antibody Antagonist That Targets the Porcine Growth Hormone Receptor

    Directory of Open Access Journals (Sweden)

    Huanzhong Cui

    2016-10-01

    Full Text Available A series of antagonists specifically targeting growth hormone receptors (GHR in different species, such as humans, rats, bovines, and mice, have been designed; however, there are currently no antagonists that target the porcine growth hormone (GH. Therefore, in this study, we developed and characterized a porcine GHR (pGHR antibody antagonist (denoted by AN98 via the hybridoma technique. The results from enzyme-linked immunosorbent assay, fluorescence activated cell sorter, indirect immunoinfluscent assay, and competitive receptor binding analysis showed that AN98 could specifically recognize pGHR, and further experiments indicated that AN98 could effectively inhibit pGH-induced signalling in CHO-pGHR cells and porcine hepatocytes. In addition, AN98 also inhibited GH-induced insulin-like growth factor-1 (IGF-1 secretion in porcine hepatocytes. In summary, these findings indicated that AN98, as a pGHR-specific antagonist, has potential applications in pGH-pGHR-related research on domestic pigs.

  17. Mycoplasma infection of cell lines can simulate the expression of Fc receptors by binding of the carbohydrate moiety of antibodies.

    Science.gov (United States)

    Lemke, H; Krausse, R; Lorenzen, J; Havsteen, B

    1985-05-01

    During the production of Fc receptor (FcR)-bearing hybridomas it was observed with a particular monoclonal anti-sheep red blood cell antibody (anti-SRBC 1/5, IgG1) that the contamination with Mycoplasma arginini of in vitro cultured cell lines leads to an apparent FcR activity. This property did not correspond with the serological typing since other antibodies of the same isotype could not support FcR rosette formation. Another mycoplasma strain M. orale lacked this property. Analysis of the binding reaction revealed that M. arginini contains a lectin which binds the carbohydrate moiety of the anti-SRBC 1/5 antibody, i.e. anti-SRBC 1/5 synthesized under the influence of tunicamycin or deglycosylated by NaIO4 oxidation did not support rosette formation. These data suggest that binding of antibodies to certain mycoplasma strains may be a pathogenic factor during mycoplasma infections by masking the microorganisms with the host's own defense molecules. The experiments with M. arginini-infected cell lines gain immunological importance since we obtained identical results with staphylococcal protein A, as another bacteriological FcR, and cell lines expressing intrinsic membrane FcR. Although it is an open question whether the glycoconjugates are directly bound by the FcR or else by influencing the three-dimensional structure of the antibodies, it seems possible that FcR in general may be lectins.

  18. Development of therapeutic antibodies to G protein-coupled receptors and ion channels: Opportunities, challenges and their therapeutic potential in respiratory diseases.

    Science.gov (United States)

    Douthwaite, Julie A; Finch, Donna K; Mustelin, Tomas; Wilkinson, Trevor C I

    2017-01-01

    The development of recombinant antibody therapeutics continues to be a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Therapeutic drug targets such as soluble cytokines, growth factors and single transmembrane spanning receptors have been successfully targeted by recombinant monoclonal antibodies and the development of new product candidates continues. Despite this growth, however, certain classes of important disease targets have remained intractable to therapeutic antibodies due to the complexity of the target molecules. These complex target molecules include G protein-coupled receptors and ion channels which represent a large target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these important regulators of cell function. Given this opportunity, a significant effort has been applied to address the challenges of targeting these complex molecules and a number of targets are linked to the pathophysiology of respiratory diseases. In this review, we provide a summary of the importance of GPCRs and ion channels involved in respiratory disease and discuss advantages offered by antibodies as therapeutics at these targets. We highlight some recent GPCRs and ion channels linked to respiratory disease mechanisms and describe in detail recent progress made in the strategies for discovery of functional antibodies against challenging membrane protein targets such as GPCRs and ion channels. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin

    Directory of Open Access Journals (Sweden)

    Felley-Bosco Emanuela

    2007-10-01

    Full Text Available Abstract Background The incidence of malignant pleural mesothelioma (MPM is associated with exposure to asbestos, and projections suggest that the yearly number of deaths in Western Europe due to MPM will increase until 2020. Despite progress in chemo- and in multimodality therapy, MPM remains a disease with a poor prognosis. Inducing apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or agonistic monoclonal antibodies which target TRAIL-receptor 1 (TRAIL-R1 or TRAIL-R2 has been thought to be a promising cancer therapy. Results We have compared the sensitivity of 13 MPM cell lines or primary cultures to TRAIL and two fully human agonistic monoclonal antibodies directed to TRAIL-R1 (Mapatumumab and TRAIL-R2 (Lexatumumab and examined sensitization of the MPM cell lines to cisplatin-induced by the TRAIL-receptor antibodies. We found that sensitivity of MPM cells to TRAIL, Mapatumumab and Lexatumumab varies largely and is independent of TRAIL-receptor expression. TRAIL-R2 contributes more than TRAIL-R1 to death-receptor mediated apoptosis in MPM cells that express both receptors. The combination of cisplatin with Mapatumumab or Lexatumumab synergistically inhibited the cell growth and enhanced apoptotic death. Furthermore, pre-treatment with cisplatin followed by Mapatumumab or Lexatumumab resulted in significant higher cytotoxic effects as compared to the reverse sequence. Combination-induced cell growth inhibition was significantly abrogated by pre-treatment of the cells with the antioxidant N-acetylcysteine. Conclusion Our results suggest that the sequential administration of cisplatin followed by Mapatumumab or Lexatumumab deserves investigation in the treatment of patients with MPM.

  20. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    International Nuclear Information System (INIS)

    Miao, H.-Q.; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping

    2006-01-01

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies

  1. Clinical Value of Thyrotropin Receptor Antibodies for the Differential Diagnosis of Interferon Induced Thyroiditis.

    Science.gov (United States)

    Benaiges, D; Garcia-Retortillo, M; Mas, A; Cañete, N; Broquetas, T; Puigvehi, M; Chillarón, J J; Flores-Le Roux, J A; Sagarra, E; Cabrero, B; Zaffalon, D; Solà, R; Pedro-Botet, J; Carrión, J A

    2016-01-01

    The clinical value of thyrotropin receptor antibodies for the differential diagnosis of thyrotoxicosis induced by pegylated interferon-alpha remains unknown. We analyzed the diagnostic accuracy of thyrotropin receptor antibodies in the differential diagnosis of thyrotoxicosis in patients with chronic hepatitis C (CHC) receiving pegylated interferon-alpha plus ribavirin. Retrospective analysis of 274 patients with CHC receiving pegylated interferon-alpha plus ribavirin. Interferon-induced thyrotoxicosis was classified according to clinical guidelines as Graves disease, autoimmune and non- autoimmune destructive thyroiditis. 48 (17.5%) patients developed hypothyroidism, 17 (6.2%) thyrotoxicosis (6 non- autoimmune destructive thyroiditis, 8 autoimmune destructive thyroiditis and 3 Graves disease) and 22 "de novo" thyrotropin receptor antibodies (all Graves disease, 2 of the 8 autoimmune destructive thyroiditis and 17 with normal thyroid function). The sensitivity and specificity of thyrotropin receptor antibodies for Graves disease diagnosis in patients with thyrotoxicosis were 100 and 85%, respectively. Patients with destructive thyroiditis developed hypothyroidism in 87.5% of autoimmune cases and in none of those with a non- autoimmune etiology (pthyroid scintigraphy for the differential diagnosis of thyrotoxicosis in CHC patients treated with pegylated interferon. © Georg Thieme Verlag KG Stuttgart · New York.

  2. A monoclonal antibody for G protein-coupled receptor crystallography

    DEFF Research Database (Denmark)

    Day, Peter W; Rasmussen, Søren Gøgsig Faarup; Parnot, Charles

    2007-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural inf...

  3. Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain

    International Nuclear Information System (INIS)

    Eisen, L.P.; Reichman, M.E.; Thompson, E.B.; Gametchu, B.; Harrison, R.W.; Eisen, H.J.

    1985-01-01

    The region of the glucocorticoid receptor that reacted with a monoclonal antibody (BUGR-1) was identified. In order to identify the immunoreactive region, the rat liver glucocorticoid receptor was subjected to limited proteolysis; immunoreactive fragments were identified by Western blotting. The monoclonal antibody reacted with both the undigested Mr approximately 97,000 receptor subunit and a Mr approximately 45,000 fragment containing the steroid-binding and DNA-binding domains. Digestion by trypsin also produced two steroid-binding fragments of Mr approximately 27,000 and 31,000 which did not react with the antibody and an immunoreactive Mr approximately 16,000 fragment. This Mr approximately 16,000 fragment was shown to bind to DNA-cellulose, indicating that it contained a DNA-binding domain of the receptor. The undigested receptor must have steroid associated with it to undergo activation to a DNA-binding form. However, the Mr approximately 16,000 immunoreactive fragment binds to DNA-cellulose even if it is obtained by digestion of the steroid-free holoreceptor which does not itself bind to DNA

  4. Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients

    DEFF Research Database (Denmark)

    Meyer, C N; Svenson, M; Schade Larsen, C

    2000-01-01

    of HIV-infected patients were therefore assessed by radioimmunoassay and radioreceptor assay. IgG from 4/505 HIV patients and 9/2000 healthy controls (p>0.05) bound rMIP-1alpha and rMIP-1beta, but not rRANTES. No other plasma factors bound the chemokines. The antibodies inhibited receptor binding of both...... chemokines. There was no association between presence of antibodies and disease stage or HIV progression rate. Three of 11 patients treated with rIL-2 developed IgG antibodies suppressing cellular binding and growth promotion of rIL-2. Hence, circulating factors, including antibodies MIP-1alpha/MIP-1beta...

  5. Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor

    DEFF Research Database (Denmark)

    List, K; Høyer-Hansen, G; Rønne, E

    1999-01-01

    Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interfer......Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance......) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former...

  6. Graves' Disease Mechanisms: The Role of Stimulating, Blocking, and Cleavage Region TSH Receptor Antibodies

    Science.gov (United States)

    Morshed, S. A.; Davies, T. F.

    2016-01-01

    The immunologic processes involved in Graves' disease (GD) have one unique characteristic – the autoantibodies to the TSH receptor (TSHR) – which have both linear and conformational epitopes. Three types of TSHR antibodies (stimulating, blocking, and cleavage) with different functional capabilities have been described in GD patients, which induce different signaling effects varying from thyroid cell proliferation to thyroid cell death. The establishment of animal models of GD by TSHR antibody transfer or by immunization with TSHR antigen has confirmed its pathogenic role and, therefore, GD is the result of a breakdown in TSHR tolerance. Here we review some of the characteristics of TSHR antibodies with a special emphasis on new developments in our understanding of what were previously called “neutral” antibodies and which we now characterize as autoantibodies to the “cleavage” region of the TSHR ectodomain. PMID:26361259

  7. A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

    Science.gov (United States)

    Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

    1990-09-01

    A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

  8. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    Science.gov (United States)

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  9. Changes in epidermal growth factor receptor expression during chemotherapy in non-small cell lung cancer

    DEFF Research Database (Denmark)

    Jakobsen, Jan Nyrop; Santoni-Rugiu, Eric; Sørensen, Jens Benn

    2014-01-01

    BACKGROUND: Antibodies targeting epidermal growth factor receptor (EGFR), such as cetuximab, may potentially improve outcome in non-small cell lung cancer (NSCLC) patients with high EGFR expression. The EGFR expression may be heterogeneously distributed within tumors, and small biopsies may thus...

  10. Synthetic α subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    International Nuclear Information System (INIS)

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

    1986-01-01

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) α subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 μg of peptide (T α 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR α subunit. Peptide H α 125-147 differs from T α 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound 125 I-labelled Hα 125-147 antibody bound Hα 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither Hα 125-147 nor T α 125-147. Rats immunized with H α 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR α subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man

  11. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    Energy Technology Data Exchange (ETDEWEB)

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

    1986-03-05

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  12. Blockade of human P2X7 receptor function with a monoclonal antibody.

    Science.gov (United States)

    Buell, G; Chessell, I P; Michel, A D; Collo, G; Salazzo, M; Herren, S; Gretener, D; Grahames, C; Kaur, R; Kosco-Vilbois, M H; Humphrey, P P

    1998-11-15

    A monoclonal antibody (MoAb) specific for the human P2X7 receptor was generated in mice. As assessed by flow cytometry, the MoAb labeled human blood-derived macrophage cells natively expressing P2X7 receptors and cells transfected with human P2X7 but not other P2X receptor types. The MoAb was used to immunoprecipitate the human P2X7 receptor protein, and in immunohistochemical studies on human lymphoid tissue, P2X7 receptor labeling was observed within discrete areas of the marginal zone of human tonsil sections. The antibody also acted as a selective antagonist of human P2X7 receptors in several functional studies. Thus, whole cell currents, elicited by the brief application of 2',3'-(4-benzoyl)-benzoyl-ATP in cells expressing human P2X7, were reduced in amplitude by the presence of the MoAb. Furthermore, preincubation of human monocytic THP-1 cells with the MoAb antagonized the ability of P2X7 agonists to induce the release of interleukin-1beta.

  13. Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

    International Nuclear Information System (INIS)

    Newsom-Davis, J.; Willcox, N.; Calder, L.

    1981-01-01

    We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlated with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases

  14. Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Newsom-Davis, J.; Willcox, N.; Calder, L.

    1981-11-26

    We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlated with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases.

  15. Blockade of epidermal growth factor receptors chemosensitizes breast cancer cells through up-regulation of Bnip3L

    NARCIS (Netherlands)

    Real, PJ; Benito, A; Cuevas, J; Berciano, MT; de Juan, A; Coffer, P; Gomez-Roman, J; Lafarga, M; Lopez-Vega, JM; Fernandez-Luna, JL

    2005-01-01

    Epidermal growth factor receptor-1 (EGFR) and EGFR-2 (HER2) have become major targets for cancer treatment. Blocking antibodies and small-molecule inhibitors are being used to silence the activity of these receptors in different tumors with varying efficacy. Thus, a better knowledge on the signaling

  16. Diagnostic sensitivity of two radio receptor assays (TRAK Assay and TRAK Dyno human) for the detection of TSH receptor antibodies

    International Nuclear Information System (INIS)

    Paunkovic, N.; Paunkovic, J.

    2003-01-01

    Radio receptor assays for the detection of TSH receptor antibodies in serum are typically based on binding the competition of TSH-R antibodies and 125I -labelled-TSH for membrane preparation of thyrocytes (TBII tests). The sensitivity of the available tests utilizing porcine cell membranes was found to be around 80%. A new test (TRAK Dyno human, BRAHMS) utilizes human recombinant TSH receptor and human standard material that is supposed to improve the performance of the test. We have compared the results of these two assays. The sensitivity of the TRAK Assay tested in 356 patients with untreated Grave's disease was found to be 85%, and 97.5% for TRAK Dyno human in 111 newly diagnosed patients. Both tests were performed from the same serum specimen for 60 of the investigated patients. The TRAK Assay was positive in 50 patients (83.2%) and TRAK Dyno human in 59 patients (98.3%). The specificity of the new radio receptor assay was also improved. (author)

  17. Clinical Utility of Acetylcholine Receptor Antibody Testing in Ocular Myasthenia Gravis.

    Science.gov (United States)

    Peeler, Crandall E; De Lott, Lindsey B; Nagia, Lina; Lemos, Joao; Eggenberger, Eric R; Cornblath, Wayne T

    2015-10-01

    The sensitivity of acetylcholine receptor (AChR) antibody testing is thought to be lower in ocular myasthenia gravis (OMG) compared with generalized disease, although estimates in small-scale studies vary. There is little information in the literature about the implications of AChR antibody levels and progression from OMG to generalized myasthenia gravis. To test the hypothesis that serum AChR antibody testing is more sensitive in OMG than previously reported and to examine the association between AChR antibody levels and progression from OMG to generalized myasthenia gravis. A retrospective, observational cohort study was conducted of 223 patients (mean [SD] age, 59.2 [16.4] years; 139 [62.3%] male) diagnosed with OMG between July 1, 1986, and May 31, 2013, at 2 large, academic medical centers. Baseline characteristics, OMG symptoms, results of AChR antibody testing, and progression time to generalized myasthenia gravis (if this occurred) were recorded for each patient. Multiple logistic regression was used to measure the association between all clinical variables and antibody result. Kaplan-Meier survival analysis was performed to examine time to generalization. Among the 223 participants, AChR antibody testing results were positive in 158 participants (70.9%). In an adjusted model, increased age at diagnosis (odds ratio [OR], 1.03; 95% CI, 1.01-1.04; P = .007) and progression to generalized myasthenia gravis (OR, 2.92; 95% CI, 1.18-7.26; P = .02) were significantly associated with positive antibody test results. Women were less likely to have a positive antibody test result (OR, 0.36; 95% CI, 0.19-0.68; P = .002). Patients who developed symptoms of generalized myasthenia gravis had a significantly higher mean (SD) antibody level than those who did not develop symptoms of generalized myasthenia gravis (12.7 [16.5] nmol/L vs 4.2 [7.9] nmol/L; P = .002). We demonstrate a higher sensitivity of AChR antibody testing than previously reported in the

  18. Coagulation factor VII variants resistant to inhibitory antibodies.

    Science.gov (United States)

    Branchini, Alessio; Baroni, Marcello; Pfeiffer, Caroline; Batorova, Angelika; Giansily-Blaizot, Muriel; Schved, Jean F; Mariani, Guglielmo; Bernardi, Francesco; Pinotti, Mirko

    2014-11-01

    Replacement therapy is currently used to prevent and treat bleeding episodes in coagulation factor deficiencies. However, structural differences between the endogenous and therapeutic proteins might increase the risk for immune complications. This study was aimed at identifying factor (F)VII variants resistant to inhibitory antibodies developed after treatment with recombinant activated factor VII (rFVIIa) in a FVII-deficient patient homozygous for the p.A354V-p.P464Hfs mutation, which predicts trace levels of an elongated FVII variant in plasma. We performed fluorescent bead-based binding, ELISA-based competition as well as fluorogenic functional (activated FX and thrombin generation) assays in plasma and with recombinant proteins. We found that antibodies displayed higher affinity for the active than for the zymogen FVII (half-maximal binding at 0.54 ± 0.04 and 0.78 ± 0.07 BU/ml, respectively), and inhibited the coagulation initiation phase with a second-order kinetics. Isotypic analysis showed a polyclonal response with a large predominance of IgG1. We hypothesised that structural differences in the carboxyl-terminus between the inherited FVII and the therapeutic molecules contributed to the immune response. Intriguingly, a naturally-occurring, poorly secreted and 5-residue truncated FVII (FVII-462X) escaped inhibition. Among a series of truncated rFVII molecules, we identified a well-secreted and catalytically competent variant (rFVII-464X) with reduced binding to antibodies (half-maximal binding at 0.198 ± 0.003 BU/ml) as compared to the rFVII-wt (0.032 ± 0.002 BU/ml), which led to a 40-time reduced inhibition in activated FX generation assays. Taken together our results provide a paradigmatic example of mutation-related inhibitory antibodies, strongly support the FVII carboxyl-terminus as their main target and identify inhibitor-resistant FVII variants.

  19. The Roles of the TSH Receptor Antibodies in Autoimmune Thyroid Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Koh, Chang Soon [Seoul National University College of Medicine, Seoul (Korea, Republic of)

    1986-09-15

    patients with Graves disease relapsed within 1 year after discontinuation of antithyroid drugs. The positive rate of TBII at the end of antithyroid drug treatment in relapse group (n=33) was significantly higher than those in remission group (n=26) (63.6% vs 23,1%; P<0.05). The mean value of TBII activities at the end of antithyroid drug treatment in relapse group was significantly elevated (29.7+21.4% vs 14.7+11.1%; P<0.05). Positive predictive value of TRII for relapse was 77.8%, which was not different from those of TRH nonresponsiveness(78.6%). The frequencies of detectable TBII in 68 patients with Hashimoto's thyroiditis, 10 patients with painless thyroiditis and 5 patients subacute thyroiditis were 14.7%, 20% and 0%, respectively. However in 25 patients with primary nongoitrous myxedema, 11 patients (44%) showed TBII activities in their sera. 9 out of 11 patients who had TBII activities in their sera showed high TBIl activities(more than 70'/ binding inhibition) and their IgG concentrations showing 50% binding inhibition of {sup 1}'2{sup 5}I-bTSH to the TSH receptor were ranges of 0.1-2.6 mg/dl. One patient who had high titer of TBII in her serum delivered a hypothyroid baby due to transplacental transfer of maternal TBII. These findings suggested that 1) TSH receptor antibodies are closely related to a pathogenetic factor of Graves' hyperthyroidism and of some patients with primary nongoitrous myxedema, 2) measurement of TSH receptor antibodies is helpful m evaluating the clinical outcome of patients with Graves disease during antithyroid drug treatment and in predicting the neonatal transient hypothyroidism of baby delivered from primary myxedema patients. 3) there are 2 or more different types of TSH receptor antibodies in autoimmune thyroid diseases including one which stimulates thyroid by binding to the TSH receptor and another which blocks adenylate cyclase stimulation by TSH.

  20. Growth factor receptors as molecular targets for cancer diagnosis and therapy

    International Nuclear Information System (INIS)

    Zalutsky, M. R.

    1997-01-01

    Growth factor receptors are of great interest as molecular targets for the diagnosis and treatment of cancer. Growth factor receptors are frequently over expressed on malignant cell populations since many cellular oncogenes encode either growth factors of their receptors. The wild-type epidermal growth factor receptor has a molecular weight of 170 kD and is over expressed on gliomas, bladder tumors, squamous cells carcinomas and breast carcinomas. Another growth factor oncogene, c-erb B-2, encodes a 185-kD glycoprotein found on the surface of gliomas, breast and ovarian cancers as well as other carcinomas of epithelial origin. In addition to causing over expression, oncogenic transformation also can result in genomic re-arrangements. An important example from the perspective of targeting is EGFRvIII, a deletion mutant which lacks amino acids 6-273 in the extracellular domain of the epiderma growth factor receptor. The EGFRvIII molecule (145 kD) may be of great value for targeting because it appears to be tumor-specific. Antibodies have been developed with specific reactivity with these growth factor receptors. Since these antibodies are internalized into the cell after receptor binding, it is necessary to use radiolabeling methods which residualize the radioactivity in the tumor cell after intracellular catabolism. To investigate this problem they have evaluated the effect of radioiodination method on the in vitro an in vivo properties of an anti-EGFRvIII antibody. Methods studied were Iodogen, tyramine-cellobiose, and N-succinimidyl 5-iodo-3-pyridine-carboxylate with the last offering optimal localization in a human xenograft model

  1. Immunogenicity of anti-tumor necrosis factor antibodies-toward improved methods of anti-antibody measurement.

    Science.gov (United States)

    Aarden, Lucien; Ruuls, Sigrid R; Wolbink, Gertjan

    2008-08-01

    To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been questioned and sometimes minimized, as few antibody responses to TmAbs (HACA or HAHA) were reported. However, the methods to detect and quantify such antibodies used in the past have been problematic. Only recently, methods have been developed that have adequate sensitivity and are not seriously disturbed by false-positive reactions caused by rheumatoid factors, natural antibodies to Fab or F(ab')2 fragments, or Fc interactions of IgG4. The large number of treated patients, in combination with these new assays, presents a unique opportunity to study the anti-antibody immune response in man, possibly allowing us to manipulate immunogenicity in the future.

  2. Evaluation of the specificity of antibodies raised against cannabinoid receptor type 2 in the mouse retina

    DEFF Research Database (Denmark)

    Cécyre, Bruno; Thomas, Sébastien; Ptito, Maurice

    2014-01-01

    Cannabinoid receptors (CB1R and CB2R) are among the most abundant G protein-coupled receptors in the central nervous system. The endocannabinoid system is an attractive therapeutic target for immune system modulation and peripheral pain management. While CB1R is distributed in the nervous system......, CB2R has traditionally been associated to the immune system. This dogma is currently a subject of debate since the discovery of CB2R expression in neurons using antibody-based methods. The localization of CB2R in the central nervous system (CNS) could have a significant impact on drug development...... because it would mean that in addition to its effects on the peripheral pain pathway, CB2R could also mediate some central effects of cannabinoids. In an attempt to clarify the debate over CB2R expression in the CNS, we tested several commercially or academically produced CB2R antibodies using Western...

  3. Drug of the year: programmed death-1 receptor/programmed death-1 ligand-1 receptor monoclonal antibodies.

    Science.gov (United States)

    Robert, Caroline; Soria, Jean-Charles; Eggermont, Alexander M M

    2013-09-01

    Programmed death-1 receptor (PD-1)/its ligand (PD-L1) antibodies have changed the landscape in oncology in 2013. The most mature results have been obtained in advanced melanoma patients. They indicate important response rates and high quality responses or prolonged duration. Also in renal cancer and in lung cancer remarkable activity has been demonstrated. Thus it is clear that these antibodies have a very broad potential and trials in many tumour types are being initiated. Breaking tolerance at the tumour site is a potent phenomenon and the potential for synergy with other checkpoint inhibitors such as ipilimumab have also been demonstrated in 2013. Long term tumour control now seems achievable and thus the concept of a clinical cure is emerging by modulation of the immune system. These antibodies bring immunotherapy to the forefront and indicate that immune-modulation will be a key component of therapeutic strategies from now on. Because of all these reasons PD-1/PD-L1 antibodies are considered 'drug of the year'. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay.

    Science.gov (United States)

    Sorsa-Leslie, Tarja; Mason, Helen D; Harris, William J; Fowler, Paul A

    2005-09-26

    We aimed to combine the generation of "artificial" antibodies with a rat pituitary bioassay as a new strategy to overcome 20 years of difficulties in the purification of gonadotrophin surge-attenuating factor (GnSAF). A synthetic single-chain antibody (Tomlinson J) phage display library was bio-panned with partially purified GnSAF produced by cultured human granulosa/luteal cells. The initial screening with a simple binding immunoassay resulted in 8 clones that were further screened using our in-vitro rat monolayer bioassay for GnSAF. Initially the antibodies were screened as pooled phage forms and subsequently as individual, soluble, single-chain antibody (scAbs) forms. Then, in order to improve the stability of the scAbs for immunopurification purposes, and to widen the range of labelled secondary antibodies available, these were engineered into full-length human immunoglobulins. The immunoglobulin with the highest affinity for GnSAF and a previously described rat anti-GnSAF polyclonal antiserum was then used to immunopurify bioactive GnSAF protein. The two purified preparations were electrophoresed on 1-D gels and on 7 cm 2-D gels (pH 4-7). The candidate GnSAF protein bands and spots were then excised for peptide mass mapping. Three of the scAbs recognised GnSAF bioactivity and subsequently one clone of the purified scAb-derived immunoglobulin demonstrated high affinity for GnSAF bioactivity, also binding the molecule in such as way as to block its bioactivity. When used for repeated immunopurification cycles and then Western blot, this antibody enabled the isolation of a GnSAF-bioactive protein band at around 66 kDa. Similar results were achieved using the rat anti-GnSAF polyclonal antiserum. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was human serum albumin precursor and variants. This study demonstrates that the combination of bioassay and phage display technologies is a powerful tool in the

  5. An Antibody Blocking Activin Type II Receptors Induces Strong Skeletal Muscle Hypertrophy and Protects from Atrophy

    Science.gov (United States)

    Minetti, Giulia C.; Sheppard, KellyAnn; Ibebunjo, Chikwendu; Feige, Jerome N.; Hartmann, Steffen; Brachat, Sophie; Rivet, Helene; Koelbing, Claudia; Morvan, Frederic; Hatakeyama, Shinji

    2014-01-01

    The myostatin/activin type II receptor (ActRII) pathway has been identified to be critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass. We have developed a novel, human anti-ActRII antibody (bimagrumab, or BYM338) to prevent binding of ligands to the receptors and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts and counteracts the inhibition of differentiation induced by myostatin or activin A. BYM338 prevents myostatin- or activin A-induced atrophy through inhibition of Smad2/3 phosphorylation, thus sparing the myosin heavy chain from degradation. BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin, detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin mutant mice, further confirming a beneficial effect on muscle growth beyond myostatin inhibition alone through blockade of ActRII ligands. BYM338 protects muscles from glucocorticoid-induced atrophy and weakness via prevention of muscle and tetanic force losses. These data highlight the compelling therapeutic potential of BYM338 for the treatment of skeletal muscle atrophy and weakness in multiple settings. PMID:24298022

  6. Antitumor efficacy of triple monoclonal antibody inhibition of epidermal growth factor receptor (EGFR) with MM151 in EGFR-dependent and in cetuximab-resistant human colorectal cancer cells

    Science.gov (United States)

    Napolitano, Stefania; Martini, Giulia; Martinelli, Erika; Della Corte, Carminia Maria; Morgillo, Floriana; Belli, Valentina; Cardone, Claudia; Matrone, Nunzia; Ciardiello, Fortunato; Troiani, Teresa

    2017-01-01

    Purpose We investigated the effect of triple monoclonal antibody inhibition of EGFR to overcome acquired resistance to first generation of anti-EGFR inhibitors. Experimental design MM151 is a mixture of three different monoclonal IgG1 antibodies directed toward three different, non-overlapping, epitopes of the EGFR. We performed an in vivo study by using human CRC cell lines (SW48, LIM 1215 and CACO2) which are sensitive to EGFR inhibitors, in order to evaluate the activity of MM151 as compared to standard anti-EGFR mAbs, such as cetuximab, as single agent or in a sequential strategy of combination MM151 with irinotecan (induction therapy) followed by MM151 with a selective MEK1/2 inhibitor (MEKi) (maintenance therapy). Furthermore, the ability of MM151 to overcome acquired resistance to cetuximab has been also evaluated in cetuximab-refractory CRC models. Results MM151 shown stronger antitumor activity as compared to cetuximab. The maintenance treatment with MM151 plus MEKi resulted the most effective therapeutic modality. In fact, this combination caused an almost complete suppression of tumor growth in SW48, LIM 1215 and CACO2 xenografts model at 30 week. Moreover, in this treatment group, mice with no evidence of tumor were more than double as compared to single agent treated mice. Its superior activity has also been demonstrated, in cetuximab-refractory CRC models. Conclusions These results provide experimental evidence that more efficient and complete EGFR blockade may determine better antitumor activity and could contribute to prevent and/or overcome acquired resistance to EGFR inhibitors. PMID:29137301

  7. Epidermal Growth Factor Receptor in Pancreatic Cancer

    International Nuclear Information System (INIS)

    Oliveira-Cunha, Melissa; Newman, William G.; Siriwardena, Ajith K.

    2011-01-01

    Pancreatic cancer is the fourth leading cause of cancer related death. The difficulty in detecting pancreatic cancer at an early stage, aggressiveness and the lack of effective therapy all contribute to the high mortality. Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein, which is expressed in normal human tissues. It is a member of the tyrosine kinase family of growth factors receptors and is encoded by proto-oncogenes. Several studies have demonstrated that EGFR is over-expressed in pancreatic cancer. Over-expression correlates with more advanced disease, poor survival and the presence of metastases. Therefore, inhibition of the EGFR signaling pathway is an attractive therapeutic target. Although several combinations of EGFR inhibitors with chemotherapy demonstrate inhibition of tumor-induced angiogenesis, tumor cell apoptosis and regression in xenograft models, these benefits remain to be confirmed. Multimodality treatment incorporating EGFR-inhibition is emerging as a novel strategy in the treatment of pancreatic cancer

  8. A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Guorui; Lam, Kwok-ho; Weisemann, Jasmin; Peng, Lisheng; Krez, Nadja; Perry, Kay; Shoemaker, Charles B.; Dong, Min; Rummel, Andreas; Jin, Rongsheng (BCH); (Cornell); (Tufts CTSI); (UCI); (MHH)

    2017-08-07

    Antibody treatment is currently the only available countermeasure for botulism, a fatal illness caused by flaccid paralysis of muscles due to botulinum neurotoxin (BoNT) intoxication. Among the seven major serotypes of BoNT/A-G, BoNT/A poses the most serious threat to humans because of its high potency and long duration of action. Prior to entering neurons and blocking neurotransmitter release, BoNT/A recognizes motoneurons via a dual-receptor binding process in which it engages both the neuron surface polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Previously, we identified a potent neutralizing antitoxin against BoNT/A1 termed ciA-C2, derived from a camelid heavy-chain-only antibody (VHH). In this study, we demonstrate that ciA-C2 prevents BoNT/A1 intoxication by inhibiting its binding to neuronal receptor SV2. Furthermore, we determined the crystal structure of ciA-C2 in complex with the receptor-binding domain of BoNT/A1 (HCA1) at 1.68 Å resolution. The structure revealed that ciA-C2 partially occupies the SV2-binding site on HCA1, causing direct interference of HCA1 interaction with both the N-glycan and peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical trials, despite that ciA-C2 is more than 10-times smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy.

  9. Development of cell-penetrating bispecific antibodies targeting the N-terminal domain of androgen receptor for prostate cancer therapy.

    Science.gov (United States)

    Goicochea, Nancy L; Garnovskaya, Maria; Blanton, Mary G; Chan, Grace; Weisbart, Richard; Lilly, Michael B

    2017-12-01

    Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody. The resulting 3E10-AR441 bispecific antibody enters human LNCaP prostate cells and accumulates in the nucleus. The antibody binds to wild-type, mutant and splice variant androgen receptor. Binding affinity of 3E10-AR441 to androgen receptor (284 nM) was lower than that of the parental AR441 mAb (4.6 nM), but could be improved (45 nM) through alternative placement of the affinity tags, and ordering of the VH and VK domains. The 3E10-AR441 bispecific antibody blocked genomic signaling by wild-type or splice variant androgen receptor in LNCaP cells. It also blocked non-genomic signaling by the wild-type receptor. Furthermore, bispecific antibody inhibited the growth of C4-2 prostate cancer cells under androgen-stimulated conditions. The 3E10-AR441 biAb can enter prostate cancer cells and inhibits androgen receptor function in a ligand-independent manner. It may be an attractive prototype agent for prostate cancer therapy. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Clinical significance of detection of antibodies to fetal and adult acetylcholine receptors in myasthenia gravis

    Institute of Scientific and Technical Information of China (English)

    Qi-Guang Shi; Zhi-Hong Wang; Xiao-Wei Ma; Da-Qi Zhang; Chun-Sheng Yang; Fu-Dong Shi; Li Yang

    2012-01-01

    Objective To evaluate the frequency,distribution and clinical significance of the antibodies to the fetal and/or adult acetylcholine receptor (AChR) in patients with myasthenia gravis (MG).Methods AChR antibodies were detected by cell-based assay in the serum of ocular MG (OMG) (n =90) and generalized MG (GMG) patients (n =110).The fetaltype (2α∶ β∶ γ∶ δ) and adult-type (2α∶ β∶ ε∶ δ) AChR were used as antigens,and their relevance to disease presentation was assessed.Results The overall frequencies of anti-adult and anti-fetal AChR antibodies were similar in all 200 patients examined,with 14 having serum specific to the AChR-γ subunit,and 22 to the AChR-ε subunit.The overall sensitivity when using the fetal and adult AChR antibodies was higher than that when using the fetal AChR antibody only (P =0.015).Compared with OMG patients,the mean age at disease onset and the positive ratio of antibodies to both isoforms of the AChR were significantly higher in patients who subsequently progressed to GMG.Older patients and patients with both anti-fetal and anti-adult AChR antibodies had a greater risk for developing generalized disease [odds ratio (OR),1.03;95% confidence interval (CI),1.01-1.06 and OR,5.09;95% CI,2.23-11.62].Conclusion Using both fetal-and adulttype AChRs as the antigens may be more sensitive than using either subtype.Patients with serum specific to both isoforms are at a greater risk of progressing to GMG.Patients with disease onset at an advanced age appear to have a higher frequency of GMG conversion.

  11. Lupus erythematosus cell preparation, antinuclear factor and antideoxyribonucleic acid antibody incongruity in systemic lupus erythematosus.

    Science.gov (United States)

    Chee, Y C

    1983-01-01

    'Total antinuclear antibody' (ANF) is detected by the fluorescent antinuclear antibody technique which is a screening test, positive in 99% of systemic lupus erythematosus (SLE) sera. The LE factor (positive in 75% of SLE sera), like the anti-DNA antibody, is an antinuclear antibody but directed against DNA-histone. ANF-negative SLE is a clinical entity with absence of these antibodies. A false negative ANF, in the presence of high titre anti-DNA antibody and/or LE cells, is illustrated in two cases of SLE. Postulated mechanisms for this phenomenon are interference in ANF detection by rheumatoid factor, and the prozone effect on the immunofluorescent tests.

  12. Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

    Directory of Open Access Journals (Sweden)

    Heger Christopher D

    2010-12-01

    Full Text Available Abstract Background Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. Methods Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. Results We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76% of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72% and 27% had only low levels of expression. Conclusions Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in

  13. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    International Nuclear Information System (INIS)

    Tao, Jun; Xiang, Jun-Jian; Li, Dan; Deng, Ning; Wang, Hong; Gong, Yi-Ping

    2010-01-01

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10 -9 M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  14. Shark Variable New Antigen Receptor (VNAR Single Domain Antibody Fragments: Stability and Diagnostic Applications

    Directory of Open Access Journals (Sweden)

    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.

  15. Ocular Safety of Intravitreal Connective Tissue Growth Factor Neutralizing Antibody.

    Science.gov (United States)

    Motevasseli, Tahmineh; Daftarian, Narsis; Kanavi, Mozhgan Rezaei; Ahmadieh, Hamid; Bagheri, Abouzar; Hosseini, Seyed Bagher; Ansari, Shabnam; Soheili, Zahra-Soheila

    2017-08-01

    To detect the safety of intravitreal injection of anti-connective tissue growth factor (CTGF) (IVAC) in rat eyes in order to apply this neutralizing antibody for experimental animal studies. Forty-five Lister Hooded male pigmented rats were divided into five groups that received IVAC (2 μl) corresponding to the doses of 10 (B), 20 (C), 50 (D), and 100 μg/ml (E), equal to 1.25, 2.5, 6.25, and 12.5 µg/ml of antibody concentration in rat vitreous, respectively. The sham group (A) received 2 μl of normal saline. Full field electroretinography (ERG) was performed at baseline and on days 7 and 28 after IVAC. The animals were euthanized and the corresponding eyes were subjected to routine histopathology, immunohistochemistry for glial fibrillary acidic protein (GFAP), and terminal transferase dUTP nick end-labeling (TUNEL) assay. Scotopic rod b-wave amplitude and maximal combined b-wave amplitude were 111.89 ± 71.2 and 178.57 ± 55.58 μV, respectively, at baseline which significantly reduced to 79.31 ± 52.59 and 128.73 ± 41.61 μV, respectively, after 28 days in group E (p < 0.05). There was no significant reduction of amplitudes in other groups with lower doses of anti-CTGF antibody. Retinal ganglion cells were significantly decreased in group E as compared to other groups. GFAP immune reactivity was not significant in any of the groups. TUNEL test showed inner retinal neural cell apoptosis only in group E. ERG, histopathologic, and apoptotic assays revealed no toxic effects of 10-50 μg/ml of IVAC in rat eyes. Using 100 μg/ml IVAC led to a significant toxic effect in terms of functional, histopathologic, and TUNEL findings.

  16. Focal status epilepticus and progressive dyskinesia: A novel phenotype for glycine receptor antibody-mediated neurological disease in children.

    Science.gov (United States)

    Chan, D W S; Thomas, T; Lim, M; Ling, S; Woodhall, M; Vincent, A

    2017-03-01

    Antibody-associated disorders of the central nervous system are increasingly recognised in adults and children. Some are known to be paraneoplastic, whereas in others an infective trigger is postulated. They include disorders associated with antibodies to N-methyl-d-aspartate receptor (NMDAR), voltage-gated potassium channel-complexes (VGKC-complex), GABA B receptor or glycine receptor (GlyR). With antibodies to NMDAR or VGKC-complexes, distinct clinical patterns are well characterised, but as more antibodies are discovered, the spectra of associated disorders are evolving. GlyR antibodies have been detected in patients with progressive encephalopathy with rigidity and myoclonus (PERM), or stiff man syndrome, both rare but disabling conditions. We report a case of a young child with focal seizures and progressive dyskinesia in whom GlyR antibodies were detected. Anticonvulsants and immunotherapy were effective in treating both the seizures and movement disorder with good neurological outcome and with a decline in the patient's serum GlyR-Ab titres. Glycine receptor antibodies are associated with focal status epilepticus and seizures, encephalopathy and progressive dyskinesia and should be evaluated in autoimmune encephalitis. Copyright © 2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

  17. Developmental regulation of human truncated nerve growth factor receptor

    Energy Technology Data Exchange (ETDEWEB)

    DiStefano, P.S.; Clagett-Dame, M.; Chelsea, D.M.; Loy, R. (Abbott Laboratories, Abbott Park, IL (USA))

    1991-01-01

    Monoclonal antibodies (designated XIF1 and IIIG5) recognizing distinct epitopes of the human truncated nerve growth factor receptor (NGF-Rt) were used in a two-site radiometric immunosorbent assay to monitor levels of NGF-Rt in human urine as a function of age. Urine samples were collected from 70 neurologically normal subjects ranging in age from 1 month to 68 years. By using this sensitive two-site radiometric immunosorbent assay, NGF-Rt levels were found to be highest in urine from 1-month old subjects. By 2.5 months, NGF-Rt values were half of those seen at 1 month and decreased more gradually between 0.5 and 15 years. Between 15 and 68 years, urine NGF-Rt levels were relatively constant at 5% of 1-month values. No evidence for diurnal variation of adult NGF-Rt was apparent. Pregnant women in their third trimester showed significantly elevated urine NGF-Rt values compared with age-matched normals. Affinity labeling of NGF-Rt with 125I-NGF followed by immunoprecipitation with ME20.4-IgG and gel autoradiography indicated that neonatal urine contained high amounts of truncated receptor (Mr = 50 kd); decreasingly lower amounts of NGF-Rt were observed on gel autoradiograms with development, indicating that the two-site radiometric immunosorbent assay correlated well with the affinity labeling technique for measuring NGF-Rt. NGF-Rt in urines from 1-month-old and 36-year-old subjects showed no differences in affinities for NGF or for the monoclonal antibody IIIG5. These data show that NGF-Rt is developmentally regulated in human urine, and are discussed in relation to the development and maturation of the peripheral nervous system.

  18. Developmental regulation of human truncated nerve growth factor receptor

    International Nuclear Information System (INIS)

    DiStefano, P.S.; Clagett-Dame, M.; Chelsea, D.M.; Loy, R.

    1991-01-01

    Monoclonal antibodies (designated XIF1 and IIIG5) recognizing distinct epitopes of the human truncated nerve growth factor receptor (NGF-Rt) were used in a two-site radiometric immunosorbent assay to monitor levels of NGF-Rt in human urine as a function of age. Urine samples were collected from 70 neurologically normal subjects ranging in age from 1 month to 68 years. By using this sensitive two-site radiometric immunosorbent assay, NGF-Rt levels were found to be highest in urine from 1-month old subjects. By 2.5 months, NGF-Rt values were half of those seen at 1 month and decreased more gradually between 0.5 and 15 years. Between 15 and 68 years, urine NGF-Rt levels were relatively constant at 5% of 1-month values. No evidence for diurnal variation of adult NGF-Rt was apparent. Pregnant women in their third trimester showed significantly elevated urine NGF-Rt values compared with age-matched normals. Affinity labeling of NGF-Rt with 125I-NGF followed by immunoprecipitation with ME20.4-IgG and gel autoradiography indicated that neonatal urine contained high amounts of truncated receptor (Mr = 50 kd); decreasingly lower amounts of NGF-Rt were observed on gel autoradiograms with development, indicating that the two-site radiometric immunosorbent assay correlated well with the affinity labeling technique for measuring NGF-Rt. NGF-Rt in urines from 1-month-old and 36-year-old subjects showed no differences in affinities for NGF or for the monoclonal antibody IIIG5. These data show that NGF-Rt is developmentally regulated in human urine, and are discussed in relation to the development and maturation of the peripheral nervous system

  19. Molecular and Therapeutic Characterization of Anti-ectodysplasin A Receptor (EDAR) Agonist Monoclonal Antibodies*

    Science.gov (United States)

    Kowalczyk, Christine; Dunkel, Nathalie; Willen, Laure; Casal, Margret L.; Mauldin, Elizabeth A.; Gaide, Olivier; Tardivel, Aubry; Badic, Giovanna; Etter, Anne-Lise; Favre, Manuel; Jefferson, Douglas M.; Headon, Denis J.; Demotz, Stéphane; Schneider, Pascal

    2011-01-01

    The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC50 of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments. PMID:21730053

  20. Expression, purification, and characterization of a diabody against the most important angiogenesis cell receptor: Vascular endothelial growth factor receptor 2

    Directory of Open Access Journals (Sweden)

    Mahdi Behdani

    2012-01-01

    Full Text Available Antibodies and their derivative fragments have long been used as tools in a variety of applications, in fundamental research work, biotechnology, diagnosis, and therapy. Camels produce single heavy-chain antibodies (VHH in addition to usual antibodies. These minimal-sized binders are very robust and bind the antigen with high affinity in a monomeric state. Vascular endothelial growth factor recepror-2 (VEGFR2 is an important tumor-associated receptor that blockade of its signaling can lead to the inhibition of neovascularization and tumor metastasis. Here, we describe the construction, expression, and purification VEGFR2-specific Diabody. Two variable fragments of a same camel anti-VEGFR2 antibody were linked together by the upper hinge segment of antibody to make a diabody. We showed the ability of diabody to recognition of VEGFR2 on the cell surface by FACS. Diabodies can be produced in the low-cost prokaryotic expression system, so they are suitable molecules for diagnostic and therapeutic issues.

  1. Negative correlation between bone mineral density and TSH receptor antibodies in long-term euthyroid postmenopausal women with treated Graves’ disease

    DEFF Research Database (Denmark)

    Ercolano, Monica A; Drnovsek, Monica L; Croome, Maria C

    2013-01-01

    Thyrotoxicosis is a cause of secondary osteoporosis. High concentrations of triiodotironine (T3) in Graves' disease stimulate bone turnover, but it is unclear if euthyroidism will always normalize bone metabolism. Thyrotropin (TSH) is known to affect directly the bone metabolism through the TSH...... receptor and TSH receptor antibodies (TRAb) may have an important role in bone turn-over.The aim of our study was to determine, in pre and postmenopausal euthyroidism patients with previous overt hyperthyroidism due to Graves' disease the bone mineral density (BMD) as well as factors that could affect BMD...

  2. Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

    International Nuclear Information System (INIS)

    Xu, Ling; Hausmann, Martin; Dietmaier, Wolfgang; Kellermeier, Silvia; Pesch, Theresa; Stieber-Gunckel, Manuela; Lippert, Elisabeth; Klebl, Frank; Rogler, Gerhard

    2010-01-01

    Cholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Expression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation. CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab

  3. Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Kellermeier Silvia

    2010-06-01

    Full Text Available Abstract Background Cholangiocarcinoma (CC is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Methods Expression of EGFR (epithelial growth factor receptor, HGFR (hepatocyte growth factor receptor IGF1R (insulin-like growth factor 1 receptor, IGF2R (insulin-like growth factor 2 receptor and VEGFR1-3 (vascular endothelial growth factor receptor 1-3 were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1. The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. Results EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml, with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D. HuH28, OZ and TFK-1 lacked KRAS mutation. Conclusion CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab.

  4. Personalized Radiation Oncology: Epidermal Growth Factor Receptor and Other Receptor Tyrosine Kinase Inhibitors.

    Science.gov (United States)

    Higgins, Geoff S; Krause, Mechthild; McKenna, W Gillies; Baumann, Michael

    Molecular biomarkers are currently evaluated in preclinical and clinical studies in order to establish predictors for treatment decisions in radiation oncology. The receptor tyrosine kinases (RTK) are described in the following text. Among them, the most data are available for the epidermal growth factor receptor (EGFR) that plays a major role for prognosis of patients after radiotherapy, but seems also to be involved in mechanisms of radioresistance, specifically in repopulation of tumour cells between radiotherapy fractions. Monoclonal antibodies against the EGFR improve locoregional tumour control and survival when applied during radiotherapy, however, the effects are heterogeneous and biomarkers for patient selection are warranted. Also other RTK´s such as c-Met and IGF-1R seem to play important roles in tumour radioresistance. Beside the potential to select patients for molecular targeting approaches combined with radiotherapy, studies are also needed to evluate radiotherapy adaptation approaches for selected patients, i.e. adaptation of radiation dose, or, more sophisticated, of target volumes.

  5. Immunogenicity of anti-tumor necrosis factor antibodies - toward improved methods of anti-antibody measurement

    NARCIS (Netherlands)

    Aarden, Lucien; Ruuls, Sigrid R.; Wolbink, Gertjan

    2008-01-01

    To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been

  6. Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

    Science.gov (United States)

    Lee, Gregory; Ge, Bixia

    2010-07-01

    As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

  7. Thyrotropin receptor antibody activities significantly correlate with the outcome of radioiodine (131I) therapy for hyperthyroid Graves' disease

    International Nuclear Information System (INIS)

    Kaise, Kazuro; Kaise, Nobuko; Yoshida, Katsumi; Fukazawa, Hiroshi; Mori, Koki; Yamamoto, Makiko; Sakurada, Toshiro; Saito, Shintaro; Yoshinaga, Kaoru

    1991-01-01

    The outcome of 131 I therapy for 109 patients with Graves' disease was analysed according to pretreatment laboratory data including thyrotropin receptor antibody (TRAb) activities. Forty-five percent of patients became euthyroid, and 13% of patients became hypothyroid within one year after 131 I therapy. Forty-two percent of patients remained hyperthyroid one year after 131 I therapy. Pretreatment values for serum T 4 , T 3 , and the estimated weight of the thyroid were significantly higher in the hyperthyroid group. The mean for the TRAb index of the hyperthyroid group was significantly higher than that of the euthyroid group. Life table analysis revealed a significant effect of the TRAb index on the rate of hyperthyroidism after 3 months or later. These results appear to suggest that the TRAb index is one of the factors which influence the outcome of 131 I therapy for Graves' disease. (author)

  8. Identification of endogenous opioid receptor components in rat brain using a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bero, L.A.; Roy, S.; Lee, N.M.

    1988-11-01

    A monoclonal antibody generated against the tertiary structure of a partially purified opioid binding protein was used to probe the structure of the dynorphin and beta-endorphin receptors. The Fab fragment 3B4F11 inhibited completely the binding of 125I-beta-endorphin and (3H)dynorphin to rat brain P2 membranes with IC50 values of 26 ng/ml and 40 ng/ml, respectively. To explore further the interaction of 3B4F11 with the beta-endorphin receptor, the effect of the Fab fragment on 125I-beta-endorphin cross-linking to rat brain membranes was examined. 125I-beta-endorphin was covalently bound to three major species of approximate molecular weights 108,000, 73,000, and 49,000. The delta-selective ligand D-Pen2, D-pen5enkephalin was least effective at inhibiting the cross-linking of beta-endorphin, whereas the micro-selective ligand Tyr-D-Ala-Gly-NMe-Phe-Gly-ol and kappa-selective ligand U50488 inhibited beta-endorphin cross-linking to the 108,000 and 73,000 Da species. Both 3B4F11 and beta-endorphin prevented the covalent binding of 125I-beta-endorphin to all three labeled species. These findings suggest that micro and kappa receptor types might have some structural similarities, whereas the delta receptor type might differ in molecular size. In addition, the micro, kappa, and delta ligands might have different primary sequences, whereas their tertiary structures might share regions of molecular homology with all three receptor constituents labeled by 125I-beta-endorphin. 3B4F11 will be a valuable tool for the purification and isolation of the several components of the beta-endorphin receptor complex.

  9. A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining.

    Science.gov (United States)

    Staudt, Nicole; Müller-Sienerth, Nicole; Fane-Dremucheva, Alla; Yusaf, Shahnaz P; Millrine, David; Wright, Gavin J

    2015-01-02

    Cell surface receptors and secreted proteins play important roles in neural recognition processes, but because their site of action can be a long distance from neuron cell bodies, antibodies that label these proteins are valuable to understand their function. The zebrafish embryo is a popular vertebrate model for neurobiology, but suffers from a paucity of validated antibody reagents. Here, we use the entire ectodomain of neural zebrafish cell surface or secreted proteins expressed in mammalian cells to select monoclonal antibodies to ten different antigens. The antibodies were characterised by Western blotting and the sensitivity of their epitopes to formalin fixation was determined. The rearranged antigen binding regions of the antibodies were amplified and cloned which enabled expression in a recombinant form from a single plasmid. All ten antibodies gave specific staining patterns within formalin-treated embryonic zebrafish brains, demonstrating that this generalised approach is particularly efficient to elicit antibodies that stain native antigen in fixed wholemount tissue. Finally, we show that additional tags can be easily added to the recombinant antibodies for convenient multiplex staining. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Hormonal receptors and vascular endothelial growth factor in juvenile nasopharyngeal angiofibroma: immunohistochemical and tissue microarray analysis.

    Science.gov (United States)

    Liu, Zhuofu; Wang, Jingjing; Wang, Huan; Wang, Dehui; Hu, Li; Liu, Quan; Sun, Xicai

    2015-01-01

    This work demonstrated that juvenile nasopharyngeal angiofibromas (JNAs) express high levels of hormone receptors and vascular endothelial growth factor (VEGF) compared with normal nasal mucosa. The interaction between hormone receptors and VEGF may be involved in the initiation and growth of JNA. JNA is a rare benign tumor that occurs almost exclusively in male adolescents. Although generally regarded as a hormone-dependent tumor, this has not been proven in previous studies. The aim of this study was to investigate the role of hormone receptors in JNA and the relationship with clinical characteristics. Standard immunohistochemical microarray analysis was performed on 70 JNA samples and 10 turbinate tissue samples. Specific antibodies for androgen receptor (AR), estrogen receptor-α (ER-α), estrogen receptor-β (ER-β), progesterone receptor (PR), and VEGF were examined, and the relationships of receptor expression with age, tumor stage, and bleeding were evaluated. RESULTS showed that JNA expressed ER-α (92.9%), ER-β (91.4%), AR (65.7%), PR (12.8%), and VEGF (95.7%) at different levels. High level of VEGF was linked to elevated ER-α and ER-β. There was no significant relationship between hormonal receptors and age at diagnosis, tumor stage or bleeding. However, overexpression of ER-α was found to be an indicator of poor prognosis (p = 0.031).

  11. Antibodies against the melanocortin-4 receptor act as inverse agonists in vitro and in vivo.

    Science.gov (United States)

    Peter, Jean-Christophe; Nicholson, Janet R; Heydet, Déborah; Lecourt, Anne-Catherine; Hoebeke, Johan; Hofbauer, Karl G

    2007-06-01

    Functionally active antibodies (Abs) against central G-protein-coupled receptors have not yet been reported. We selected the hypothalamic melanocortin-4 receptor (MC4-R) as a target because of its crucial role in the regulation of energy homeostasis. A 15 amino acid sequence of the N-terminal (NT) domain was used as an antigen. This peptide showed functional activity in surface plasmon resonance experiments and in studies on HEK-293 cells overexpressing the human MC4-R (hMC4-R). Rats immunized against the NT peptide produced specific antibodies, which were purified and characterized in vitro. In HEK-293 cells, rat anti-NT Abs showed specific immunofluorescence labeling of hMC4-R. They reduced the production of cAMP under basal conditions and after stimulation with a synthetic MC4-R agonist. Rats immunized against the NT peptide developed a phenotype consistent with MC4-R blockade, that is, increased food intake and body weight, increased liver and fat pad weight, and elevated plasma triglycerides. In a separate experiment in rats, an increase in food intake could be produced after injection of purified Abs into the third ventricle. Similar results were obtained in rats injected with anti-NT Abs raised in rabbits. Our data show for the first time that active immunization of rats against the NT sequence of the MC4-R results in specific Abs, which appear to stimulate food intake by acting as inverse agonists in the hypothalamus.

  12. The Anti-Acetylcholine Receptor Antibody Test in Suspected Ocular Myasthenia Gravis

    Directory of Open Access Journals (Sweden)

    Jung Jin Lee

    2014-01-01

    Full Text Available Aim. To estimate the clinical significance of anti-acetylcholine receptor antibody (anti-AChR-Ab levels in suspected ocular myasthenia gravis. Methods. In total, 144 patients complaining of fluctuating diplopia and ptosis were evaluated for serum levels of anti-acetylcholine receptor antibody and their medical charts were retrospectively reviewed. Subjects were classified into three groups: variable diplopia only, ptosis only, and both variable diplopia and ptosis. We investigated serum anti-AChR-Ab titer levels and performed thyroid autoantibody tests. Results. Patients’ chief complaints were diplopia (N=103, ptosis (N=12, and their concurrence (N=29. Abnormal anti-AChR-Ab was observed in 21 of 144 patients (14.1%. Between the three groups, mean age, number of seropositive patients, and mean anti-AChR-Ab level were not significantly different (P=0.224, 0.073, and 0.062, resp.. Overall, 27.5% of patients had abnormal thyroid autoantibodies. Conclusion. The sensitivity of anti-AChR-Ab was 14.1% in suspected ocular myasthenia gravis and seropositivity in myasthenia gravis patients showed a high correlation with the presence of thyroid autoantibodies.

  13. The clinical value of serum thyrotrophin receptor antibody level in patients with Graves ophthalmopathy

    International Nuclear Information System (INIS)

    Wang Chaodian; Shi Yuhong; Yan Bing

    2013-01-01

    Objective: To explore the value of serum thyrotrophin receptor antibody (TRAb) on the pathological mechanism of Graves ophthalmopathy. Methods: Two hundred and nineteen newly diagnosed Graves disease patients who were divided into Graves ophthalmopathy group (n=121) and without Graves ophthalmopathy group (n=98) were tested serum concentration with thyroid function, thyroperoxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb) and TRAb. According to the consensus statement of the European Group on Graves ophthalmopathy, clinical activity score (CAS) and severity evaluation were carried out on Graves ophthalmopathy patients. Results: There was no significant difference in serum concentration of free thyroxine (FT 4 ), free triiodothyronine (FT 3 ), TPOAb and TRAb between the Graves ophthalmopathy group and the without Graves ophthalmopathy group. Serum concentration of TRAb was not correlated with the severity and CAS of Graves ophthalmopathy. Conclusions: The CAS and the severity of Graves ophthalmopathy were irrelevant to the serum concentration of TRAb. Therefore, the correlation between TRAb and Graves ophthalmopathy still needs further study. (authors)

  14. ANTIBODIES TO LEUKEMIA DIFFERENTIATION FACTOR (HLDF IN PATIENTS WITH GASTROINTESTINAL CANCER

    Directory of Open Access Journals (Sweden)

    A. I. Autenshlus

    2010-01-01

    Full Text Available Antibodies to leukemia differentiation factor (HLDF in patients with gastrointestinal cancer Abstract. Patients with gastric cancer exhibit higher levels of IgG4-antibodies to human leukemia differentiation factor (HLDF, as compared with healthy individuals, whereas, in patients with colorectal cancer, one may detect high levels of IgA anti-HDLF antibodies, along with lower levels of IgG1 class antibodies against HLDF than in control group. Among patients with gastrointestinal cancer, a positive correlation is revealed between contents of highly differentiated cells in the tumor, and IgM antibodies to HDLF. Meanwhile, a reverse relationship is noted between low differentiation of tumor cells and levels of IgG2 antibodies to HDLF in gastric cancer patients, or IgG3 antibodies to HDLF in patients with colorectal cancer.

  15. Comparison of monoclonal antibodies and tritiated ligands for estrogen receptor assays in 241 breast cancer cytosols

    International Nuclear Information System (INIS)

    Goussard, J.; Lechevrel, C.; Martin, P.M.; Roussel, G.

    1986-01-01

    Estrogen receptor determinations have been performed on 241 cytosols from 160 breast cancer tumors using both radioactive ligands ([ 3 H]-estradiol, [3H]R2858) and monoclonal antibodies (Abbott ER-EIA Kit) to compare the two methods and to evaluate the clinical usefulness of the new immunological, simplified assay. Intra- and interassay reproducibility of the enzyme immunoassay (EIA) method was studied during a 6-month period on 35 standard curves with 4 different batches of monoclonal antibodies. Intraassay coefficients of variation studied on duplicates were smaller than 5% in most cases and reproducibility of the curves showed coefficients of variation lower than 10% except for standard 0 and 5 fmol/ml. Pooled cytosols used as control for the dextran coated charcoal method had interassay variation coefficients between 3.8 and 11.4%. Reproducibility has been studied on clinical specimens assayed twice at two different periods with either EIA or dextran coated charcoal methods. Slopes obtained were 1.05 and 0.96, respectively. A good stability of EIA results was obtained with protein concentrations in the range 4-0.15 mg/ml cytosol. No significant effects of dithiothreitol or monothioglycerol (1 mM) on EIA and dextran coated charcoal assay were observed. Eighty breast cancer cytosols were assayed with both EIA and Scatchard analysis. The slope of the regression curve obtained was 1.04 (r = 0.963). Cytosols were assayed by EIA and by a saturating concentration of tritiated ligand (5 nM). With 153 cytosols the EIA/5 nM slope was 1.34 (r = 0.978). This slope can be compared with the slope Scatchard/5 nM obtained with 90 cytosols: 1.29 (r = 0.985). Absence of cross-reactivity of monoclonal ER antibodies with progesterone receptor was observed

  16. Cold Antibodies: An uncommon factor in transfusion safety in a ...

    African Journals Online (AJOL)

    Background Cold reacting antibodies with a thermal optimum at 0°C are an uncommon occurrence, and the clinical manifestations are rarely observed in the warm climate of the tropical countries of sub-Saharan Africa. Objective The objective of this presentation is to report two cases in which cold-reacting antibodies were ...

  17. Pattern of hormone receptors and human epidermal growth factor ...

    African Journals Online (AJOL)

    Introduction: Breast cancer is the most common cancer among women globally. With immunohistochemistry (IHC), breast cancer is classified into four groups based on IHC profile of estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2/neu) expression, positive (+) and/or ...

  18. Molecular analysis of the nerve growth factor receptor

    International Nuclear Information System (INIS)

    Hempstead, B.; Patil, N.; Olson, K.; Chao, M.

    1988-01-01

    An essential molecule in the translocation of information by nerve growth factor (NGF) to responsive cells is the cell-surface receptor for NGF. This paper presents information on the genomic structure of the NGF receptor gene, NGF receptor models, and transfection of NGF receptors. Equilibrium binding of [ 125 I]NGF to cells reveals two distinct affinity states for the NGF receptor. The human NGF receptor gene is a single-copy gene, consisting of six exons that span 23 kb. The receptor gene is capable of being transferred to fibroblast cells from human genomic DNA and expressed at high levels. The constitutive nature of the receptor promoter sequence is a partial explanation of why this tissue-specific gene is expressed efficiently in a variety of nonneuronal cells after genomic gene transfer. The two kinetic forms of the NGF receptor appear to be encoded by the same protein, which is the product of a single gene

  19. Field flow fractionation for assessing neonatal Fc receptor and Fcγ receptor binding to monoclonal antibodies in solution.

    Science.gov (United States)

    Pollastrini, Joey; Dillon, Thomas M; Bondarenko, Pavel; Chou, Robert Y-T

    2011-07-01

    Analysis of the strength and stoichiometry of immunoglobulin G (IgG) binding to neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR) is important for evaluating the pharmacokinetics and effector functions of therapeutic monoclonal antibody (mAb) products, respectively. The current standard for assessing FcγR and FcRn binding is composed of cell-based and surface plasmon resonance (SPR) assays. In this work, asymmetrical flow field flow fractionation (AF4) was evaluated to establish the true stoichiometry of IgG binding in solution. AF4 and liquid chromatography-mass spectrometry (LC-MS) were applied to directly observe IgG/FcγR and IgG/FcRn complexes, which were not observed using nonequilibrium size exclusion chromatography (SEC) analysis. Human serum albumin (HSA), an abundant component of human blood and capable of binding FcRn, was studied in combination with FcRn and IgG. AF4 demonstrated that the majority of large complexes of IgG/FcRn/HSA were at an approximate 1:2:1 molar ratio. In addition, affinity measurements of the complex were performed in the sub-micromolar affinity range. A significant decrease in binding was detected for IgG molecules with increased oxidation in the Fc region. AF4 was useful in detecting weak binding between full-length IgG/Fc fragments and Fc receptors and the effect of chemical modifications on binding. AF4 is a useful technique in the assessment of mAb product quality attributes. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Interaction of epidermal growth factor receptors with the cytoskeleton is related to receptor clustering

    NARCIS (Netherlands)

    van Belzen, N.; Spaargaren, M.; Verkleij, A. J.; Boonstra, J.

    1990-01-01

    Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction,

  1. Recent Advances of Colony-Stimulating Factor-1 Receptor (CSF-1R) Kinase and Its Inhibitors.

    Science.gov (United States)

    El-Gamal, Mohammed I; Al-Ameen, Shahad K; Al-Koumi, Dania M; Hamad, Mawadda G; Jalal, Nouran A; Oh, Chang-Hyun

    2018-01-17

    Colony stimulation factor-1 receptor (CSF-1R), which is also known as FMS kinase, plays an important role in initiating inflammatory, cancer, and bone disorders when it is overstimulated by its ligand, CSF-1. Innate immunity, as well as macrophage differentiation and survival, are regulated by the stimulation of the CSF-1R. Another ligand, interlukin-34 (IL-34), was recently reported to activate the CSF-1R receptor in a different manner. The relationship between CSF-1R and microglia has been reviewed. Both CSF-1 antibodies and small molecule CSF-1R kinase inhibitors have now been tested in animal models and in humans. In this Perspective, we discuss the role of CSF-1 and IL-34 in producing cancer, bone disorders, and inflammation. We also review the newly discovered and improved small molecule kinase inhibitors and monoclonal antibodies that have shown potent activity toward CSF-1R, reported from 2012 until 2017.

  2. Characterization of a purified nicotinic receptor from rat brain by using idiotypic and anti-idiotypic antibodies

    International Nuclear Information System (INIS)

    Abood, L.G.; Langone, J.J.; Bjercke, R.; Lu, X.; Banerjee, S.

    1987-01-01

    The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified from rat brain by affinity chromatography and to provide a highly sensitive assay for determining [ 3 H]nicotine binding to the purified material. An enantiomeric analogue of nicotine. (-)-6-hydroxymethylnicotine, was used to prepare the affinity column. In addition, with the use of an anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex composed of two components with molecular masses of 62 and 57 kDa. It was also demonstrated that the same two proteins could be purified by immunoaffinity chromatography with the use of an anti-idiotypic monoclonal antibody. With the use of the anti-nicotine antibody to measure [ 3 H]nicotine binding, the purified material was shown to bind 250 pmol/mg of protein. By utilizing a procedure in which the purified receptor protein was conjugated to membranes by disulfide bonds, a binding activity of 80 pmol/mg was obtained. With the availability of sterospecific monoclonal antibodies to (-)-nicotine as well as monoclonal anti-idiotypic antibodies derived when the anti-nicotine antibodies were used as immunogens, additional procedures became available for the further characterization of the purified nicotine receptor and examining its (-)-[ 3 H]nicotine-binding characteristics

  3. Anti-PDGF receptor β antibody-conjugated squarticles loaded with minoxidil for alopecia treatment by targeting hair follicles and dermal papilla cells.

    Science.gov (United States)

    Aljuffali, Ibrahim A; Pan, Tai-Long; Sung, Calvin T; Chang, Shu-Hao; Fang, Jia-You

    2015-08-01

    This study developed lipid nanocarriers, called squarticles, conjugated with anti-platelet-derived growth factor (PDGF)-receptor β antibody to determine whether targeted Minoxidil (MXD) delivery to the follicles and dermal papilla cells (DPCs) could be achieved. Squalene and hexadecyl palmitate (HP) were used as the matrix of the squarticles. The PDGF-squarticles showed a mean diameter and zeta potential of 195 nm and -46 mV, respectively. Nanoparticle encapsulation enhanced MXD porcine skin deposition from 0.11 to 0.23 μg/mg. The antibody-conjugated nanoparticles ameliorated follicular uptake of MXD by 3-fold compared to that of the control solution in the in vivo mouse model. Both vertical and horizontal skin sections exhibited a wide distribution of nanoparticles in the follicles, epidermis, and deeper skin strata. The encapsulated MXD moderately elicited proliferation of DPCs and vascular endothelial growth factor (VEGF) expression. The active targeting of PDGF-squarticles may be advantageous to improving the limited success of alopecia therapy. Topical use of minoxidil is only one of the very few treatment options for alopecia. Nonetheless, the current delivery method is far from ideal. In this article, the authors developed lipid nanocarriers with anti-platelet-derived growth factor receptor ? antibody to target dermal papilla cells, and showed enhanced uptake of minoxidil. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Visualization of Tumor Angiogenesis Using MR Imaging Contrast Agent Gd-DTPA-anti-VEGF Receptor 2 Antibody Conjugate in a Mouse Tumor Model

    International Nuclear Information System (INIS)

    Jun, Hong Young; Yin, Hong Hua; Kim, Sun Hee; Park, Seong Hoon; Kim, Hun Soo; Yoon Kwon Ha Yoon

    2010-01-01

    To visualize tumor angiogenesis using the MRI contrast agent, Gd- DTPA-anti-VEGF receptor 2 antibody conjugate, with a 4.7-Tesla MRI instrument in a mouse model. We designed a tumor angiogenesis-targeting T1 contrast agent that was prepared by the bioconjugation of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) and an anti-vascular endothelial growth factor receptor-2 (VEGFR2) antibody. The specific binding of the agent complex to cells that express VEGFR2 was examined in cultured murine endothelial cells (MS-1 cells) with a 4.7-Tesla magnetic resonance imaging scanner. Angiogenesis-specific T1 enhancement was imaged with the Gd-DTPA-anti-VEGFR2 antibody conjugate using a CT-26 adenocarcinoma tumor model in eight mice. As a control, the use of the Gd-DTPA-anti-rat immunoglobulin G (Gd-DTPA-anti-rat IgG) was imaged with a tumor model in eight mice. Statistical significance was assessed using the Mann-Whitney test. Tumor tissue was examined by immunohistochemical analysis. The Gd-DTPA-anti-VEGFR2 antibody conjugate showed predominant binding to cultured endothelial cells that expressed a high level of VEGFR2. Signal enhancement was approximately three-fold for in vivo T1-weighted MR imaging with the use of the Gd-DTPA-anti-VEGFR2 antibody conjugate as compared with the Gd-DTPA-rat IgG in the mouse tumor model (p < 0.05). VEGFR2 expression in CT-26 tumor vessels was demonstrated using immunohistochemical staining. MR imaging using the Gd-DTPA-anti-VEGFR2 antibody conjugate as a contrast agent is useful in visualizing noninvasively tumor angiogenesis in a murine tumor model

  5. Visualization of Tumor Angiogenesis Using MR Imaging Contrast Agent Gd-DTPA-anti-VEGF Receptor 2 Antibody Conjugate in a Mouse Tumor Model

    Energy Technology Data Exchange (ETDEWEB)

    Jun, Hong Young; Yin, Hong Hua; Kim, Sun Hee; Park, Seong Hoon; Kim, Hun Soo; Yoon Kwon Ha Yoon [Wonkwang University School of Medicine, Iksan (Korea, Republic of)

    2010-08-15

    To visualize tumor angiogenesis using the MRI contrast agent, Gd- DTPA-anti-VEGF receptor 2 antibody conjugate, with a 4.7-Tesla MRI instrument in a mouse model. We designed a tumor angiogenesis-targeting T1 contrast agent that was prepared by the bioconjugation of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) and an anti-vascular endothelial growth factor receptor-2 (VEGFR2) antibody. The specific binding of the agent complex to cells that express VEGFR2 was examined in cultured murine endothelial cells (MS-1 cells) with a 4.7-Tesla magnetic resonance imaging scanner. Angiogenesis-specific T1 enhancement was imaged with the Gd-DTPA-anti-VEGFR2 antibody conjugate using a CT-26 adenocarcinoma tumor model in eight mice. As a control, the use of the Gd-DTPA-anti-rat immunoglobulin G (Gd-DTPA-anti-rat IgG) was imaged with a tumor model in eight mice. Statistical significance was assessed using the Mann-Whitney test. Tumor tissue was examined by immunohistochemical analysis. The Gd-DTPA-anti-VEGFR2 antibody conjugate showed predominant binding to cultured endothelial cells that expressed a high level of VEGFR2. Signal enhancement was approximately three-fold for in vivo T1-weighted MR imaging with the use of the Gd-DTPA-anti-VEGFR2 antibody conjugate as compared with the Gd-DTPA-rat IgG in the mouse tumor model (p < 0.05). VEGFR2 expression in CT-26 tumor vessels was demonstrated using immunohistochemical staining. MR imaging using the Gd-DTPA-anti-VEGFR2 antibody conjugate as a contrast agent is useful in visualizing noninvasively tumor angiogenesis in a murine tumor model

  6. The Role of Natural Antibodies to CC Chemokine Receptor 5 in HIV Infection

    Directory of Open Access Journals (Sweden)

    Assunta Venuti

    2017-10-01

    Full Text Available The CC chemokine receptor 5 (CCR5 is responsible for immune and inflammatory responses by mediation of chemotactic activity in leukocytes, although it is expressed on different cell types. It has been shown to act as co-receptor for the human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV. Natural reactive antibodies (Abs recognizing first loop (ECL1 of CCR5 have been detected in several pools of immunoglobulins from healthy donors and from several cohorts of either HIV-exposed but uninfected subjects (ESN or HIV-infected individuals who control disease progression (LTNP as well. The reason of development of anti-CCR5 Abs in the absence of autoimmune disease is still unknown; however, the presence of these Abs specific for CCR5 or for other immune receptors and mediators probably is related to homeostasis maintenance. The majority of anti-CCR5 Abs is directed to HIV binding site (N-terminus and ECL2 of the receptor. Conversely, it is well known that ECL1 of CCR5 does not bind HIV; thus, the anti-CCR5 Abs directed to ECL1 elicit a long-lasting internalization of CCR5 but not interfere with HIV binding directly; these Abs block HIV infection in either epithelial cells or CD4+ T lymphocytes and the mechanism differs from those ones described for all other CCR5-specific ligands. The Ab-mediated CCR5 internalization allows the formation of a stable signalosome by interaction of CCR5, β-arrestin2 and ERK1 proteins. The signalosome degradation and the subsequent de novo proteins synthesis determine the CCR5 reappearance on the cell membrane with a very long-lasting kinetics (8 days. The use of monoclonal Abs to CCR5 with particular characteristics and mode of action may represent a novel mode to fight viral infection in either vaccinal or therapeutic strategies.

  7. Radioimmunodetection of human leukemia with anti-interleukin-2 receptor antibody in severe combined immunodeficiency mice

    International Nuclear Information System (INIS)

    Hosono, Makoto; Takaori-Kondo, Akifumi; Zheng-Sheng, Yao; Kobayashi, Hisataka; Hosono, Masako N.; Sakahara, Harumi; Imada, Kazunori; Okuma, Minoru; Uchiyama, Takashi; Konishi, Junji

    1995-01-01

    Anti-Tac monoclonal antibody recognizes human interleukin-2 receptor, which is overexpressed in leukemic cells of most adult T-cell leukemia (ATL) patients. To examine the potency of anti-Tac for targeting of ATL, biodistributions of intravenously administered 125 I- and 111 In-labeled anti-Tac were examined in severe combined immunodeficiency (SCID) mice inoculated with ATL cells. Significant amounts of radiolabeled anti-Tac were found in the spleen and thymus. The trafficking of ATL cells in SCID mice was detected using 111 In-oxine-labeled ATL cells. These results were coincident with the histologically confirmed infiltration of ATL cells. The radiolabeled anti-Tac seemed potent for targeting of ATL

  8. Circulating angiotensin type II receptor: Possible marker for antibody mediated rejection after renal transplantation?

    Science.gov (United States)

    Kimball, Pamela M; Gupta, Gaurav; McDougan, Felecia

    2017-10-01

    Presence of antibody [Ab] against angiotensin receptor [AT1R] indicates heightened risk for antibody mediated rejection [AMR] after transplantation but is insufficient as a marker. We speculated AT1R might be released systemically because of AMR and might be a useful biomarker. AT1R was measured in blood from 73 Normals and 72 renal patients pre- and post-transplantation. Patients were stratified as AMR-free [Gp1], AMR1yr [Gp3]. AT1R was higher [13±26vs.367±537, p<0.01)] and more prevalent [20% vs. 92%, p<0.01] among renal patients than Normals. Pretransplant levels were similar [p=ns] between groups. One-year posttransplant levels approached [p<0.01] normalcy for Gps1+3 but spiked during AMR and remained elevated [155±58, p<0.01] for 50% Gp2 patients. One-year AT1R levels were higher among subsequent graft failures than surviving grafts [171±267vs. 38±50, p<0.01]. Pretransplant AT1R was abnormally elevated: possibly indicating ongoing tissue injury. Pretransplant AT1R didn't predict risk for AMR. However, AT1R spiked during early AMR and sustained elevations were associated with poorer outcomes. Copyright © 2017. Published by Elsevier Inc.

  9. Human Immunodeficiency Virus Proteins Mimic Human T Cell Receptors Inducing Cross-Reactive Antibodies

    Directory of Open Access Journals (Sweden)

    Robert Root-Bernstein

    2017-10-01

    Full Text Available Human immunodeficiency virus (HIV hides from the immune system in part by mimicking host antigens, including human leukocyte antigens. It is demonstrated here that HIV also mimics the V-β-D-J-β of approximately seventy percent of about 600 randomly selected human T cell receptors (TCR. This degree of mimicry is greater than any other human pathogen, commensal or symbiotic organism studied. These data suggest that HIV may be evolving into a commensal organism just as simian immunodeficiency virus has done in some types of monkeys. The gp120 envelope protein, Nef protein and Pol protein are particularly similar to host TCR, camouflaging HIV from the immune system and creating serious barriers to the development of safe HIV vaccines. One consequence of HIV mimicry of host TCR is that antibodies against HIV proteins have a significant probability of recognizing the corresponding TCR as antigenic targets, explaining the widespread observation of lymphocytotoxic autoantibodies in acquired immunodeficiency syndrome (AIDS. Quantitative enzyme-linked immunoadsorption assays (ELISA demonstrated that every HIV antibody tested recognized at least one of twelve TCR, and as many as seven, with a binding constant in the 10−8 to 10−9 m range. HIV immunity also affects microbiome tolerance in ways that correlate with susceptibility to specific opportunistic infections.

  10. Bivalent Llama Single-Domain Antibody Fragments against Tumor Necrosis Factor Have Picomolar Potencies due to Intramolecular Interactions

    Directory of Open Access Journals (Sweden)

    Els Beirnaert

    2017-07-01

    Full Text Available The activity of tumor necrosis factor (TNF, a cytokine involved in inflammatory pathologies, can be inhibited by antibodies or trap molecules. Herein, llama-derived variable heavy-chain domains of heavy-chain antibody (VHH, also called Nanobodies™ were generated for the engineering of bivalent constructs, which antagonize the binding of TNF to its receptors with picomolar potencies. Three monomeric VHHs (VHH#1, VHH#2, and VHH#3 were characterized in detail and found to bind TNF with sub-nanomolar affinities. The crystal structures of the TNF–VHH complexes demonstrate that VHH#1 and VHH#2 share the same epitope, at the center of the interaction area of TNF with its TNFRs, while VHH#3 binds to a different, but partially overlapping epitope. These structures rationalize our results obtained with bivalent constructs in which two VHHs were coupled via linkers of different lengths. Contrary to conventional antibodies, these bivalent Nanobody™ constructs can bind to a single trimeric TNF, thus binding with avidity and blocking two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities.

  11. A radioisotope dilution assay for unlabelled vitamin B12-intrinsic factor complex employing the binding intrinsic factor antibody: probable evidence for two types of binding antibody

    International Nuclear Information System (INIS)

    Jacob, E.; O'Brien, H.A.W.; Mollin, D.L.

    1977-01-01

    A new radioisotope dilution assay for vitamin B 12 -intrinsic factor complex is described. The method is based on the use of the binding type intrinsic antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B 12 complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B 12 -intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for at least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B 12 complex and one which combines equally well with this complex or with free intrinsic factor. (author)

  12. New-Onset Headache in Patients With Autoimmune Encephalitis Is Associated With anti-NMDA-Receptor Antibodies.

    Science.gov (United States)

    Schankin, Christoph J; Kästele, Fabian; Gerdes, Lisa Ann; Winkler, Tobias; Csanadi, Endy; Högen, Tobias; Pellkofer, Hannah; Paulus, Walter; Kümpfel, Tania; Straube, Andreas

    2016-06-01

    We tested the hypotheses (i) that autoimmune encephalitis is associated with new-onset headache, and (ii) that the occurrence of headache is associated with the presence of anti-N-methyl-D-aspartate (NMDA)-receptor antibodies. Autoimmune encephalitis presents with cognitive dysfunction as well as neuro-psychiatric symptoms. Its pathophysiology might involve antibody-mediated dysfunction of the glutamatergic system as indicated by the presence of anti-NMDA-receptor antibodies in some patients. In this cross-sectional study, patients with autoimmune encephalitis were assessed with a standardized interview for previous headache and headache associated with autoimmune encephalitis. Headache was classified according to the International Classification of Headache Disorders, second edition. Clinical and paraclinical findings were correlated with the occurrence of headache. Of 40 patients with autoimmune encephalitis, 19 did not have a history of headache. Of those, nine suffered from encephalitis-associated headache. Seven of these nine had anti-NMDA-receptor antibodies in contrast to only two among the remaining 10 patients without new-onset headache (P = .023, odds ratio: 14, 95% confidence interval: 1.5; 127). In most patients headache occurred in attacks on more than 15 days/month, was severe, and of short duration (less than 4 hours). International Headache Society criteria for migraine were met in three patients. New-onset headache is a relevant symptom in patients with autoimmune encephalitis who have no history of previous headache, especially in the subgroup with anti-NMDA-receptor antibodies. This indicates a thorough investigation for secondary headaches including anti-NMDA-R antibodies for patients with new-onset headache and neuropsychiatric findings. Glutamatergic dysfunction might be important for the generation of head pain but may only occasionally be sufficient to trigger migraine-like attacks in nonmigraineurs. © 2016 American Headache Society.

  13. Retrieval of estradiol receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies.

    Science.gov (United States)

    Sierralta, W D; Thole, H H

    1996-05-01

    The unmasking of estradiol receptor in paraffin sections of Bouin's-fixed uterine tissue from ovariectomized gilts was attained with microwave treatment. Immunocytochemistry of the receptor was performed using a polyclonal or five monoclonal antibodies, two of which are commercially available, reacting with different domains of the protein and an amplified-peroxidase system for detection. With five of the antibodies, a predominance of nuclear staining was observed in cells of endometrial glands, while one monoclonal antibody (13H2), reacting with the receptor's domain E, showed a preference for the cytoplasmic receptor. In stroma, all antibodies detected more receptor in nuclei than in cytoplasm. In epithelium, the commercially available antibody H222, our monoclonals 13H2 and HT65, and the polyclonal antibody 402 demonstrated more receptor in cytoplasmic than in nuclear areas. In myometrium, the nuclei from longitudinal and ring muscles were definitely stained with the antibodies. We conclude that the accessibilities of the antibody epitopes of the receptor differ according to the functional uterine cell type.

  14. A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines.

    Science.gov (United States)

    Smolarek, Dorota; Hattab, Claude; Hassanzadeh-Ghassabeh, Gholamreza; Cochet, Sylvie; Gutiérrez, Carlos; de Brevern, Alexandre G; Udomsangpetch, Rachanee; Picot, Julien; Grodecka, Magdalena; Wasniowska, Kazimiera; Muyldermans, Serge; Colin, Yves; Le Van Kim, Caroline; Czerwinski, Marcin; Bertrand, Olivier

    2010-10-01

    Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

  15. Assessment of estrogen receptor-monoclonal antibody interaction by high performance liquid chromatography

    International Nuclear Information System (INIS)

    Brandt, D.W.; Wittliff, J.L.

    1986-01-01

    To define the interrelationships between the various isoforms of the estrogen receptors (ER), a monoclonal antibody-horse radish peroxidase conjugate H222 was used as a probe in conjunction with HPIEC (Synchrom AX-1000) and HPSEC (TSK-3000 SW Toyo Soda) columns. ER from breast tumors was assessed using [16α- 125 I]-iodoestradiol-17β (3nM) +/-200 fold excess estradiol-17β and excess H222. When ER was analyzed by HPSEC (size and shape), with 400 mM KCl which caused the dissociation of ER into 4S isoforms, a shift in retension time to higher molecular weight species was seen. The H222 appeared to interact with most isoforms of ER. However, when ER was analyzed by HPIEC (surface charge) with H222, a shift in virtually all of the high salt (150mM) isoform to the flow-through was observed with only 46% shift in elution of the low salt (60-70mM) isoforms. H222 did not alter total ER binding capacity. These data suggest that H222 recognized discrete forms of the ER. Therefore, modification in the receptor may have occurred which masks or removes the antigenic determinant limiting the specificity of H222. These results indicate that H222 may be employed as a tool to elucidate the interrelationships between these ER species

  16. [N-methyl-D-aspartate receptor antibody encephalitis: value of immunomodulatory therapy].

    Science.gov (United States)

    Le Moigno, L; Ternant, D; Paintaud, G; Thibault, G; Cloarec, S; Tardieu, M; Lagrue, E; Castelnau, P

    2014-06-01

    Anti-N-methyl-D-aspartate receptor (NMDA-R) encephalitis is little known to pediatricians and likely underdiagnosed. The child's vital and cognitive prognosis is at stake. The use of immunomodulatory drugs, such as rituximab has led to spectacular results, but many questions remain about its mode of action in this type of pathology. We report the case of a 6-year-old girl with no medical history, admitted for status epilepticus preceded by behavior symptoms and sleep disorders. Gradually, the child became bedridden, mute, and animated by predominantly orofacial dyskinesia. Examinations were normal (cerebrospinal fluid [CSF] analysis, brain MRI). The diagnosis was established by the presence of NMDA-R antibodies in the CSF. After exclusion of a tumor-associated syndrome, treatment was started initially by intravenous immunoglobulins, then by plasma exchange, and finally rituximab. The patient was cured with rituximab despite an unusually early recovery of the B-cell pool. Anti-N-methyl-D-aspartate receptor (NMDA-R) encephalitis is a severe but potentially reversible neurologic disorder only recently described, even in childhood. It may be reversible without sequelae if diagnosed and treated early. The use of immunomodulatory therapy, such as rituximab seemingly improves the outcome. Immunological monitoring is needed to better understand its mechanism of action in autoimmune diseases of the nervous system in childhood. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  17. Urokinase-type plasminogen activator receptor (uPAR), tissue factor (TF) and epidermal growth factor receptor (EGFR)

    DEFF Research Database (Denmark)

    Christensen, Anders; Kiss, Katalin; Lelkaitis, Giedrius

    2017-01-01

    Background: Tumor-specific biomarkers are a prerequisite for the development of targeted imaging and therapy in oral squamous cell carcinoma (OSCC). urokinase-type Plasminogen Activator Receptor (uPAR), Tissue Factor (TF) and Epidermal Growth Factor Receptor (EGFR) are three biomarkers that exhib...... with a reduced survival. uPAR seems to be a prognostic biomarker in oral cancer....

  18. In-vivo fluorescence detection of breast cancer growth factor receptors by fiber-optic probe

    Science.gov (United States)

    Bustamante, Gilbert; Wang, Bingzhi; DeLuna, Frank; Sun, LuZhe; Ye, Jing Yong

    2018-02-01

    Breast cancer treatment options often include medications that target the overexpression of growth factor receptors, such as the proto-oncogene human epidermal growth factor receptor 2 (HER2/neu) and epidermal growth factor receptor (EGFR) to suppress the abnormal growth of cancerous cells and induce cancer regression. Although effective, certain treatments are toxic to vital organs, and demand assurance that the pursued receptor is present at the tumor before administration of the drug. This requires diagnostic tools to provide tumor molecular signatures, as well as locational information. In this study, we utilized a fiber-optic probe to characterize in vivo HER2 and EGFR overexpressed tumors through the fluorescence of targeted dyes. HER2 and EGFR antibodies were conjugated with ICG-Sulfo-OSu and Alexa Fluor 680, respectively, to tag BT474 (HER2+) and MDA-MB-468 (EGFR+) tumors. The fiber was inserted into the samples via a 30-gauge needle. Different wavelengths of a supercontinuum laser were selected to couple into the fiber and excite the corresponding fluorophores in the samples. The fluorescence from the dyes was collected through the same fiber and quantified by a time-correlated single photon counter. Fluorescence at different antibody-dye concentrations was measured for calibration. Mice with subcutaneous HER2+ and/or EGFR+ tumors received intravenous injections of the conjugates and were later probed at the tumor sites. The measured fluorescence was used to distinguish between tumor types and to calculate the concentration of the antibody-dye conjugates, which were detectable at levels as low as 40 nM. The fiber-optic probe presents a minimally invasive instrument to characterize the molecular signatures of breast cancer in vivo.

  19. Monoclonal antibodies directed to human insulin-like growth factor I (IGF I)

    International Nuclear Information System (INIS)

    Laubli, U.K.; Baier, W.; Celio, M.R.; Binz, H.; Humbel, R.E.

    1982-01-01

    Mouse hybridomas secreting antibodies to human insulin-like growth factor I (IGF I) were produced by fusion of spleen cells of hyperimmunised mice with FO mouse-myeloma cells. Eight clones producing antibodies against human IGF I have been isolated, two of which have been characterised. One was used in a radioimmunoassay, the other for immunopurification of IGF. (Auth.)

  20. Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman

    2015-01-01

    The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest...... in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation...... to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found...

  1. Immediate and Catastrophic Antibody-Mediated Rejection in a Lung Transplant Recipient With Anti-Angiotensin II Receptor Type 1 and Anti-Endothelin-1 Receptor Type A Antibodies.

    Science.gov (United States)

    Cozzi, E; Calabrese, F; Schiavon, M; Feltracco, P; Seveso, M; Carollo, C; Loy, M; Cardillo, M; Rea, F

    2017-02-01

    Preexisting donor-specific anti-HLA antibodies (DSAs) have been associated with reduced survival of lung allografts. However, antibodies with specificities other than HLA may have a detrimental role on the lung transplant outcome. A young man with cystic fibrosis underwent lung transplantation with organs from a suitable deceased donor. At the time of transplantation, there were no anti-HLA DSAs. During surgery, the patient developed a severe and intractable pulmonary hypertension associated with right ventriular dysfunction, which required arteriovenous extracorporeal membrane oxygenation. After a brief period of clinical improvement, a rapid deterioration in hemodynamics led to the patient's death on postoperative day 5. Postmortem studies showed that lung specimens taken at the end of surgery were compatible with antibody-mediated rejection (AMR), while terminal samples evidenced diffuse capillaritis, blood extravasation, edema, and microthrombi, with foci of acute cellular rejection (A3). Immunological investigations demonstrated the presence of preexisting antibodies against the endothelin-1 receptor type A (ET A R) and the angiotensin II receptor type 1 (AT 1 R), two of the most potent vasoconstrictors reported to date, whose levels slightly rose after transplantation. These data suggest that preexisting anti-ET A R and anti-AT 1 R antibodies may have contributed to the onset of AMR and to the catastrophic clinical course of this patient. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

  2. Isotypes of Epstein-Barr Virus Antibodies in Rheumatoid Arthritis: Association with Rheumatoid Factors and Citrulline-Dependent Antibodies

    Directory of Open Access Journals (Sweden)

    Marie Wulff Westergaard

    2015-01-01

    Full Text Available In order to study the humoral immune response against Epstein-Barr virus (EBV in patients with rheumatoid arthritis (RA and to compare it with the two major autoantibody types in RA, plasma samples from 77 RA patients, 28 patients with systemic lupus erythematosus (SLE, and 28 healthy controls (HCs were investigated by enzyme-linked immunosorbent assays (ELISA. Increased percentages of positives and concentrations of IgG/IgA/IgM antibodies against the latent EBV nuclear antigen-1 (EBNA-1 were observed in RA patients compared to SLE patients and HCs. Increased concentrations and percentages of positives of IgG/IgA/IgM against the early lytic EBV antigen diffuse (EAD were also found in RA patients compared to HCs but were highest in SLE patients. Furthermore, associations between the elevated EBNA-1 IgA and EBNA-1 IgM levels and the presence of IgM and IgA rheumatoid factors (RFs and anti-citrullinated protein antibodies (ACPAs, IgG and between elevated IgA concentrations against EAD and the presence of RFs and ACPAs in RA patients were found. Thus, RA patients had elevated antibodies of all isotypes characteristic of latent EBV infection (whereas SLE patients had elevated antibodies characteristic of lytic EBV infection. Notably, for IgM and IgA (but not IgG, these were associated with the presence of characteristic RA autoantibodies.

  3. Panel of monoclonal antibodies to sperm surface proteins as a tool for monitoring localization and identification of sperm-zona pellucida receptors

    Czech Academy of Sciences Publication Activity Database

    Zigo, Michal; Dorosh, Andriy; Pohlová, Alžběta; Jonáková, Věra; Šulc, Miroslav; Maňásková-Postlerová, Pavla

    March, č. 359 (2015), s. 895-908 ISSN 0302-766X R&D Projects: GA ČR(CZ) GA14-05547S; GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GAP503/12/1834 Institutional research plan: CEZ:AV0Z50520701 Institutional support: RVO:86652036 ; RVO:61388971 Keywords : zona pellucida-binding receptors * monoclonal antibodies against sperm surface proteins * sperm surface proteins * RAB-2A * lactahedrin P47 Subject RIV: CE - Biochemistry Impact factor: 2.948, year: 2015

  4. A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

    DEFF Research Database (Denmark)

    Owens, T

    1988-01-01

    on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2......Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect...... fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed...

  5. Clonal relationships between thyroid-stimulating hormone receptor-stimulating antibodies illustrate the effect of hypermutation on antibody function

    DEFF Research Database (Denmark)

    Padoa, Carolyn J; Larsen, Sanne L; Hampe, Christiane S

    2009-01-01

    relationship and derivation from a single precursor B-cell clone. The IGHV-region genes of the two mAbs underwent high degrees of somatic hypermutation by sharing numerous mutations before diverging, while the IGLV genes evolved separately. Interestingly, the mutations were present in both the complementarity......, in experimentally immunized mice, multiple pathogenic antibodies to TSHR can arise from a single clone by a series of somatic mutations in the V-region genes and may give an insight into how such antibodies develop spontaneously in autoimmune Graves' disease....

  6. Tumor necrosis factor alpha antibody (infliximab) therapy profoundly down-regulates the inflammation in Crohn's ileocolitis

    NARCIS (Netherlands)

    Baert, F. J.; D'Haens, G. R.; Peeters, M.; Hiele, M. I.; Schaible, T. F.; Shealy, D.; Geboes, K.; Rutgeerts, P. J.

    1999-01-01

    Anti-tumor necrosis factor alpha monoclonal antibody treatment (infliximab) reduces clinical signs and symptoms in patients with Crohn's disease. The effects of infliximab on mucosal histopathologic abnormalities in Crohn's ileocolitis were studied. Thirteen patients with steroid-refractory Crohn's

  7. Prevention and Reversal of Antibody Responses Against Factor IX in Gene Therapy for Hemophilia B

    Directory of Open Access Journals (Sweden)

    Sushrusha eNayak

    2011-12-01

    Full Text Available Intramuscular (IM administration of an adeno-associated viral (AAV vector represents a simple and safe method of gene transfer for treatment of the X-linked bleeding disorder hemophilia B (factor IX, F.IX, deficiency. However, the approach is hampered by an increased risk of immune responses against F.IX. Previously, we demonstrated that the drug cocktail of immune suppressants rapamycin, IL-10, and a specific peptide (encoding a dominant CD4+ T cell epitope caused an induction of regulatory T cells (Treg with a concomitant apoptosis of antigen-specific effector T cells (J. Thromb. Haemost. 7:1523, 2009. This protocol was effective in preventing inhibitory antibody formation against human F.IX (hF.IX in muscle gene transfer to C3H/HeJ hemophilia B mice (with targeted F9 gene deletion. Here, we show that this protocol can also be used to reverse inhibitor formation. IM injection of AAV1-hF.IX vector resulted in inhibitors of on average 8-10 BU within 1 month. Subsequent treatment with the tolerogenic cocktail accomplished a rapid reduction of hF.IX-specific antibodies to <2 BU, which lasted for >4.5 months. Systemic hF.IX expression increased from undetectable to >200 ng/ml, and coagulation times improved. In addition, we developed an alternative prophylactic protocol against inhibitor formation that did not require knowledge of T cell epitopes, consisting of daily oral administration of rapamycin for 1-month combined with frequent, low-dose intravenous injection of hF.IX protein. Experiments in T cell receptor transgenic mice showed that the route and dosing schedule of drug administration substantially affected Treg induction. When combined with intravenous antigen administration, oral delivery of rapamycin had to be performed daily in order to induce Treg, which were suppressive and phenotypically comparable to natural Treg.

  8. Myasthenic Crisis Complicated with Myxedema, Positive for Both Anti-acetylcholine Receptor and Anti-muscle-specific Tyrosine Kinase Antibodies.

    Science.gov (United States)

    Horiuchi, Kazuhiro; Nagai, Azusa; Wakita, Masahiro; Ito, Shotaro; Takamura, Kei; Houzen, Hideki

    2018-01-15

    We herein report the case of myasthenic crisis occurring in a 51-year-old man. He had experienced ptosis, increased body weight with edema, and fatigue with dyspnea. He presented at our emergency department with disturbed consciousness. He was originally diagnosed with myxedema coma, and he required artificial respiration. Because his weakness persisted and he was positive for anti-acetylcholine receptor antibodies and anti-muscle-specific tyrosine kinase antibodies, we diagnosed myasthenic crisis after various examinations. His clinical response to treatment was good and he was discharged in an ambulatory status 3 months after admission. This case demonstrates that myasthenic crisis may occur in association with myxedema.

  9. Receptors for insulin-like growth factors I and II: autoradiographic localization in rat brain and comparison to receptors for insulin

    International Nuclear Information System (INIS)

    Lesniak, M.A.; Hill, J.M.; Kiess, W.; Rojeski, M.; Pert, C.B.; Roth, J.

    1988-01-01

    Receptors for insulin-like growth factor I (IGF-I) in rat brain were visualized using autoradiography with [125I]IGF-I. The binding of the labeled peptide was competed for fully by high concentrations of unlabeled IGF-I. At intermediate concentrations of unlabeled peptide the binding of [125I]IGF-I was competed for by unlabeled IGF-I more effectively than by IGF-II or insulin, which is typical of receptors for IGF-I. Essentially every brain section shows specific binding of IGF-I, and the pattern of binding of IGF-I to its receptors correlated well with the cytoarchitectonic structures. In parallel studies we showed that [125I]IGF-II was bound to tissue sections of rat brain and that the binding was competed for by an excess of unlabeled IGF-II. However, intermediate concentrations of unlabeled peptides gave inconclusive results. To confirm that the binding of [125I]IGF-II was to IGF-II receptors, we showed that antibodies specific for the IGF-II receptor inhibited the binding of labeled IGF-II. Furthermore, the binding of the antibody to regions of the brain section, visualized by the application of [125I]protein-A, gave patterns indistinguishable from those obtained with [125I]IGF-II alone. Again, the binding was very widely distributed throughout the central nervous system, and the patterns of distribution corresponded well to the underlying neural structures. Densitometric analysis of the receptors enabled us to compare the distribution of IGF-I receptors with that of IGF-II receptors as well as retrospectively with that of insulin receptors

  10. Steroid hormone and epidermal growth factor receptors in meningiomas.

    Science.gov (United States)

    Horsfall, D J; Goldsmith, K G; Ricciardelli, C; Skinner, J M; Tilley, W D; Marshall, V R

    1989-11-01

    A prospective study of steroid hormone and epidermal growth factor receptor expression in 57 meningiomas is presented. Scatchard analysis of radioligand binding identified 20% of meningiomas as expressing classical oestrogen receptors (ER) at levels below that normally accepted for positivity, the remainder being negative. ER could not be visualized in any meningioma using immunocytochemistry. Alternatively, 74% of meningiomas demonstrated the presence of progesterone receptors (PR) by Scatchard analysis, the specificity of which could not be attributed to glucocorticoid or androgen receptors. Confirmation of classical PR presence was determined by immunocytochemical staining. The presence of epidermal growth factor receptor (EGFR) was demonstrated in 100% of meningiomas using immunocytochemical staining. These data are reviewed in the context of previously reported results and are discussed in relation to the potential for medical therapy as an adjunct to surgery.

  11. Factors of Innate and Adaptive Local Immunity in Children with Primary Deficiencies of Antibody Formation

    Directory of Open Access Journals (Sweden)

    L.I. Chernyshova

    2013-10-01

    Full Text Available In 40 children with various types of primary immunodeficiencies (PID of antibody formation we examined factors of local immunity in saliva. It is found that in the saliva of children with PID of antibody formation in comparison with immunocompetent children the concentration of factors of adaptive immunity is significantly reduced. Lack of adaptive immunity in the PID of antibody formation to some extent is compensated by increased concentrations of innate immune factors on the mucous membranes — the free Sc, as well as lactoferrin in selective immunodeficiency of IgA. At PID of antibody formation we observed increased TNF-α level in the saliva, which may indicate the persistence of local inflammation on the membranes of the respiratory tract.

  12. Ah receptor mediated suppression of the antibody response in mice is primarily dependent on the Ah phenotype of lymphoid tissue

    International Nuclear Information System (INIS)

    Silkworth, J.B.; Antrim, L.A.; Sack, G.

    1986-01-01

    Halogenated aromatic hydrocarbons act through the aromatic hydrocarbon (Ah) receptor in mice to produce a series of toxic effects of the immune system. The receptor protein is a product of the Ah gene locus. Ah responsive (Ahb/Ahb) mice express a high affinity receptor in both lymphoid and nonlymphoid tissues whereas nonresponsive Ahd/Ahd mice express a poor affinity receptor. To determine the role of the Ah receptor of lymphoid tissue relative to that of nonlymphoid tissue in the induction of immune impairment, bone marrow was used to reconstitute lethally irradiated mice of the same or opposite Ah phenotype. All mice were given 3,3',4,4'-tetrachlorobiphenyl (35 and 350 mumol/kg) ip 2 days before immunization with sheep erythrocytes (SRBC). The immune response to this T dependent antigen and organ weights were determined 5 or 7 days later in normal or chimeric mice, respectively. Monoclonal Lyt 1.1 and Lyt 1.2 antibodies were used to establish the origin of the cells which repopulated the chimeric thymuses. The immune responses of both BALB/cBy (Ahb/Ahb) and the BALB/cBy X DBA/2 hybrid, CByD2F1 (Ahb/Ahd), were significantly suppressed but DBA/2 mice were unaffected. The immune responses of chimeric BALB/cBy----BALB/cBy and BALB/cBy----DBA/2 (donor----recipient) mice were also significantly suppressed and thymic atrophy was observed in both cases. The serum anti-SRBC antibody titers of DBA/2----BALB/cBy chimeras were also significantly decreased although not to the same extent as in BALB/cBy----DBA/2 mice. Chimeric DBA/2----DBA/2 mice were not affected. These results indicate that the sensitivity to Ah receptor mediated suppression of the antibody response is primarily determined by the Ah phenotype of the lymphoid tissue

  13. Andrographolide regulates epidermal growth factor receptor and transferrin receptor trafficking in epidermoid carcinoma (A-431) cells

    Science.gov (United States)

    Tan, Y; Chiow, KH; Huang, D; Wong, SH

    2010-01-01

    Background and purpose: Andrographolide is the active component of Andrographis paniculata, a plant used in both Indian and Chinese traditional medicine, and it has been demonstrated to induce apoptosis in different cancer cell lines. However, not much is known about how it may affect the key receptors implicated in cancer. Knowledge of how andrographolide affects receptor trafficking will allow us to better understand new mechanisms by which andrographolide may cause death in cancer cells. Experimental approach: We utilized the well-characterized epidermal growth factor receptor (EGFR) and transferrin receptor (TfR) expressed in epidermoid carcinoma (A-431) cells as a model to study the effect of andrographolide on receptor trafficking. Receptor distribution, the total number of receptors and surface receptors were analysed by immunofluorescence, Western blot as well as flow-cytometry respectively. Key results: Andrographolide treatment inhibited cell growth, down-regulated EGFRs on the cell surface and affected the degradation of EGFRs and TfRs. The EGFR was internalized into the cell at an increased rate, and accumulated in a compartment that co-localizes with the lysosomal-associated membrane protein in the late endosomes. Conclusion and implications: This study sheds light on how andrographolide may affect receptor trafficking by inhibiting receptor movement from the late endosomes to lysosomes. The down-regulation of EGFR from the cell surface also indicates a new mechanism by which andrographolide may induce cancer cell death. PMID:20233216

  14. Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)-21 receptor-blocking therapeutic antibody.

    Science.gov (United States)

    Xue, L; Hickling, T; Song, R; Nowak, J; Rup, B

    2016-01-01

    Reliable risk assessment for biotherapeutics requires accurate evaluation of risk factors associated with immunogenicity. Immunogenicity risk assessment tools were developed and applied to investigate the immunogenicity of a fully human therapeutic monoclonal antibody, ATR-107 [anti-interleukin (IL)-21 receptor] that elicited anti-drug antibodies (ADA) in 76% of healthy subjects in a Phase 1 study. Because the ATR-107 target is expressed on dendritic cells (DCs), the immunogenicity risk related to engagement with DC and antigen presentation pathways was studied. Despite the presence of IL-21R on DCs, ATR-107 did not bind to the DCs more extensively than the control therapeutic antibody (PF-1) that had elicited low clinical ADA incidence. However, ATR-107, but not the control therapeutic antibody, was translocated to the DC late endosomes, co-localized with intracellular antigen-D related (HLA-DR) molecules and presented a dominant T cell epitope overlapping the complementarity determining region 2 (CDR2) of the light chain. ATR-107 induced increased DC activation exemplified by up-regulation of DC surface expression of CD86, CD274 (PD-L1) and CD40, increased expansion of activated DC populations expressing CD86(hi), CD40(hi), CD83(hi), programmed death ligand 1 (PD-L1)(hi), HLA-DR(hi) or CCR7(hi), as well as elevated secretion of tumour necrosis factor (TNF)-α by DCs. DCs exposed to ATR-107 stimulated an autologous T cell proliferative response in human donor cells, in concert with the detection of immunoglobulin (Ig)G-type anti-ATR-107 antibody response in clinical samples. Collectively, the enhanced engagement of antigen presentation machinery by ATR-107 was suggested. The approaches and findings described in this study may be relevant to identifying lower immunogenicity risk targets and therapeutic molecules. © 2015 British Society for Immunology.

  15. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: Evidence for more than one receptor class

    International Nuclear Information System (INIS)

    Gronwald, R.G.K.; Grant, F.J.; Haldeman, B.A.; Hart, C.E.; O'Hara, P.J.; Hagen, F.S.; Ross, R.; Bowen-Pope, D.F.; Murray, M.J.

    1988-01-01

    The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A) + RNA from human dermal fibroblasts detects a major and a minor transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an ∼ 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF. Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class

  16. [Contributions of neuropsychology to anti-NMDA receptor antibody encephalitis: a literature review].

    Science.gov (United States)

    Luna-Lario, P; Hernaez-Goni, P; Tirapu-Ustarroz, J

    2016-05-01

    Limbic encephalitis generated by anti-N-methyl-D-aspartate (NMDA) receptor antibodies is an acute and severe neurological entity, which is more prevalent in young females and is associated to an underlying tumour. Since it leads to severe cognitive impairment, thought needs to be given to the contributions of neuropsychology to the diagnosis, development and treatment of the disease, which have received little attention from researchers to date. A review is conducted of the prior literature, evaluating the measurement of the cognitive symptoms (predominantly mnemonic and executive) associated to this disease. Valid, reliable neuropsychological instruments are proposed, and it is suggested that neuropsychological measures may be used as parameters to follow up these patients which help monitor their functionality in daily living once they have recovered from the acute phase. Similarly they can become a basis on which to assemble rehabilitation programmes that favour the accomplishment of personal autonomy and the patients' reintegration in the community. Nevertheless, we stress the need to include neuropsychologists and neuropsychiatrists in not only the detection but also the treatment of these patients so as to enable them to recover their personal independence and re-adapt to their natural settings.

  17. Clinical diagnosis of Graves’ or non-Graves’ hyperthyroidism compared to TSH receptor antibody test

    Directory of Open Access Journals (Sweden)

    Lauren Bell

    2018-04-01

    Full Text Available Background: TSH receptor antibody (TRAb is considered the gold standard diagnostic test for the autoimmunity of Graves’ disease (GD, which is commonly diagnosed clinically. Aim: To evaluate the true positive (sensitivity and true negative (specificity rates of clinical diagnosis of GD or non-GD hyperthyroidism compared to the TRAb test. Setting: University teaching hospital in North West England. Participants: Patients in the Endocrinology service who had a TRAb measurement between December 2009 and October 2015. Methods: Electronic patient records were studied retrospectively for a pre-TRAb clinical diagnosis of GD or non-GD hyperthyroidism. We examined descriptive statistics and binary classification tests; Fisher exact test was used to analyse contingency tables. Results: We identified 316 patients with a mean age of 45 (range, 17–89 years; 247 (78% were women. Compared to the TRAb result, clinical diagnosis had a sensitivity of 88%, specificity 66%, positive predictive value 72%, negative predictive value 84%, false negative rate 12%, false positive rate 34%, positive likelihood ratio 2.6 and negative likelihood ratio 0.2 (P < 0.0001. Conclusions: Clinicians were liable to both over- and under-diagnose GD. The TRAb test can help reduce the number of incorrect or unknown diagnoses in the initial clinical assessment of patients presenting with hyperthyroidism.

  18. Signal transduction by the platelet-derived growth factor receptor

    International Nuclear Information System (INIS)

    Williams, L.T.; Escobedo, J.A.; Keating, M.T.; Coughlin, S.R.

    1988-01-01

    The mitogenic effects of platelet-derived growth factor (PDGF) are mediated by the PDGF receptor. The mouse PDGF receptor was recently purified on the basis of its ability to become tyrosine phosphorylated in response to the A-B human platelet form of PDGF, and the receptor amino acid sequence was determined from a full-length cDNA clone. Both the human and mouse receptor cDNA sequences have been expressed in Chinese hamster ovary fibroblast (CHO) cells that normally lack PDGF receptors. This paper summarizes recent results using this system to study signal transduction by the PDGF receptor. Some of the findings show that the KI domain of the PDGF receptor plays an important role in the stimulation of DNA synthesis by PDGF. Surprisingly, the kinase insert region is not essential for PDGF stimulation of PtdIns turnover, pH change, increase in cellular calcium, and receptor autophosphorylation. In addition, PDGF stimulates a conformational change in the receptor

  19. Simultaneous Vascular Targeting and Tumor Targeting of Cerebral Breast Cancer Metastases Using a T-Cell Receptor Mimic Antibody

    Science.gov (United States)

    2014-05-01

    in May 2013, the difference between nude mice (which lack T- cells , but still have a partially functional adaptive and innate immune system) and NSG...Mangada J, Greiner DL, Handgretinger R. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human...Targeting of Cerebral Breast Cancer Metastases Using a T- Cell Receptor Mimic Antibody PRINCIPAL INVESTIGATOR: Ulrich Bickel

  20. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  1. Murine B cell development and antibody responses to model antigens are not impaired in the absence of the TNF receptor GITR.

    Directory of Open Access Journals (Sweden)

    Lenka Sinik Teodorovic

    Full Text Available The Glucocorticoid-Induced Tumor necrosis factor Receptor GITR, a member of the tumor necrosis factor receptor superfamily, has been shown to be important in modulating immune responses in the context of T cell immunity. B lymphocytes also express GITR, but a role of GITR in humoral immunity has not been fully explored. To address this question, we performed studies to determine the kinetics of GITR expression on naïve and stimulated B cells and the capacity of B cells to develop and mount antibody responses in GITR(-/- mice. Results of our studies indicate that all mature B cells express GITR on the cell surface, albeit at different levels. Expression of GITR on naïve mature B cells is upregulated by BCR signaling, but is counteracted by helper T cell-related factors and other inflammatory signals in vitro. In line with these findings, expression of GITR on germinal center and memory B cells is lower than that on naïve B cells. However, the expression of GITR is strongly upregulated in plasma cells. Despite these differences in GITR expression, the absence of GITR has no effect on T cell-dependent and T cell-independent antibody responses to model antigens in GITR(-/- mice, or on B cell activation and proliferation in vitro. GITR deficiency manifests only with a slight reduction of mature B cell numbers and increased turnover of naïve B cells, suggesting that GITR slightly contributes to mature B cell homeostasis. Overall, our data indicate that GITR does not play a significant role in B cell development and antibody responses to T-dependent and independent model antigens within the context of a GITR-deficient genetic background.

  2. Stereotypic Movements in Case of Sporadic Creutzfeldt-Jakob Disease: Possible Role of Anti-NMDA Receptor Antibodies

    Directory of Open Access Journals (Sweden)

    Michelle Molina

    2012-12-01

    Full Text Available Sporadic Creutzfeldt-Jakob disease (sCJD and anti-NMDA receptor antibody encephalitis (NMDAE can both produce a rapidly progressive dementia with resulting state of catatonia or akinetic mutism. Both are associated with movement disorders. In published case series, myoclonus appears to be the most frequent movement disorder in sCJD, while stereotypic, synchronized, one-cycle-per-second movements such as arm or leg elevation, jaw opening, grimacing, head turning, and eye deviation are seen in NMDAE. We report a case of a 59-year-old woman with rapidly worsening cognitive disturbance leading to a nearly catatonic state interrupted by stereotypic movements. sCJD was diagnosed via periodic sharp wave complexes on EEG as well as cerebrospinal fluid (CSF 14-3-3 and tau protein elevation. Characteristic movement disorder of NMDAE was present in absence of ovarian mass or CSF pleiocytosis. Given prior case reports of presence of anti-NMDA receptor antibodies in sCJD, we propose that the movement disorder in this case was caused by anti-NMDA receptor antibodies whose formation was secondary to neuronal damage from prion disease. It is important to consider sCJD even in cases that have some clinical features suggestive of NMDAE.

  3. Aspirin reduces serum anti-melanocyte antibodies and soluble interleukin-2 receptors in vitiligo patients

    International Nuclear Information System (INIS)

    Zailaie, Mohamad Z.

    2005-01-01

    Increased serum levels of certain immunologic markers including immunoglobulin G (IgG) anti-melanocyte/ vitiligo antibodies (V-IgG) and soluble interleukin-2 receptors (sIL-2R) are associated with augmented humoral and cellular immunity involved in melanocyte cytotoxicity during the active phase of non-segmental vitiligo. Recent reports have shown that, aspirin possesses a wide range of immunomodulatory and antioxidant properties. Therefore, the aim of the present study is to investigate the effect of long-term treatment of vitiligo patients with low-dose oral aspirin on serum V-IgG activity and sIL-2R concentration. The present study was carried out at the Vitiligo Unit, King Abdul-Aziz University Medical Center, Jeddah, Kingdom of Saudi Arabia between March and October 2003. Eighteen female and 14 male patients with a recent onset of non-segmental vitiligo were divided into 2 equal groups. One group received a daily single dose of oral aspirin (300 mg) and the second group received only placebo for a period of 12 weeks. Serum V-IgG activity and sIL-2R concentration were determined before and at the end of treatment period. The V-IgG activity was measured using cellular enzyme-linked immunosorbent assay (ELISA) following incubation of IgG antibodies with an adult cultured melanocytes. Serum sIL-2R concentration was measured using the highly sensitive quantitative sandwich ELISA utilizing a commercially available kit. As expected, the serum V-IgG activity and sIL-2R concentration of the active vitiligo patients (0.81 +/- 0.23 optical density (O.D.), 1428 +/- 510 pg/ml) were significantly increased compared with that of controls (0.27 +/- 0.1 O.D., 846 +/- 312 pg/ml; p<0.05, p<0.01). Aspirin-treated vitiligo patients showed significant decrease in serum V-IgG activity and sIL-2R concentration (0.32 +/- 0.08 O.D., 756 +/- 216 pg/ml) compared with that of placebo-treated patients (0.83 +/- 0.19 O.D., 1327 +/- 392 pg/ml; p<0.01). Low-dose oral aspirin treatment of

  4. The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

    OpenAIRE

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai; Hizi, Amnon

    2013-01-01

    Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening s...

  5. Novel Drosophila receptor that binds multiple growth factors

    International Nuclear Information System (INIS)

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-01-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10 -6 to 10 -8 M. The 100 kDa protein can be affinity-labeled with these 125 I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by 125 I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors

  6. Assembly and activation of neurotrophic factor receptor complexes.

    Science.gov (United States)

    Simi, Anastasia; Ibáñez, Carlos F

    2010-04-01

    Neurotrophic factors play important roles in the development and function of both neuronal and glial elements of the central and peripheral nervous systems. Their functional diversity is in part based on their ability to interact with alternative complexes of receptor molecules. This review focuses on our current understanding of the mechanisms that govern the assembly and activation of neurotrophic factor receptor complexes. The realization that many, if not the majority, of these complexes exist in a preassembled form at the plasma membrane has forced the revision of classical ligand-mediated oligomerization models, and led to the discovery of novel mechanisms of receptor activation and generation of signaling diversity which are likely to be shared by many different classes of receptors.

  7. Monoclonal antibodies to human factor VII: a one step immunoradiometric assay for VII:Ag.

    OpenAIRE

    Takase, T; Tuddenham, E G; Chand, S; Goodall, A H

    1988-01-01

    Three mouse monoclonal antibodies (RFF-VII/1, RFF-VII/2, and RFF-VII/3) which bind specifically to different epitopes on human factor VII antigen were raised. Two of the antibodies, RFF-VII/1 and RFF-VII/2, bound strongly to factor VII antigen (VII:Ag), but only RFF-VII/1 and RFF-VII/3 were potent inhibitors of factor VII coagulation activity (VII:C). RFF-VII/1 and RFF-VII/2 were used in a one step, double monoclonal immunoradiometric assay for VII:Ag. This was highly reproducible and detecte...

  8. Prostaglandin D2 Receptor DP1 Antibodies Predict Vaccine-induced and Spontaneous Narcolepsy Type 1: Large-scale Study of Antibody Profiling

    Directory of Open Access Journals (Sweden)

    Helle Sadam

    2018-03-01

    Full Text Available Background: Neuropathological findings support an autoimmune etiology as an underlying factor for loss of orexin-producing neurons in spontaneous narcolepsy type 1 (narcolepsy with cataplexy; sNT1 as well as in Pandemrix influenza vaccine-induced narcolepsy type 1 (Pdmx-NT1. The precise molecular target or antigens for the immune response have, however, remained elusive. Methods: Here we have performed a comprehensive antigenic repertoire analysis of sera using the next-generation phage display method - mimotope variation analysis (MVA. Samples from 64 children and adolescents were analyzed: 10 with Pdmx-NT1, 6 with sNT1, 16 Pandemrix-vaccinated, 16 H1N1 infected, and 16 unvaccinated healthy individuals. The diagnosis of NT1 was defined by the American Academy of Sleep Medicine international criteria of sleep disorders v3. Findings: Our data showed that although the immunoprofiles toward vaccination were generally similar in study groups, there were also striking differences in immunoprofiles between sNT1 and Pdmx-NT1 groups as compared with controls. Prominent immune response was observed to a peptide epitope derived from prostaglandin D2 receptor (DP1, as well as peptides homologous to B cell lymphoma 6 protein. Further validation confirmed that these can act as true antigenic targets in discriminating NT1 diseased along with a novel epitope of hemagglutinin of H1N1 to delineate exposure to H1N1. Interpretation: We propose that DP1 is a novel molecular target of autoimmune response and presents a potential diagnostic biomarker for NT1. DP1 is involved in the regulation of non-rapid eye movement (NREM sleep and thus alterations in its functions could contribute to the disturbed sleep regulation in NT1 that warrants further studies. Together our results also show that MVA is a helpful method for finding novel peptide antigens to classify human autoimmune diseases, possibly facilitating the design of better therapies. Keywords: Narcolepsy type 1

  9. Antisperm antibodies as a factor of male infertility. Relevance, modern methods of diagnosis and treatment

    Directory of Open Access Journals (Sweden)

    O. A. Nikiforov

    2017-08-01

    Full Text Available According to WHO statistics 40 % of childless marriage is due to factors of male infertility. One of them is the presence of antisperm antibodies in the male organism, which may be in blood serum, on the surface of spermatozoids and seminal plasma. Aim. Оn the grounds of specialized literature analysis, to show the relevance of this problem in Reproductive Medicine, to descript Basic methods of Modern treatment and diagnosis of this pathology in the body of infertile males. The most common methods of antisperm antibodies identifying are: MAR-test sample Shuvarskiy–Sims–Hyuner, Kurtsrok–Miller test, the method of latex agglutination, solid-phase immunoenzymatic blood test. Indications for antisperm antibodies determining are: modified indices, deviations in post-coital test, a negative test of sperm and cervical mucus interaction in vitro, unexplained infertility in the married couples, failure or low indices during IVF (in vitro fertilization and of course, the exclusion of other causes of infertility. When antisperm antibodies are detected, the strategy of treatment may be destined to reduction of their titer for further pregnancy. Such types of therapy can be used: contraceptive (long-term use contraception barrier to reduce antisperm antibodies titer in women, plasmapheresis, artificial insemination with pretreated from antisperm antibodies husband's sperm, methods of assisted reproductive technologies. Conclusoins. The formation of antisperm antibodies leads to infertility of immunological genesis (in 20 % of couples with unexplained infertility. To confirm their presence in the male body it is necessary to perform the MAR-test, Shuvarsky test, other tests and, of course, the exclusion of other causes of infertility. Men of reproductive age with an immunological factor of infertility provides for a comprehensive treatment, including elimination of all possible causative and contributing factors of infertility (infection of the male

  10. Mouse Hepatitis Virus Strain A59 and Blocking Antireceptor Monoclonal Antibody Bind to the N-Terminal Domain of Cellular Receptor

    Science.gov (United States)

    Dveksler, Gabriela S.; Pensiero, Michael N.; Dieffenbach, Carl W.; Cardellichio, Christine B.; Basile, Alexis A.; Elia, Patrick E.; Holmes, Kathryn V.

    1993-03-01

    Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.

  11. Investigation of a panel of monoclonal antibodies and polyclonal sera against anthrax toxins resulted in identification of an anti-lethal factor antibody with disease-enhancing characteristics.

    Science.gov (United States)

    Kulshreshtha, Parul; Tiwari, Ashutosh; Priyanka; Joon, Shikha; Sinha, Subrata; Bhatnagar, Rakesh

    2015-12-01

    Hybridomas were created using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor). Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immnized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies secreted by all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H10 and H8) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). Single chain variable fragment (LETscFv) was derived from H10 hybridoma. H11 was found to have disease-enhancing property. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature. This in vitro abrogation of disease-enhancement provides the proof of concept that in polyclonal sera the disease enhancing character of a fraction of antibodies is overshadowed by the protective nature of the rest of the antibodies generated on active immunization. Copyright © 2015. Published by Elsevier Ltd.

  12. Engineering of PDMS surfaces for use in microsystems for capture and isolation of complex and biomedically important proteins: epidermal growth factor receptor as a model system.

    Science.gov (United States)

    Lowe, Aaron M; Ozer, Byram H; Wiepz, Gregory J; Bertics, Paul J; Abbott, Nicholas L

    2008-08-01

    Elastomers based on poly(dimethylsiloxane) (PDMS) are promising materials for fabrication of a wide range of microanalytical systems due to their mechanical and optical properties and ease of processing. To date, however, quantitative studies that demonstrate reliable and reproducible methods for attachment of binding groups that capture complex receptor proteins of relevance to biomedical applications of PDMS microsystems have not been reported. Herein we describe methods that lead to the reproducible capture of a transmembrane protein, the human epidermal growth factor (EGF) receptor, onto PDMS surfaces presenting covalently immobilized antibodies for EGF receptor, and subsequent isolation of the captured receptor by mechanical transfer of the receptor onto a chemically functionalized surface of a gold film for detection. This result is particularly significant because the physical properties of transmembrane proteins make this class of proteins a difficult one to analyze. We benchmark the performance of antibodies to the human EGF receptor covalently immobilized on PDMS against the performance of the same antibodies physisorbed to conventional surfaces utilized in ELISA assays through the use of EGF receptor that was (32)P-radiolabeled in its autophosphorylation domain. These results reveal that two pan-reactive antibodies for the EGF receptor (clones H11 and 111.6) and one phosphospecific EGF receptor antibody (clone pY1068) capture the receptor on both PDMS and ELISA plates. When using H11 antibody to capture EGF receptor and subsequent treatment with a stripping buffer (NaOH and sodium dodecylsulfate) to isolate the receptor, the signal-to-background obtained using the PDMS surface was 82 : 1, exceeding the signal-to-background measured on the ELISA plate (<48 : 1). We also characterized the isolation of captured EGF receptor by mechanical contact of the PDMS surface with a chemically functionalized gold film. The efficiency of mechanical transfer of the

  13. Prevalence of elevated serum anti-N-methyl-D-aspartate receptor antibody titers in patients presenting exclusively with psychiatric symptoms: a comparative follow-up study.

    Science.gov (United States)

    Ando, Yoshihito; Shimazaki, Haruo; Shiota, Katsutoshi; Tetsuka, Syuichi; Nakao, Koichi; Shimada, Tatsuhiro; Kurata, Kazumi; Kuroda, Jinichi; Yamashita, Akihiro; Sato, Hayato; Sato, Mamoru; Eto, Shinkichi; Onishi, Yasunori; Tanaka, Keiko; Kato, Satoshi

    2016-07-08

    Increasing numbers of patients with elevated anti-N-methyl-D-aspartate (NMDA) receptor antibody titers presenting exclusively with psychiatric symptoms have been reported. The aim of the present study was to clarify the prevalence of elevated serum anti-NMDA receptor antibody titers in patients with new-onset or acute exacerbations of psychiatric symptoms. In addition, the present study aimed to investigate the association between elevated anti-NMDA receptor titers and psychiatric symptoms. The present collaborative study included 59 inpatients (23 male, 36 female) presenting with new-onset or exacerbations of schizophrenia-like symptoms at involved institutions from June 2012 to March 2014. Patient information was collected using questionnaires. Anti-NMDA receptor antibody titers were measured using NMDAR NR1 and NR2B co-transfected human embryonic kidney (HEK) 293 cells as an antigen (cell-based assay). Statistical analyses were performed for each questionnaire item. The mean age of participants was 42.0 ± 13.7 years. Six cases had elevated serum anti-NMDA antibody titers (10.2 %), four cases were first onset, and two cases with disease duration >10 years presented with third and fifth recurrences. No statistically significant difference in vital signs or major symptoms was observed between antibody-positive and antibody-negative groups. However, a trend toward an increased frequency of schizophrenia-like symptoms was observed in the antibody-positive group. Serum anti-NMDA receptor antibody titers may be associated with psychiatric conditions. However, an association with specific psychiatric symptoms was not observed in the present study. Further studies are required to validate the utility of serum anti-NMDA receptor antibody titer measurements at the time of symptom onset.

  14. Monoclonal antibodies to human factor VII: production of immunodepleted plasma for VII:C assays.

    OpenAIRE

    Takase, T; Tuddenham, E G; Chand, S; Goodall, A H

    1988-01-01

    A high affinity monoclonal antibody to factor VII (RFF-VII/1), coupled to sepharose, was used to immunodeplete factor VII from normal plasma. The plasma could be used as a substrate in a one stage coagulation assay and performed as well as, or better than, commercially available factor VII deficient plasma or plasma from congenitally deficient factor VII patients. Plasma from normal donors (n = 20), patients with liver disease (n = 20), and patients receiving warfarin (n = 20), or congenitall...

  15. Blood-brain barrier drug delivery of IgG fusion proteins with a transferrin receptor monoclonal antibody.

    Science.gov (United States)

    Pardridge, William M

    2015-02-01

    Biologic drugs are large molecules that do not cross the blood- brain barrier (BBB). Brain penetration is possible following the re-engineering of the biologic drug as an IgG fusion protein. The IgG domain is a MAb against an endogenous BBB receptor such as the transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse to ferry the fused biologic drug into the brain via receptor-mediated transport on the endogenous BBB TfR. This review discusses TfR isoforms, models of BBB transport of transferrin and TfRMAbs, and the genetic engineering of TfRMAb fusion proteins, including BBB penetrating IgG-neurotrophins, IgG-decoy receptors, IgG-lysosomal enzyme therapeutics and IgG-avidin fusion proteins, as well as BBB transport of bispecific antibodies formed by fusion of a therapeutic antibody to a TfRMAb targeting antibody. Also discussed are quantitative aspects of the plasma pharmacokinetics and brain uptake of TfRMAb fusion proteins, as compared to the brain uptake of small molecules, and therapeutic applications of TfRMAb fusion proteins in mouse models of neural disease, including Parkinson's disease, stroke, Alzheimer's disease and lysosomal storage disorders. The review covers the engineering of TfRMAb-avidin fusion proteins for BBB targeted delivery of biotinylated peptide radiopharmaceuticals, low-affinity TfRMAb Trojan horses and the safety pharmacology of chronic administration of TfRMAb fusion proteins. The BBB delivery of biologic drugs is possible following re-engineering as a fusion protein with a molecular Trojan horse such as a TfRMAb. The efficacy of this technology will be determined by the outcome of future clinical trials.

  16. Anti-N-methyl-D-aspartate receptor encephalitis with serum anti-thyroid antibodies and IgM antibodies against Epstein-Barr virus viral capsid antigen: a case report and one year follow-up

    Directory of Open Access Journals (Sweden)

    Xu Chun-Ling

    2011-11-01

    Full Text Available Abstract Background Anti-N-methyl-D-aspartate receptor encephalitis is an increasingly common autoimmune disorder mediated by antibodies to certain subunit of the N-methyl-D-aspartate receptor. Recent literatures have described anti-thyroid and infectious serology in this encephalitis but without follow-up. Case presentation A 17-year-old Chinese female patient presented with psychiatric symptoms, memory deficits, behavioral problems and seizures. She then progressed through unresponsiveness, dyskinesias, autonomic instability and central hypoventilation during treatment. Her conventional blood work on admission showed high titers of IgG antibodies to thyroglobulin, thyroid peroxidase and IgM antibodies to Epstein-Barr virus viral capsid antigen. An immature ovarian teratoma was found and removal of the tumor resulted in a full recovery. The final diagnosis of anti-N-methyl-D-aspartate receptor encephalitis was made by the identification of anti-N-methyl-D-aspartate receptor antibodies in her cerebral spinal fluid. Pathology studies of the teratoma revealed N-methyl-D-aspartate receptor subunit 1 positive ectopic immature nervous tissue and Epstein-Barr virus latent infection. She was discharged with symptoms free, but titers of anti-thyroid peroxidase and anti-thyroglobulin antibodies remained elevated. One year after discharge, her serum remained positive for anti-thyroid peroxidase and anti-N-methyl-D-aspartate receptor antibodies, but negative for anti-thyroglobulin antibodies and IgM against Epstein-Barr virus viral capsid antigen. Conclusions Persistent high titers of anti-thyroid peroxidase antibodies from admission to discharge and until one year later in this patient may suggest a propensity to autoimmunity in anti- N-methyl-D-aspartate receptor encephalitis and support the idea that neuronal and thyroid autoimmunities represent a pathogenic spectrum. Enduring anti-N-methyl-D-aspartate receptor antibodies from admission to one year

  17. Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

    Science.gov (United States)

    Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

    1984-09-01

    In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the

  18. Evaluation of pre-existing antibody presence as a risk factor for posttreatment anti-drug antibody induction: analysis of human clinical study data for multiple biotherapeutics.

    Science.gov (United States)

    Xue, Li; Rup, Bonita

    2013-07-01

    Biotherapeutic-reactive antibodies in treatment-naïve subjects (i.e., pre-existing antibodies) have been commonly detected during clinical immunogenicity assessments; however information on pre-existing antibody prevalence, physiological effects, and impact on posttreatment anti-drug antibody (ADA) induction remains limited. In this analysis, pre-existing antibody prevalence and impact on posttreatment ADA induction were determined using ADA data from 12 biotherapeutics analyzed in 32 clinical studies. Approximately half (58%) of the biotherapeutics were associated with some level of pre-existing antibodies and 67% of those were associated with posttreatment ADA induction. Across all studies, 5.6% of study subjects demonstrated presence of pre-existing antibodies, among which, 17% of the individual subjects had posttreatment increases in their ADA titers while 16% had decreased titers and 67% had no change in titers. However, in studies conducted in the rheumatoid arthritis (RA) population, 14.8% of RA patients were associated with pre-existing antibodies and 30% of those had posttreatment titer increases. The results suggest that in most study subjects, pre-existing antibodies pose a low risk for posttreatment ADA induction. That said, the high risk of induction implicated for RA patients, primarily observed in treatments evaluating novel antibody-based constructs, indicates that further understanding of the contribution of product and disease-specific factors is needed. Cross-industry efforts to collect and analyze a larger data set would enhance understanding of the prevalence, nature, and physiological consequences of pre-existing antibodies, better inform the immunogenicity risk profiles of products associated with these antibodies and lead to better fit-for-purpose immunogenicity management and mitigation strategies.

  19. Topical administration of adrenergic receptor pharmaceutics and nerve growth factor

    Directory of Open Access Journals (Sweden)

    Jena J Steinle

    2010-06-01

    Full Text Available Jena J SteinleDepartments of Ophthalmology and Anatomy and Neurobiology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN 38163, USAAbstract: Topical application of nerve growth factor (NGF and adrenergic receptor pharmaceutics are currently in use for corneal ulcers and glaucoma. A recent interest in the neuroprotective abilities of NGF has led to a renewed interest in NGF as a therapeutic for retinal and choroidal diseases. NGF can promote cell proliferation through actions of the TrkA receptor or promote apoptosis through receptor p75NTR. This understanding has led to novel interest in the role of NGF for diseases of the posterior eye. The role of β-adrenergic receptor agonists and antagonists for treatments of glaucoma, diabetic retinopathy, and their potential mechanisms of action, are still under investigation. This review discusses the current knowledge and applications of topical NGF and adrenergic receptor drugs for ocular disease.Keywords: NGF, β-adrenergic receptor agents, α-adrenergic receptor agents, retina, cornea, glaucoma

  20. Expression analysis and specific blockade of the receptor for human thymic stromal lymphopoietin (TSLP) by novel antibodies to the human TSLPRα receptor chain.

    Science.gov (United States)

    Borowski, Andreas; Vetter, Tina; Kuepper, Michael; Wohlmann, Andreas; Krause, Sebastian; Lorenzen, Thomas; Virchow, Johann Christian; Luttmann, Werner; Friedrich, Karlheinz

    2013-02-01

    Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine with a pivotal role in development and maintenance of atopic diseases such as allergic asthma and atopic dermatitis. Moreover, recent studies show an involvement of TSLP in the progression of various cancers. TSLP signaling is mediated by the TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor. It consists of the IL-7 receptor alpha chain (IL-7Rα), which is shared with the IL-7 receptor, and the TSLPRα chain as a specific subunit. Blocking signal release by TSLP without affecting IL-7 function is a potentially interesting option for the treatment of atopic diseases or certain tumors. By employing the extracellular domain of human TSLPRα chain (hTSLPRα(ex)) as an antigen, we generated a set of monoclonal antibodies. Several binders to native and/or denatured receptor protein were identified and characterized by cytometry and Western blot analysis. A screen based on a STAT3-driven reporter gene assay in murine pro-B cells expressing a functional hTSLPR yielded two hybridoma clones with specific antagonistic properties towards hTSLP, but not IL-7. Kinetic studies measuring blockade of hTSLP-dependent STAT phosphorylation in a TSLP-responsive cell line revealed an inhibitory constant in the nanomolar range. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    Directory of Open Access Journals (Sweden)

    Vanesa Alonso-Camino

    2013-01-01

    Full Text Available A human single-chain variable fragment (scFv antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs. The repertoire was fused to a first-generation T cell receptor ζ (TCRζ-based chimeric antigen receptor (CAR. We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2 bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR and the selection context (cell synapse, which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

  2. "Light switch" mental status changes and irritable insomnia are two particularly salient features of anti-NMDA receptor antibody encephalitis.

    Science.gov (United States)

    DeSena, Allen D; Greenberg, Benjamin M; Graves, Donna

    2014-07-01

    Anti-N-methyl-D-aspartate antibody encephalitis is becoming increasingly recognized as a cause of acute and subacute encephalopathy in both adults and children. The typical features of this disorder include some degree of encephalopathy, seizures, and often a movement disorder component. However, there is wide variability in its presentation, and diagnosis based on clinical features alone is often delayed. We report a series of four of 12 patients observed at our children's hospital between 2011 and 2013 that we chose as particularly representative examples of two distinct clinical features. In these individuals with anti-N-methyl-D-aspartate receptor antibody encephalitis, we note a very rapid on-off state between responsiveness and nonresponsiveness and/or insomnia accompanied by extreme irritability. We describe the abrupt mental status shift as "light switch" because the patients can awaken in seconds from a completely nonresponsive state. The insomnia noted in our patients was also impressive and often present early in the patients' courses. Light switch mental status changes and irritable insomnia are important early features of anti-N-methyl-D-aspartate receptor antibody encephalitis that can signal the presence of this disorder. The exact pathophysiology of these two symptoms has not been fully elucidated, and we feel that presence of one or both of these symptoms early in the disease course should prompt immediate concern for this disorder. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Simultaneous application of bevacizumab and anti-CTGF antibody effectively suppresses proangiogenic and profibrotic factors in human RPE cells.

    Science.gov (United States)

    Bagheri, Abouzar; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Samiei, Shahram; Sheibani, Nader; Astaneh, Shamila Darvishalipour; Kanavi, Mozhgan Rezaei; Mohammadian, Azam

    2015-01-01

    Retinal pigment epithelial (RPE) cells play key roles in the development of choroidal neovascularization and subsequent fibrosis. We investigated the impact of bevacizumab, antihuman vascular endothelial growth factor (VEGF) antibody, and anticonnective tissue growth factor (anti-CTGF) neutralizing antibody, individually or in combination, on proangiogenic and profibrotic properties of RPE cells. Primary cultures of human RPE cells were incubated with different concentrations of bevacizumab (0.25, 0.5, and 0.8 mg/ml) and/or anti-CTGF (10 μg/ml), and cell proliferation and apoptosis were determined. Expression and activity of proangiogenic and profibrotic genes including matrix metalloproteinases (MMP)-2 and 9, VEGFA, CTGF, vascular endothelial growth factor receptor-1 (VEGFR-1), cathepsin D, tissue inhibitor of metalloproteinases (TIMP) -1 and -2, and alpha smooth muscle actin (α-SMA) were assessed with slot blot, real-time RT-PCR, and zymography. Bevacizumab alone inhibited proliferation of RPE cells while anti-CTGF or bevacizumab and anti-CTGF combined had no inhibitory effect in this regard. Bevacizumab increased MMP-2, MMP-9, and cathepsin D but decreased VEGFA and VEGFR-1 expression. The CTGF level was increased by using 0.25 mg/ml bevacizumab but decreased at the 0.8 mg/ml concentration of bevacizumab. Treatment with anti-CTGF antibody decreased MMP-2 expression whereas combined treatment with bevacizumab and anti-CTGF resulted in decreased expression of MMP-2, TIMP-1, cathepsin D, VEGFA, CTGF, and α-SMA in the treated cultures. Treatment of RPE cells with the combination of bevacizumab and anti-CTGF could effectively suppress the proangiogenic and profibrotic activity of RPE cells.

  4. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Yi, Ka Hee; Kim, Chang Min

    1996-12-01

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves' patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves' disease will be elucidated. (author). 25 refs

  5. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ka Hee; Kim, Chang Min [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1996-12-01

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves` patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves` disease will be elucidated. (author). 25 refs.

  6. Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01

    DEFF Research Database (Denmark)

    Geisler, C; Plesner, T; Pallesen, G

    1988-01-01

    A murine monoclonal antibody (MoAb) F101.01 reacting with the T cell receptor (TCR)-T3 complex is presented. Immunohistological studies showed that F101.01 specifically stains T-zone lymphocytes in lymph nodes, tonsils, and splenic tissue. Two-colour immunofluorescence and flow cytometry...... demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20......), and myeloid cells (CD13 and CD33) were excluded. The target antigen of F101.01 co-modulated with the CD3-defined antigen (T3) and the TCR recognized by the MoAb WT-31. CD3 antibody and WT-31 both blocked binding of F101.01. F101.01 precipitated the TCR-T3 complex from lysates of 125I-labelled peripheral blood...

  7. Development and validation of receptor occupancy pharmacodynamic assays used in the clinical development of the monoclonal antibody vedolizumab.

    Science.gov (United States)

    Wyant, Tim; Estevam, Jose; Yang, Lili; Rosario, Maria

    2016-03-01

    Vedolizumab is a monoclonal antibody approved for use in ulcerative colitis and Crohn's disease. By specifically binding to α4 β7 integrin, vedolizumab prevents trafficking of lymphocytes to the gut, thereby interfering with disease pathology. During the clinical development program, the pharmacodynamic effect of vedolizumab was evaluated by 2 flow cytometry receptor occupancy assays: act-1 (ACT-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Here we describe the development and validation of these assays. The ACT-1 assay is a receptor occupancy free-site assay that uses a monoclonal antibody with the same binding epitope as vedolizumab to detect free (unbound) sites on α4 β7 integrin. The MAdCAM-1 assay used a soluble version of the natural ligand for α4 β7 integrin to detect free sites. The assays were validated using a fit-for-purpose approach throughout the clinical development of vedolizumab. Both the ACT-1 assay and the MAdCAM-1 assay demonstrated acceptable reproducibility and repeatability. The assays were sufficiently stable to allow for clinical use. During clinical testing the assays demonstrated that vedolizumab was able to saturate peripheral cells at all doses tested. Two pharmacodynamic receptor occupancy assays were developed and validated to assess the effect of vedolizumab on peripheral blood cells. The results of these assays demonstrated the practical use of flow cytometry to examine pharmacodynamic response in clinical trials. © 2015 International Clinical Cytometry Society.

  8. An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

    Directory of Open Access Journals (Sweden)

    Marcela V Maus

    2016-01-01

    Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

  9. Monoclonal antibodies in rheumatoid arthritis: comparative effectiveness of tocilizumab with tumor necrosis factor inhibitors

    Directory of Open Access Journals (Sweden)

    Tanaka T

    2014-04-01

    Full Text Available Toshio Tanaka,1,2 Yoshihiro Hishitani,3 Atsushi Ogata2,3 1Department of Clinical Application of Biologics, Osaka University Graduate School of Medicine, Osaka University, Osaka, Japan; 2Department of Immunopathology, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan; 3Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Osaka University Graduate School of Medicine, Osaka University, Osaka, Japan Abstract: Rheumatoid arthritis (RA is a chronic inflammatory disease characterized by persistent joint inflammation, systemic inflammation, and immunological abnormalities. Because cytokines such as tumor necrosis factor (TNF-α and interleukin (IL-6 play a major role in the development of RA, their targeting could constitute a reasonable novel therapeutic strategy for treating RA. Indeed, worldwide clinical trials of TNF inhibiting biologic disease modifying antirheumatic drugs (bDMARDs including infliximab, adalimumab, golimumab, certolizumab pegol, and etanercept as well as the humanized anti-human IL-6 receptor antibody, tocilizumab, have demonstrated outstanding clinical efficacy and tolerable safety profiles, resulting in worldwide approval for using these bDMARDs to treat moderate to severe active RA in patients with an inadequate response to synthetic disease modifying antirheumatic drugs (sDMARDs. Although bDMARDs have elicited to a paradigm shift in the treatment of RA due to the prominent efficacy that had not been previously achieved by sDMARDs, a substantial percentage of patients failed primary or secondary responses to bDMARD therapy. Because RA is a heterogeneous disease in which TNF-α and IL-6 play overlapping but distinct pathological roles, further studies are required to determine the best use of TNF inhibitors and tocilizumab in individual RA patients. Keywords: interleukin-6, rheumatoid arthritis, adalimumab, biologic

  10. Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex

    DEFF Research Database (Denmark)

    Stryhn, A; Andersen, P S; Pedersen, L O

    1996-01-01

    Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide...... each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T...... cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody....

  11. Nature of the bifunctional chelating agent used for radioimmunotherapy with yttrium-88 monoclonal antibodies: critical factors in determining in vivo survival and organ toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Kozak, R.W.; Raubitschek, A.; Mirzadeh, S.; Brechbiel, M.W.; Junghaus, R.; Gansow, O.A.; Waldmann, T.A. (Center for Biologics Evaluation and Research, FDA, Bethesda, MD (USA))

    1989-05-15

    One factor that is critical to the potential effectiveness of radioimmunotherapy is the design of radiometal-chelated antibodies that will be stable in vivo. Stability in vivo depends on the condition that both the chelate linkage and radiolabeling procedures not alter antibody specificity and biodistribution. In addition, synthesis and selection of the chelating agent is critical for each radiometal in order to prevent inappropriate release of the radiometal in vivo. In the present study, we compare the in vivo stability of seven radioimmunoconjugates that use different polyaminocarboxylate chelating agents to complex yttrium-88 to the mouse anti-human interleukin-2 receptor monoclonal antibody, anti-Tac. Chelate linkage and radiolabeling procedures did not alter the immunospecificity of anti-Tac. In order to assess whether yttrium was inappropriately released from the chelate-coupled antibody in vivo, iodine-131-labeled and yttrium-88 chelate-coupled antibodies were simultaneously administered to the same animals to correlate the decline in yttrium and radioiodinated antibody activity. The four stable yttrium-88 chelate-coupled antibodies studied displayed similar iodine-131 and yttrium-88 activity, indicating minimal elution of yttrium-88 from the complex. In contrast, the unstable yttrium-88 chelate-coupled antibodies had serum yttrium-88 activities that declined much more rapidly than their iodine-131 activities, suggesting loss of the radiolabel yttrium-88 from the chelate. Furthermore, high rates of yttrium-88 elution correlated with deposition in bone. Four chelating agents emerged as promising immunotherapeutic reagents: isothiocyanate benzyl DTPA and its derivatives 1B3M, MX, and 1M3B.

  12. Maintenance immunosuppression with intermittent intravenous IL-2 receptor antibody therapy in renal transplant recipients.

    Science.gov (United States)

    Gabardi, Steven; Catella, Jennifer; Martin, Spencer T; Perrone, Ronald; Chandraker, Anil; Magee, Colm C; McDevitt-Potter, Lisa M

    2011-09-01

    To report what we believe to be the first 2 cases of long-term (>24 months) intermittent intravenous interleukin-2 receptor antibody (IL-2RA) therapy for maintenance immunosuppression following renal transplantation. The first patient is a 52-year-old female with a history of intolerance to calcineurin inhibitors (CNIs) and sirolimus. Following her second transplant, the patient received mycophenolate mofetil 100 mg twice daily, a tapering corticosteroid regimen (initial dose of methylprednisolone 500 mg tapered over 1 week to prednisone 30 mg/day), and biweekly intravenous daclizumab 1-1.2 mg/kg/dose; 33 months after transplant the IL-2RA was changed to intravenous basiliximab 40 mg once a month. At 40 months after transplant, the patient continued to have stable renal function (estimated glomerular filtration rate 48 mL/min/1.73 m²) with excellent tolerability. The second patient is a 59-year-old female also intolerant to CNIs and sirolimus who required intermittent maintenance therapy with intravenous basiliximab 20 mg/dose. Despite an initial rejection episode, the patient tolerated more than 2 years of basiliximab therapy with good renal function (estimated glomerular filtration rate 103 months after transplant 69 mL/min/1.73 m²) and no adverse events. The IL-2RAs basiliximab and daclizumab possess several characteristics of ideal maintenance immunosuppressive agents (ie, nondepleting, long half-lives, limited adverse events). Based on a MEDLINE search (through December 31, 2010) using the search terms basiliximab, daclizumab, organ transplant, immunosuppression, and/or maintenance immunosuppression, and an advanced search in the published abstracts from the American Transplant Congress and World Transplant Congress (2000-2010), it appears that IL-2RAs have been used successfully as short-term therapy in both renal and extrarenal transplant recipients to allow for renal recovery following CNI-induced nephrotoxicity. In heart transplant recipients, the IL-2

  13. The strength of small: Improved targeting of Insulin-like Growth Factor-1 Receptor (IGF-1R) with F(ab')2-R1507 fragments in Ewing sarcomas

    NARCIS (Netherlands)

    Fleuren, Emmy D. G.; Versleijen-Jonkers, Yvonne M. H.; Heskamp, Sandra; Roeffen, Melissa H. S.; Bouwman, Wilbert H.; Molkenboer-Kuenen, Janneke D. M.; van Laarhoven, Hanneke W. M.; Oyen, Wim J. G.; Boerman, Otto C.; van der Graaf, Winette T. A.

    2013-01-01

    To investigate whether F(ab')2-fragments of the monoclonal Insulin-like Growth Factor-1 Receptor (IGF-1R) antibody R1507 (F(ab')2-R1507) can successfully target IGF-1R in Ewing sarcomas (ES). BALB/c nude mice were subcutaneously implanted with IGF-1R-expressing human ES xenografts (EW-5 and EW-8)

  14. Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor.

    Science.gov (United States)

    Vrolix, Kathleen; Fraussen, Judith; Losen, Mario; Stevens, Jo; Lazaridis, Konstantinos; Molenaar, Peter C; Somers, Veerle; Bracho, Maria Alma; Le Panse, Rozen; Stinissen, Piet; Berrih-Aknin, Sonia; Maessen, Jos G; Van Garsse, Leen; Buurman, Wim A; Tzartos, Socrates J; De Baets, Marc H; Martinez-Martinez, Pilar

    2014-08-01

    Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR-MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR-MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR-MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein-Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR-MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR-MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR-MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Intra-articular administration of an antibody against CSF-1 receptor reduces pain-related behaviors and inflammation in CFA-induced knee arthritis.

    Science.gov (United States)

    Alvarado-Vazquez, P A; Morado-Urbina, C E; Castañeda-Corral, G; Acosta-Gonzalez, R I; Kitaura, H; Kimura, K; Takano-Yamamoto, T; Jiménez-Andrade, J M

    2015-01-01

    Several studies have shown that blockade of colony stimulating factor-1 (CSF-1) or its receptor (CSF-1R) inhibits disease progression in rodent models of rheumatoid arthritis (RA); however, the role of the CSF-1/CSF-1R pathway in RA-induced pain and functional deficits has not been studied. Thus, we examined the effect of chronic intra-articular administration of a monoclonal anti-CSF-1R antibody (AFS98) on spontaneous pain, knee edema and functional disabilities in mice with arthritis. Unilateral arthritis was produced by multiple injections of complete Freund's adjuvant (CFA) into the right knee joint of adult male ICR mice. CFA-injected mice were then treated twice weekly from day 10 until day 25 with anti-CSF-1R antibody (3 and 10 μg/5 μL per joint), isotype control (rat IgG 10 μg/5 μL per joint) or PBS (5 μl/joint). Knee edema, spontaneous flinching, vertical rearing and horizontal exploratory activity were assessed at different days. Additionally, counts of peripheral leukocytes and body weight were measured to evaluate general health status. Intra-articular treatment with anti-CSF-1R antibody significantly increased horizontal exploratory activity and vertical rearing as well as reduced spontaneous flinching behavior and knee edema as compared to CFA-induced arthritis mice treated with PBS. Treatment with this antibody neither significantly affect mouse body weight nor the number of peripheral leukocytes. These results suggest that blockade of CSF-1R at the initial injury site (joint) could represent a therapeutic alternative for improving the functional disabilities and attenuating pain and inflammation in patients with RA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Characterization of the receptors for mycobacterial cord factor in Guinea pig.

    Directory of Open Access Journals (Sweden)

    Kenji Toyonaga

    Full Text Available Guinea pig is a widely used animal for research and development of tuberculosis vaccines, since its pathological disease process is similar to that present in humans. We have previously reported that two C-type lectin receptors, Mincle (macrophage inducible C-type lectin, also called Clec4e and MCL (macrophage C-type lectin, also called Clec4d, recognize the mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM. Here, we characterized the function of the guinea pig homologue of Mincle (gpMincle and MCL (gpMCL. gpMincle directly bound to TDM and transduced an activating signal through ITAM-bearing adaptor molecule, FcRγ. Whereas, gpMCL lacked C-terminus and failed to bind to TDM. mRNA expression of gpMincle was detected in the spleen, lymph nodes and peritoneal macrophages and it was strongly up-regulated upon stimulation of zymosan and TDM. The surface expression of gpMincle was detected on activated macrophages by a newly established monoclonal antibody that also possesses a blocking activity. This antibody potently suppressed TNF production in BCG-infected macrophages. Collectively, gpMincle is the TDM receptor in the guinea pig and TDM-Mincle axis is involved in host immune responses against mycobacteria.

  17. Characterization of the receptors for mycobacterial cord factor in Guinea pig.

    Science.gov (United States)

    Toyonaga, Kenji; Miyake, Yasunobu; Yamasaki, Sho

    2014-01-01

    Guinea pig is a widely used animal for research and development of tuberculosis vaccines, since its pathological disease process is similar to that present in humans. We have previously reported that two C-type lectin receptors, Mincle (macrophage inducible C-type lectin, also called Clec4e) and MCL (macrophage C-type lectin, also called Clec4d), recognize the mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM). Here, we characterized the function of the guinea pig homologue of Mincle (gpMincle) and MCL (gpMCL). gpMincle directly bound to TDM and transduced an activating signal through ITAM-bearing adaptor molecule, FcRγ. Whereas, gpMCL lacked C-terminus and failed to bind to TDM. mRNA expression of gpMincle was detected in the spleen, lymph nodes and peritoneal macrophages and it was strongly up-regulated upon stimulation of zymosan and TDM. The surface expression of gpMincle was detected on activated macrophages by a newly established monoclonal antibody that also possesses a blocking activity. This antibody potently suppressed TNF production in BCG-infected macrophages. Collectively, gpMincle is the TDM receptor in the guinea pig and TDM-Mincle axis is involved in host immune responses against mycobacteria.

  18. Total Thyroidectomy for Thyroid Cancer Followed by Thyroid Storm due to Thyrotropin Receptor Antibody Stimulation of Metastatic Thyroid Tissue

    DEFF Research Database (Denmark)

    Folkestad, Lars; Brandt, Frans; Brix, Thomas

    2017-01-01

    BACKGROUND: Graves disease (GD) is an autoimmune condition characterized by the presence of antibodies against the thyrotropin receptor (TRAB), which stimulate the thyroid gland to produce excess thyroid hormone. Theoretically, TRAB could stimulate highly differentiated thyroid cancer tissue and...... treatment continued until after the fourth RAI dose. Hypothyroidism did not occur until following the fifth RAI treatment. SUMMARY AND CONCLUSIONS: We present a patient initially diagnosed with thyrotoxicosis and subsequently with metastatic follicular variant of papillary thyroid cancer. It is suggested...... that TRAB stimulated the highly differentiated extrathyroidal metastatic thyroid tissue to produce excessive amounts of thyroid hormone, delayed diagnosis, and potential aggravation of the course of thyroid cancer....

  19. Targeting the Insulin-Like Growth Factor 1 Receptor in Ewing's Sarcoma: Reality and Expectations

    Directory of Open Access Journals (Sweden)

    David Olmos

    2011-01-01

    Full Text Available Ewing's sarcoma family of tumours comprises a group of very aggressive diseases that are potentially curable with multimodality treatment. Despite the undoubted success of current treatment, approximately 30% of patients will relapse and ultimately die of disease. The insulin-like growth factor 1 receptor (IGF-1R has been implicated in the genesis, growth, proliferation, and the development of metastatic disease in Ewing's sarcoma. In addition, IGF1-R has been validated, both in vitro and in vivo, as a potential therapeutic target in Ewing's sarcoma. Phase I studies of IGF-1R monoclonal antibodies reported several radiological and clinical responses in Ewing's sarcoma patients, and initial reports of several Phase II studies suggest that about a fourth of the patients would benefit from IGF-1R monoclonal antibodies as single therapy, with approximately 10% of patients achieving objective responses. Furthermore, these therapies are well tolerated, and thus far severe toxicity has been rare. Other studies assessing IGF-1R monoclonal antibodies in combination with traditional cytotoxics or other targeted therapies are expected. Despite, the initial promising results, not all patients benefit from IGF-1R inhibition, and consequently, there is an urgent need for the identification of predictive markers of response.

  20. Targeting the Insulin-Like Growth Factor 1 Receptor in Ewing's Sarcoma: Reality and Expectations

    Science.gov (United States)

    Olmos, David; Martins, Ana Sofia; Jones, Robin L.; Alam, Salma; Scurr, Michelle; Judson, Ian R.

    2011-01-01

    Ewing's sarcoma family of tumours comprises a group of very aggressive diseases that are potentially curable with multimodality treatment. Despite the undoubted success of current treatment, approximately 30% of patients will relapse and ultimately die of disease. The insulin-like growth factor 1 receptor (IGF-1R) has been implicated in the genesis, growth, proliferation, and the development of metastatic disease in Ewing's sarcoma. In addition, IGF1-R has been validated, both in vitro and in vivo, as a potential therapeutic target in Ewing's sarcoma. Phase I studies of IGF-1R monoclonal antibodies reported several radiological and clinical responses in Ewing's sarcoma patients, and initial reports of several Phase II studies suggest that about a fourth of the patients would benefit from IGF-1R monoclonal antibodies as single therapy, with approximately 10% of patients achieving objective responses. Furthermore, these therapies are well tolerated, and thus far severe toxicity has been rare. Other studies assessing IGF-1R monoclonal antibodies in combination with traditional cytotoxics or other targeted therapies are expected. Despite, the initial promising results, not all patients benefit from IGF-1R inhibition, and consequently, there is an urgent need for the identification of predictive markers of response. PMID:21647361

  1. Targeting the epidermal growth factor receptor in solid tumor malignancies

    DEFF Research Database (Denmark)

    Nedergaard, Mette K; Hedegaard, Chris J; Poulsen, Hans S

    2012-01-01

    been proposed as valid targets in many cancer therapy settings. Different strategies have been developed in order to either inhibit EGFR/EGFRvIII activity or to ablate EGFR/EGFRvIII-positive tumor cells. Drugs that inhibit these receptors include monoclonal antibodies (mAbs) that bind...... to the extracellular part of EGFR, blocking the binding sites for the EGFR ligands, and intracellular tyrosine kinase inhibitors (TKIs) that block the ATP binding site of the tyrosine kinase domain. Besides an EGFRvIII-targeted vaccine, conjugated anti-EGFR mAbs have been used in different settings to deliver lethal...... agents to the EGFR/EGFRvIII-positive cells; among these are radio-labelled mAbs and immunotoxins. This article reviews the current status and efficacy of EGFR/EGFRvIII-targeted therapies....

  2. Epitope mapping of functional domains of human factor V with human and mouse monoclonal antibodies

    International Nuclear Information System (INIS)

    Annamalai, A.E.; Rao, A.K.; Chiu, H.C.; Wang, D.; Dutta-Roy, A.K.; Colman, R.W.

    1986-01-01

    The authors previously described two human monoclonal antibodies (MAbs) which inactivated factor V. The authors have now purified the predominant antibody (H2) on protein A Sepharose using a pH gradient and typed it as IgG 1 ,. Immunoprecipitation of 125 I-human factor Va with H2 demonstrated specificity for the heavy chain (D), Mr = 105,000. The authors compared using ELISA the competitive binding to factor Va, of H2, H1 and two mouse MAbs, B38 (directed to E) and B10 (to activation peptide, Cl). All four antibodies recognized distinct epitopes in factor V with steric overlap in some cases. Factor Xa showed a concentration dependent competition for binding of H1, H2 and B38 but not B10 to factor V/Va in ELISA. All MAbs bound to factor V/Va in the absence of Ca ++ . However, Ca ++ at 8 mM increased the binding of H1 and H2 to 165% and 360% and did not have any effect on the binding of either mouse MAbs. Prothrombin at a concentration of up to 400 μg/ml did not inhibit binding of any of these antibodies. Thus, both the light (E) and heavy (D) chains of factor Va but not the activation peptide (Cl) interact with factor Xa as defined by the MAbs. In addition, sites on both chains for Ca ++ are recognized by particular MAbs (H1 and H2). These studies increase their knowledge of the interactions of factor V domains in the formation of prothrombinase complex

  3. Glutamate receptor antibodies in neurological diseases: anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies, anti-NMDA-NR2A/B antibodies, anti-mGluR1 antibodies or anti-mGluR5 antibodies are present in subpopulations of patients with either: epilepsy, encephalitis, cerebellar ataxia, systemic lupus erythematosus (SLE) and neuropsychiatric SLE, Sjogren's syndrome, schizophrenia, mania or stroke. These autoimmune anti-glutamate receptor antibodies can bind neurons in few brain regions, activate glutamate receptors, decrease glutamate receptor's expression, impair glutamate-induced signaling and function, activate blood brain barrier endothelial cells, kill neurons, damage the brain, induce behavioral/psychiatric/cognitive abnormalities and ataxia in animal models, and can be removed or silenced in some patients by immunotherapy.

    Science.gov (United States)

    Levite, Mia

    2014-08-01

    Glutamate is the major excitatory neurotransmitter of the Central Nervous System (CNS), and it is crucially needed for numerous key neuronal functions. Yet, excess glutamate causes massive neuronal death and brain damage by excitotoxicity--detrimental over activation of glutamate receptors. Glutamate-mediated excitotoxicity is the main pathological process taking place in many types of acute and chronic CNS diseases and injuries. In recent years, it became clear that not only excess glutamate can cause massive brain damage, but that several types of anti-glutamate receptor antibodies, that are present in the serum and CSF of subpopulations of patients with a kaleidoscope of human neurological diseases, can undoubtedly do so too, by inducing several very potent pathological effects in the CNS. Collectively, the family of anti-glutamate receptor autoimmune antibodies seem to be the most widespread, potent, dangerous and interesting anti-brain autoimmune antibodies discovered up to now. This impression stems from taking together the presence of various types of anti-glutamate receptor antibodies in a kaleidoscope of human neurological and autoimmune diseases, their high levels in the CNS due to intrathecal production, their multiple pathological effects in the brain, and the unique and diverse mechanisms of action by which they can affect glutamate receptors, signaling and effects, and subsequently impair neuronal signaling and induce brain damage. The two main families of autoimmune anti-glutamate receptor antibodies that were already found in patients with neurological and/or autoimmune diseases, and that were already shown to be detrimental to the CNS, include the antibodies directed against ionotorpic glutamate receptors: the anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies and anti-NMDA-NR2 antibodies, and the antibodies directed against Metabotropic glutamate receptors: the anti-mGluR1 antibodies and the anti-mGluR5 antibodies. Each type of these anti

  4. Factors associated with Coxiella burnetii antibody positivity in Danish dairy cows

    DEFF Research Database (Denmark)

    Paul, Suman; Agger, Jens Frederik Gramstrup; Markussen, Bo

    2012-01-01

    The aim of the study was to identify associations between the level of Coxiella burnetii (C. burnetii) antibodies in individual milk samples and cow and herd level factors in Danish dairy cows. The study, designed as a prospective cross sectional study with follow up, included 24 herds identified...

  5. Targeting the MET oncogene by concomitant inhibition of receptor and ligand via an antibody-"decoy" strategy.

    Science.gov (United States)

    Basilico, Cristina; Modica, Chiara; Maione, Federica; Vigna, Elisa; Comoglio, Paolo M

    2018-04-25

    MET, a master gene sustaining "invasive growth," is a relevant target for cancer precision therapy. In the vast majority of tumors, wild-type MET behaves as a "stress-response" gene and relies on the ligand (HGF) to sustain cell "scattering," invasive growth and apoptosis protection (oncogene "expedience"). In this context, concomitant targeting of MET and HGF could be crucial to reach effective inhibition. To test this hypothesis, we combined an anti-MET antibody (MvDN30) inducing "shedding" (i.e., removal of MET from the cell surface), with a "decoy" (i.e., the soluble extracellular domain of the MET receptor) endowed with HGF-sequestering ability. To avoid antibody/decoy interaction-and subsequent neutralization-we identified a single aminoacid in the extracellular domain of MET-lysine 842-that is critical for MvDN30 binding and engineered the corresponding recombinant decoyMET (K842E). DecoyMET K842E retains the ability to bind HGF with high affinity and inhibits HGF-induced MET phosphorylation. In HGF-dependent cellular models, MvDN30 antibody and decoyMET K842E used in combination cooperate in restraining invasive growth, and synergize in blocking cancer cell "scattering." The antibody and the decoy unbridle apoptosis of colon cancer stem cells grown in vitro as spheroids. In a preclinical model, built by orthotopic transplantation of a human pancreatic carcinoma in SCID mice engineered to express human HGF, concomitant treatment with antibody and decoy significantly reduces metastatic spread. The data reported indicate that vertical targeting of the MET/HGF axis results in powerful inhibition of ligand-dependent MET activation, providing proof of concept in favor of combined target therapy of MET "expedience." © 2018 UICC.

  6. Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy

    Directory of Open Access Journals (Sweden)

    Szlachcic A

    2016-08-01

    Full Text Available Anna Szlachcic, Malgorzata Zakrzewska, Michal Lobocki, Piotr Jakimowicz, Jacek Otlewski Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Abstract: Fibroblast growth factor receptors (FGFRs are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V, was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE, and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. Keywords: fibroblast growth factor 1, FGF receptor, targeted cancer therapy, cytotoxic conjugates, FGFR-dependent cancer, MMAE, auristatin

  7. Thyrotropin receptor antibody activities significantly correlate with the outcome of radioiodine ( sup 131 I) therapy for hyperthyroid Graves' disease

    Energy Technology Data Exchange (ETDEWEB)

    Kaise, Kazuro; Kaise, Nobuko; Yoshida, Katsumi; Fukazawa, Hiroshi; Mori, Koki; Yamamoto, Makiko; Sakurada, Toshiro; Saito, Shintaro; Yoshinaga, Kaoru (Tohoku Univ., Sendai (Japan). School of Medicine)

    1991-08-01

    The outcome of {sup 131}I therapy for 109 patients with Graves' disease was analysed according to pretreatment laboratory data including thyrotropin receptor antibody (TRAb) activities. Forty-five percent of patients became euthyroid, and 13% of patients became hypothyroid within one year after {sup 131}I therapy. Forty-two percent of patients remained hyperthyroid one year after {sup 131}I therapy. Pretreatment values for serum T{sub 4}, T{sub 3}, and the estimated weight of the thyroid were significantly higher in the hyperthyroid group. The mean for the TRAb index of the hyperthyroid group was significantly higher than that of the euthyroid group. Life table analysis revealed a significant effect of the TRAb index on the rate of hyperthyroidism after 3 months or later. These results appear to suggest that the TRAb index is one of the factors which influence the outcome of {sup 131}I therapy for Graves' disease. (author).

  8. Bevacizumab, an anti-vascular endothelial growth factor antibody, inhibits osteoarthritis

    OpenAIRE

    Nagai, Toshihiro; Sato, Masato; Kobayashi, Miyuki; Yokoyama, Munetaka; Tani, Yoshiki; Mochida, Joji

    2014-01-01

    Introduction Angiogenesis is an important factor in the development of osteoarthritis (OA). We investigated the efficacy of bevacizumab, an antibody against vascular endothelial growth factor and an inhibitor of angiogenesis, in the treatment of OA using a rabbit model of anterior cruciate ligament transection. Methods First, we evaluated the response of gene expression and histology of the normal joint to bevacizumab treatment. Next, in a rabbit model of OA induced by anterior cruciate ligam...

  9. Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling

    Science.gov (United States)

    Wang, Linlin; Schulz, Thomas C.; Sherrer, Eric S.; Dauphin, Derek S.; Shin, Soojung; Nelson, Angelique M.; Ware, Carol B.; Zhan, Mei; Song, Chao-Zhong; Chen, Xiaoji; Brimble, Sandii N.; McLean, Amanda; Galeano, Maria J.; Uhl, Elizabeth W.; D'Amour, Kevin A.; Chesnut, Jonathan D.; Rao, Mahendra S.

    2007-01-01

    Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1β (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs. PMID:17761519

  10. Extracorporeal adsorption of anti-factor VIII allo-antibodies on randomly functionalized polystyrene resins.

    Science.gov (United States)

    Huguet, Hélène-Céline; Lasne, Dominique; Rothschild, Chantal; Siali, Rosa; Jozefonvicz, Jacqueline

    2004-02-01

    The occurrence of anti-factor VIII (FVIII) allo-antibodies is a severe complication of the treatment of haemophilia A patients, leading to the inhibition of transfused FVIII activity. The effective elimination of these inhibitory antibodies plays a decisive role in the management of affected patients. To achieve this, immunoadsorption devices employing synthetic adsorbers, which selectively eliminate inhibitors, are of interest in the treatment strategy of haemophilia A patients with inhibitors. Adsorbers consisting of polystyrene-based beads substituted with sulphonate and L-tyrosyl methylester groups, which mimic part of epitope of FVIII molecule recognized by inhibitors, exhibit selective binding capacities towards anti-FVIII antibodies. The adsorption of FVIII inhibitors was investigated by simulating an extracorporeal circulation of haemophilic plasma over these functionalized resins. These innovative adsorbers are able to remove around 25% of anti-FVIII antibodies in 15 minutes depending on the plasma tested. Furthermore, they do not modify the amount of essential plasmatic proteins or residual immunoglobulins G. Experiments which were carried out using different plasmas with various inhibitor titres demonstrate a good reproducibility regarding the adsorption capacity of the synthetic resin. The characteristics of adsorption are similar on either native or regenerated resins. Both the purely synthetic nature of the resin and its easy processability demonstrate the real advantages over currently available protocols. This synthetic adsorber is a major technological advance in selective removal of FVIII inhibitory antibodies.

  11. Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics.

    Science.gov (United States)

    Tatarewicz, Suzanna; Miller, Jill M; Swanson, Steven J; Moxness, Michael S

    2010-05-31

    Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs. When using a sensitive electrochemiluminescent (ECL) bridging immunoassay, samples from subjects with rheumatoid arthritis demonstrated much higher baseline reactivity than healthy subjects. Interference was found to be dependent on the aggregation state of the therapeutic antibody that had been conjugated with the detection reagent (ruthenium). Size exclusion high performance liquid chromatography (SE-HPLC) demonstrated that of the total integrated peaks, as little as 0.55% high molecular weight aggregates (>600kDa) were sufficient to cause increased reactivity. Stability studies of the ruthenium and biotin conjugated therapeutic antibody indicated that storage time, temperature and buffer formulation were critical in maintaining the integrity of the reagents. Through careful SE-HPLC monitoring we were able to choose appropriate storage and buffer conditions which led to a reduction in the false reactivity rate in therapeutic-naïve serum from a rheumatoid arthritis population.

  12. Insulin and insulin-like growth factor receptors and responses

    International Nuclear Information System (INIS)

    Roth, R.A.; Steele-Perkins, G.; Hari, J.; Stover, C.; Pierce, S.; Turner, J.; Edman, J.C.; Rutter, W.J.

    1988-01-01

    Insulin is a member of a family of structurally related hormones with diverse physiological functions. In humans, the best-characterized members of this family include insulin, insulin-like growth factor (IGF)-I, and IGF-II. Each of these three polypeptide hormones has its own distinct receptor. The structures of each of these receptors have now been deduced from analyses of isolated cDNA clones. To study further the responses mediated through these three different receptors, the authors have been studying cells expressing the proteins encoded by these three cDNAs. The isolated cDNAs have been transfected into Chinese hamster ovary (CHO) cells, and the resulting transfected cell lines have been characterized as to the ligand-binding activities and signal-transducing activities of the expressed proteins

  13. Granulocyte-Colony Stimulating Factor Receptor, Tissue Factor, and VEGF-R Bound VEGF in Human Breast Cancer In Loco.

    Science.gov (United States)

    Wojtukiewicz, Marek Z; Sierko, Ewa; Skalij, Piotr; Kamińska, Magda; Zimnoch, Lech; Brekken, Ralf A; Thorpe, Philip E

    2016-01-01

    Doxorubicin and docetaxel-based chemotherapy regimens used in breast cancer patients are associated with high risk of febrile neutropenia (FN). Granulocyte colony-stimulating factors (G-CSF) are recommended for both treating and preventing chemotherapy-induced neutropenia. Increased thrombosis incidence in G-CSF treated patients was reported; however, the underlying mechanisms remain unclear. The principal activator of blood coagulation in cancer is tissue factor (TF). It additionally contributes to cancer progression and stimulates angiogenesis. The main proangiogenic factor is vascular endothelial growth factor (VEGF). The aim of the study was to evaluate granulocyte-colony stimulating factor receptor (G-CSFR), tissue factor (TF) expression and vascular endothelial growth factor receptor (VEGF-R) bound VEGF in human breast cancer in loco. G-CSFR, TF and VEGFR bound VEGF (VEGF: VEGFR) were assessed in 28 breast cancer tissue samples. Immunohistochemical (IHC) methodologies according to ABC technique and double staining IHC procedure were employed utilizing antibodies against G-CSFR, TF and VEGF associated with VEGFR (VEGF: VEGFR). Expression of G-CSFR was demonstrated in 20 breast cancer tissue specimens (71%). In 6 cases (21%) the expression was strong (IRS 9-12). Strong expression of TF was observed in all investigated cases (100%). Moreover, expression of VEGF: VEGFR was visualized in cancer cells (IRS 5-8). No presence of G-CSFR, TF or VEGF: VEGFR was detected on healthy breast cells. Double staining IHC studies revealed co-localization of G-CSFR and TF, G-CSFR and VEGF: VEGFR, as well as TF and VEGF: VEGFR on breast cancer cells and ECs. The results of the study indicate that GCSFR, TF and VEGF: VEGFR expression as well as their co-expression might influence breast cancer biology, and may increase thromboembolic adverse events incidence.

  14. Distribution of corticotropin-releasing factor receptors in primate brain

    International Nuclear Information System (INIS)

    Millan, M.A.; Jacobowitz, D.M.; Hauger, R.L.; Catt, K.J.; Aguilera, G.

    1986-01-01

    The distribution and properties of receptors for corticotropin-releasing factor (CRF) were analyzed in the brain of cynomolgus monkeys. Binding of [ 125 I]tyrosine-labeled ovine CRF to frontal cortex and amygdala membrane-rich fractions was saturable, specific, and time- and temperature-dependent, reaching equilibrium in 30 min at 23 0 C. Scatchard analysis of the binding data indicated one class of high-affinity sites with a K/sub d/ of 1 nM and a concentration of 125 fmol/mg. As in the rat pituitary and brain, CRF receptors in monkey cerebral cortex and amygdala were coupled to adenylate cyclase. Autoradiographic analysis of specific CRF binding in brain sections revealed that the receptors were widely distributed in the cerebral cortex and limbic system. Receptor density was highest in the pars tuberalis of the pituitary and throughout the cerebral cortex, specifically in the prefrontal, frontal, orbital, cingulate, insular, and temporal areas, and in the cerebellar cortex. A low binding density was present in the superior colliculus, locus coeruleus, substantia gelatinosa, preoptic area, septal area, and bed nucleus of the stria terminalis. These data demonstrate that receptors for CRF are present within the primate brain at areas related to the central control of visceral function and behavior, suggesting that brain CRF may serve as a neurotransmitter in the coordination of endocrine and neural mechanisms involved in the response to stress

  15. Enavatuzumab, a Humanized Anti-TWEAK Receptor Monoclonal Antibody, Exerts Antitumor Activity through Attracting and Activating Innate Immune Effector Cells

    Directory of Open Access Journals (Sweden)

    Shiming Ye

    2017-01-01

    Full Text Available Enavatuzumab is a humanized IgG1 anti-TWEAK receptor monoclonal antibody that was evaluated in a phase I clinical study for the treatment of solid malignancies. The current study was to determine whether and how myeloid effector cells were involved in postulated mechanisms for its potent antitumor activity in xenograft models. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models whose response to enavatuzumab relied on the binding of Fc of the antibody to Fcγ receptor. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in nonresponders. Consistent with the xenograft studies, human effector cells preferentially migrated toward in vivo-responsive tumor cells treated by enavatuzumab in vitro, with the majority of migratory cells being monocytes. Conditioned media from enavatuzumab-treated tumor cells contained elevated levels of chemokines, which might be responsible for enavatuzumab-triggered effector cell migration. These preclinical studies demonstrate that enavatuzumab can exert its potent antitumor activity by actively recruiting and activating myeloid effectors to kill tumor cells. Enavatuzumab-induced chemokines warrant further evaluation in clinical studies as potential biomarkers for such activity.

  16. Toll-like receptor activation enhances cell-mediated immunity induced by an antibody vaccine targeting human dendritic cells

    Directory of Open Access Journals (Sweden)

    Berger Marc A

    2007-01-01

    Full Text Available Abstract Previously, we have successfully targeted the mannose receptor (MR expressed on monocyte-derived dendritic cells (DCs using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGβ. Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several toll-like receptor (TLR-specific ligands that may synergize with MR targeting and be applicable as adjuvants in the clinic. We demonstrate that antigen-specific helper and cytolytic T cells from both healthy donors and cancer patients were effectively primed with B11-hCGβ-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DCs via TLR3 with dsRNA (poly I:C and DC TLR 7/8 with Resiquimod (R-848, respectively, elicited efficient antigen presentation-mediated by MR-targeting. We demonstrate that MR and TLRs contribute towards maturation and activation of DCs by a mechanism that may be driven by a combination of adjuvant and antibody vaccines that specifically deliver antigenic targets to DCs.

  17. Comment on "Antibodies to influenza nucleoprotein cross-react with human hypocretin receptor 2".

    OpenAIRE

    Vassalli, A.; Li, S.; Tafti, M.

    2015-01-01

    Did hypocretin receptor 2 autoantibodies cause narcolepsy with hypocretin deficiency in Pandemrix-vaccinated children, as suggested by Ahmed et al.? Using newly developed mouse models to report and inactivate hypocretin receptor expression, Vassalli et al. now show that hypocretin neurons (whose loss causes narcolepsy) do not express hypocretin autoreceptors, raising questions to the interpretation of Ahmed et al.'s findings.

  18. Platelet-derived growth factor receptors in the human central nervous system : autoradiographic distribution and receptor densities in multiple sclerosis

    NARCIS (Netherlands)

    De Keyser, J; Wilczak, N

    1997-01-01

    Platelet derived growth factor (PDGF) receptors were studied in postmortem adult human brain and cervical spinal cord using autoradiography with human recombinant I-125-PDGF-BB. PDGF-BB binds to the three different dimers of PDGF receptors (alpha alpha, alpha beta and beta beta) PDGF receptors were

  19. Antithyroglobulin antibody

    Science.gov (United States)

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  20. A case of late-onset, thymoma-associated myasthenia gravis with ryanodine receptor and titin antibodies and concomitant granulomatous myositis.

    Science.gov (United States)

    Stefanou, M I; Komorowski, L; Kade, S; Bornemann, A; Ziemann, U; Synofzik, M

    2016-09-13

    Myasthenia gravis is an autoimmune neuromuscular disorder, which has only rarely been reported to co-manifest with myositis. The diagnosis of concomitant myositis in patients with myasthenia gravis is clinically challenging, and requires targeted investigations for the differential diagnosis, including EMG, autoantibody assays, muscle biopsy and, importantly, imaging of the mediastinum for thymoma screening. This report presents a case-vignette of a 72-year-old woman with progressive proximal muscle weakness and myalgias, diagnosed with thymoma-associated myasthenia and bioptically verified granulomatous myositis, with positive autoantibody status for ryanodine receptor and titin antibodies. The diagnosis of concurrent myositis and myasthenia gravis, especially in the presence of ryanodine receptor and titin antibodies, should lead neurologists to adopt different treatment strategies compared to those applied in myasthenia or myositis alone. Moreover, further evidence is warranted that titin and, particularly, ryanodine receptor antibodies may co-occur or be pathophysiologically involved in myasthenia-myositis cases.

  1. Successful combination immunotherapy of anti-gamma aminobutyric acid (GABA)A receptor antibody-positive encephalitis with extensive multifocal brain lesions.

    Science.gov (United States)

    Fukami, Yuki; Okada, Hiroaki; Yoshida, Mari; Yamaguchi, Keiji

    2017-08-31

    A 78-year old woman who presented with akinetic mutism was admitted to our hospital. Brain MRI showed multifocal increased T 2 /FLAIR signal with extensive cortical-subcortical involvement. We suspected autoimmune encephalitis and the patient received methylprednisolone pulse. Her conscious level gradually recovered, but later relapsed again and presented with refractory status epilepticus. We treated her with intravenous immunoglobulin, plasma exchange and pulsed cyclophosphamide, with satisfactory response. A brain biopsy showed perivascular lymphocytic infiltrates and reactive gliosis. Anti-gamma aminobutyric acid (GABA) A receptor antibodies test came back to be positive after her recovery, and the diagnosis of anti-GABA A receptor antibody-positive encephalitis was made. This is a very rare case where brain biopsies were performed in a patient with anti-GABA A receptor antibody-positive encephalitis.

  2. Treatment with anti-interferon-δ monoclonal antibodies modifies experimental autoimmune encephalomyelitis in interferon-δ receptor knockout mice

    DEFF Research Database (Denmark)

    Espejo, C.; Penkowa, Milena; Saez-Torres, I.

    2001-01-01

    Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis......Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis...

  3. Direct demonstration of rapid insulin-like growth factor II receptor internalization and recycling in rat adipocytes. Insulin stimulates 125I-insulin-like growth factor II degradation by modulating the IGF-II receptor recycling process

    International Nuclear Information System (INIS)

    Oka, Y.; Rozek, L.M.; Czech, M.P.

    1985-01-01

    The photoactive insulin-like growth factor (IGF)-II analogue 4-azidobenzoyl- 125 I-IGF-II was synthesized and used to label specifically and covalently the Mr = 250,000 Type II IGF receptor. When rat adipocytes are irradiated after a 10-min incubation with 4-azidobenzoyl- 125 I-IGF-II at 10 degrees C and immediately homogenized, most of the labeled IGF-II receptors are associated with the plasma membrane fraction, indicating that receptors accessible to the labeling reagent at low temperature are on the cell surface. However, when the photolabeled cells are incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors are rapidly internalized with a half-time of 3.5 min as evidenced by a loss from the plasma membrane fraction and a concomitant appearance in the low density microsome fraction. The steady state level of cell surface IGF-II receptors in the presence or absence of IGF-II remains constant under these conditions, demonstrating that IGF-II receptors rapidly recycle back to the cell surface at the same rate as receptor internalization. Using the above methodology, it is shown that acute insulin action: 1) increases the steady state number of cell surface IGF-II receptors; 2) increases the number of ligand-bound IGF-II receptors that are internalized per unit of time; and 3) increases the rate of cellular 125 I-IGF-II degradation by a process that is blocked by anti-IGF-II receptor antibody

  4. Isotypic analysis of antibodies against activated Factor VII in patients with Factor VII deficiency using the x-MAP technology.

    Science.gov (United States)

    Pfeiffer, Caroline; Mathieu-Dupas, Eve; Logghe, Pauline; Lissalde-Lavigne, Géraldine; Balicchi, Julien; Caliskan, Umran; Valentin, Thomas; Laune, Daniel; Molina, Franck; Schved, Jean François; Giansily-Blaizot, Muriel

    2016-05-01

    While the immune response to hemophilic factors in hemophilia has been widely studied, little is known about the development of anti-Factor VII (FVII) antibodies in FVII deficiency. We developed a robust technique based on the x-MAP technology to detect the presence of antibodies against FVII and characterize their isotype and validated this method using blood samples from 100 patients with FVII deficiency (median FVII clotting activity [FVII:C]: 6%) and 95 healthy controls. Anti-FVII antibodies were detected in patients but also in some controls, although the concentration of total immunoglobulin G (IgGt) and IgG1 and IgG4 subclasses was significantly different between groups. The IgG1 subclass concentrations remained significantly different also when only untreated patients were compared with controls. This difference could partially be related to the F7 genotype, particularly in patients harboring the p.Arg139Gln mutation. This x-MAP-based method might be useful for assessing the immunogenicity of novel FVII compounds and of activated FVII (FVIIa) concentrates. Further prospective studies are needed to better understand the clinical relevance of these antibodies in the management of patients with FVII deficiency. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Epidermal growth factor receptor expression in urinary bladder cancer

    Directory of Open Access Journals (Sweden)

    Dayalu S.L. Naik

    2011-01-01

    Full Text Available Objective : To evaluate the expression pattern of epidermal growth factor receptor (EGFR in urinary bladder cancer and its association with human epidermal growth factor receptor 2 (HER2, epidermal growth factor (EGF, interleukin-6 (IL-6, and high risk human papilloma virus (HPV types 16 and 18. Materials and Methods : Thirty cases of urothelial carcinoma were analyzed. EGFR, HER2, EGF, and IL-6 expressions in the tissue were evaluated by immunohistochemical staining. For HPV, DNA from tissue samples was extracted and detection of HPV was done by PCR technique. Furthermore, evaluation of different intracellular molecules associated with EGFR signaling pathways was performed by the western blot method using lysates from various cells and tissues. Results : In this study, the frequencies of immunopositivity for EGFR, HER2, EGF, and IL-6 were 23%, 60%, 47%, and 80%, respectively. No cases were positive for HPV-18, whereas HPV-16 was detected in 10% cases. Overall, expression of EGFR did not show any statistically significant association with the studied parameters. However, among male patients, a significant association was found only between EGFR and HER2. Conclusions : Overexpression of EGFR and/or HER2, two important members of the same family of growth factor receptors, was observed in a considerable proportion of cases. Precise knowledge in this subject would be helpful to formulate a rational treatment strategy in patients with urinary bladder cancer.

  6. Association between serum antibodies to periodontal bacteria and rheumatoid factor in NHANES III

    Science.gov (United States)

    Goh, Charlene E.; Kopp, Jacob; Papapanou, Panos N.; Molitor, Jerry A.; Demmer, Ryan T.

    2016-01-01

    Objective Alterations in the microbiome, including the periodontal microbiome, may be a risk factor for rheumatoid arthritis (RA). Most studies that have analyzed this association are relatively small, focus primarily on a single periodontal pathogen (Porphyromonas gingivalis), and are not population-based. We investigated the association between elevated serum IgG antibodies to 19 periodontal species and the prevalence of rheumatoid factor (RF) in a large nationally representative sample of adults. Methods The Third National Health and Nutrition Examination Survey is a cross-sectional sample of the non-institutionalized US population (n=33,994). Our study population included all dentate participants ≥60 years, who did not have RA as defined by a modified version of the American College of Rheumatology 1987 criteria, and had complete data for both serum IgG antibodies against periodontal bacteria and serum RF antibody titer (n=2461). Results Adjusted odds ratios (ORs) (95% CI) summarizing the relationship between the 19 periodontal serum IgGs and RF seropositivity ranged from 0.53 (0.29, 0.97) to 1.27 (0.79, 2.06), and 17 of the 19 observed ORs were periodontal IgGs to be mostly unassociated with RF seropositivity in the nationally representative NHANES III. Elevated antibody levels to P. intermedia and C. ochracea were associated with lower odds of RF seropositivity. PMID:27110949

  7. Novel targeted approaches to treating biliary tract cancer: the dual epidermal growth factor receptor and ErbB-2 tyrosine kinase inhibitor NVP-AEE788 is more efficient than the epidermal growth factor receptor inhibitors gefitinib and erlotinib.

    Science.gov (United States)

    Wiedmann, Marcus; Feisthammel, Jürgen; Blüthner, Thilo; Tannapfel, Andrea; Kamenz, Thomas; Kluge, Annett; Mössner, Joachim; Caca, Karel

    2006-08-01

    Aberrant activation of the epidermal growth factor receptor is frequently observed in neoplasia, notably in tumors of epithelial origin. Attempts to treat such tumors with epidermal growth factor receptor antagonists resulted in remarkable success in recent studies. Little is known, however, about the efficacy of this therapy in biliary tract cancer. Protein expression of epidermal growth factor receptor, ErbB-2, and vascular endothelial growth factor receptor-2 was assessed in seven human biliary tract cancer cell lines by immunoblotting. In addition, histological sections from 19 patients with extrahepatic cholangiocarcinoma were analyzed for epidermal growth factor receptor, ErbB-2 and vascular endothelial growth factor receptor-2 expression by immunohistochemistry. Moreover, we sequenced the cDNA products representing the entire epidermal growth factor receptor coding region of the seven cell lines, and searched for genomic epidermal growth factor receptor amplifications and polysomy by fluorescence in-situ hybridization. Cell growth inhibition by gefitinib erlotinib and NVP-AEE788 was studied in vitro by automated cell counting. In addition, the anti-tumoral effect of erlotinib and NVP-AEE788 was studied in a chimeric mouse model. The anti-tumoral drug mechanism in this model was assessed by MIB-1 antibody staining, terminal deoxynucleotidyl transfer-mediated dUTP nick end-labelling assay, von Willebrand factor staining, and immunoblotting for p-p42/44 (p-Erk1/2, p-MAPK) and p-AKT. Immunoblotting revealed expression of epidermal growth factor receptor, ErbB-2, and vascular endothelial growth factor receptor-2 in all biliary tract cancer cell lines. EGFR was detectable in six of 19 (32%) extrahepatic human cholangiocarcinoma tissue samples, ErbB-2 in 16 of 19 (84%), and vascular endothelial growth factor receptor-2 in nine of 19 (47%). Neither epidermal growth factor receptor mutations nor amplifications or polysomy were found in the seven biliary tract cancer

  8. Specific, high affinity receptors for insulin-like growth factor II in the rat kidney glomerulus

    International Nuclear Information System (INIS)

    Haskell, J.F.; Pillion, D.J.; Meezan, E.

    1988-01-01

    Rat renal glomeruli were isolated by a technique involving kidney perfusion with a solution containing magnetic iron oxide particles, followed by homogenization, sieving, and concentration over a strong magnet. Isolated glomeruli were treated with 1% Triton X-100 to solubilize plasma membrane components, while insoluble basement membrane components were removed by centrifugation. [ 125 I]Insulin-like growth factor II (IGF-II) binding to this preparation was competitively inhibited by increasing amounts of unlabeled IGF-II, with 50% inhibition at an IGF-II concentration of 1 ng/ml. [ 125 I]IGF-II was covalently cross-linked with disuccinimidyl suberate to its receptor in rat renal glomeruli and a specific high mol wt (255,000) band could be identified on autoradiograms of dodecyl sulfate-polyacrylamide gels. [ 125 I]IGF-II binding and cross-linking to this band was inhibited by a polyclonal antibody against the type II IGF receptor. These results demonstrate for the first time that the isolated rat renal glomerulus contains a high affinity receptor for IGF-II

  9. A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients

    Directory of Open Access Journals (Sweden)

    Marzia Vezzalini

    2017-06-01

    Full Text Available Abstract Background Protein tyrosine phosphatase receptor gamma (PTPRG is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML have been reported, only one polyclonal antibody (named chPTPRG has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2 to better define PTPRG protein downregulation in CML patients. Methods TPγ B9-2 specifically recognizes PTPRG (both human and murine by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Results Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells. After effective tyrosine kinase inhibitor (TKI treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI non-responder patients, confirming that downregulation selectively occurs in primary CML cells. Conclusions The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the

  10. Diagnostic accuracy study of a factor VIII ELISA for detection of factor VIII antibodies in congenital and acquired haemophilia A.

    Science.gov (United States)

    Batty, Paul; Moore, Gary W; Platton, Sean; Maloney, James C; Palmer, Ben; Bowles, Louise; Pasi, K John; Rangarajan, Savita; Hart, Daniel P

    2015-10-01

    Antibody formation to factor VIII (FVIII) remains the greatest clinical and diagnostic challenge to the haemophilia-treating physician. Current guidance for testing for inhibitory FVIII antibodies (inhibitors) recommends the functional Nijmegen-Bethesda assay (NBA). A FVIII ELISA offers a complementary, immunological approach for FVIII antibody testing. It was the aim of this study to retrospectively evaluate the performance of a FVIII ELISA (index) for detection of FVIII antibodies, compared with the NBA (reference). All samples sent for routine FVIII antibody testing at two haemophilia Comprehensive Care Centres, were tested in parallel using the NBA and a solid-phase, indirect FVIII ELISA kit (Immucor). A total of 497 samples from 239 patients (severe haemophilia A=140, non-severe haemophilia A=85, acquired haemophilia A=14) were available for analysis. Sixty-three samples tested positive by the NBA (prevalence 12.7%, 95% confidence interval [CI], 9.9-15.9 %), with a median inhibitor titre of 1.2 BU/ml (range 0.7-978.0). The FVIII ELISA demonstrated a specificity of 94.0% (95%CI, 91.3-96.0), sensitivity of 77.8% (95%CI, 65.5-87.3), negative predictive value of 96.7% (95%CI, 94.5-98.2), positive predictive value 65.3% (95%CI, 53.5-76.0), negative likelihood ratio 0.2 (95%CI, 0.1-0.4), positive likelihood ratio 13.0 (95%CI, 8.7-19.3) and a diagnostic odds ratio of 54.9 (95%CI, 27.0-112.0). Strong positive correlation (r=0.77, pNBA (log adjusted) and FVIII ELISA optical density. In conclusion, FVIII ELISA offers a simple, specific, surveillance method enabling batch testing of non-urgent samples for the presence of FVIII antibodies.

  11. Acquired Antibody Responses against Plasmodium vivax Infection Vary with Host Genotype for Duffy Antigen Receptor for Chemokines (DARC)

    Science.gov (United States)

    Maestre, Amanda; Muskus, Carlos; Duque, Victoria; Agudelo, Olga; Liu, Pu; Takagi, Akihide; Ntumngia, Francis B.; Adams, John H.; Sim, Kim Lee; Hoffman, Stephen L.; Corradin, Giampietro; Velez, Ivan D.; Wang, Ruobing

    2010-01-01

    Background Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are ‘resistant’ to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens. Methodology/Findings We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion. Conclusion/Significance Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the

  12. Acquired antibody responses against Plasmodium vivax infection vary with host genotype for duffy antigen receptor for chemokines (DARC.

    Directory of Open Access Journals (Sweden)

    Amanda Maestre

    2010-07-01

    Full Text Available Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are 'resistant' to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens.We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1 and Duffy binding protein (PvDBP varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B. The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion.Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades

  13. Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha

    Science.gov (United States)

    DOKI, Tomoyoshi; TAKANO, Tomomi; HOHDATSU, Tsutomu

    2016-01-01

    Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2–4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2–4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2–4) by fusing the variable region of mouse mAb 2–4 to the constant region of feline antibody. The chimeric mAb 2–4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2–4 and chimeric mAb 2–4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2–4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2–4 was reduced. In contrast, in cats treated with chimeric mAb 2–4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2–4-treated cats. PMID:27264736

  14. Radioreceptor assay study of thyrotropin receptor antibody (TRAb) in Grave's diseases

    International Nuclear Information System (INIS)

    Lu Chao; Lin Xiangtong

    1989-01-01

    Here was reported the assay system using pig thyroid TSH receptor and 125 I-bTSH purified by receptor of thyroid cell membrane for the study of TRAb activity. The binding rate of ASH soluble receptor with 125 I-bTSH was 22.2 ∼ 37.4%, while as the control was only 1.0 ∼ 2.1%. TRAb was measured clinically in 48 cases of Grave's diseases and 25 normal persons. The TSH binding inhabitory index(TRII) was introduced for reflection of TRAb activity. The results showed that TBII was positure in 30 of 48 patients of Grave's diseases, the detctactibility was 79.2%

  15. Topical administration of adrenergic receptor pharmaceutics and nerve growth factor

    OpenAIRE

    Steinle, Jena

    2010-01-01

    Jena J SteinleDepartments of Ophthalmology and Anatomy and Neurobiology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN 38163, USAAbstract: Topical application of nerve growth factor (NGF) and adrenergic receptor pharmaceutics are currently in use for corneal ulcers and glaucoma. A recent interest in the neuroprotective abilities of NGF has led to a renewed interest in NGF as a therapeutic for retinal and choroidal diseases. NGF can promote cell proliferati...

  16. Antibody therapy of cancer : Fc receptor-mediated mechanisms of action

    NARCIS (Netherlands)

    Overdijk, M.B.

    2013-01-01

    Cancer, a class of malignant diseases characterized by unregulated cell growth, is still a leading cause of death worldwide. The high specificity of antibodies combined with the ability to engage multiple mechanisms of action (MoA) and minimal side-effects makes them attractive agents for targeted

  17. Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII.

    OpenAIRE

    Broze, G J; Hickman, S; Miletich, J P

    1985-01-01

    Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on We...

  18. Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors

    DEFF Research Database (Denmark)

    Sorkin, A; Helin, K; Waters, C M

    1992-01-01

    The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of ...

  19. Targeting non-small cell lung cancer cells by dual inhibition of the insulin receptor and the insulin-like growth factor-1 receptor.

    Directory of Open Access Journals (Sweden)

    Emma E Vincent

    Full Text Available Phase III trials of the anti-insulin-like growth factor-1 receptor (IGF1R antibody figitumumab in non-small cell lung cancer (NSCLC patients have been discontinued owing to lack of survival benefit. We investigated whether inhibition of the highly homologous insulin receptor (IR in addition to the IGF1R would be more effective than inhibition of the IGF1R alone at preventing the proliferation of NSCLC cells. Signalling through IGF1R and IR in the NSCLC cell lines A549 and Hcc193 was stimulated by a combination of IGF1, IGF2 and insulin. It was inhibited by antibodies that block ligand binding, αIR3 (IGF1R and IR47-9 (IR, and by the ATP-competitive small molecule tyrosine kinase inhibitors AZ12253801 and NVPAWD742 which inhibit both IGF1R and IR tyrosine kinases. The effect of inhibitors was determined by an anchorage-independent proliferation assay and by analysis of Akt phosphorylation. In Hcc193 cells the reduction in cell proliferation and Akt phosphorylation due to anti-IGF1R antibody was enhanced by antibody-mediated inhibition of the IR whereas in A549 cells, with a relatively low IR:IGF1R expression ratio, it was not. In each cell line proliferation and Akt phosphorylation were more effectively inhibited by AZ12253801 and NVPAWD742 than by combined αIR3 and IR47-9. When the IGF1R alone is inhibited, unencumbered signalling through the IR can contribute to continued NSCLC cell proliferation. We conclude that small molecule inhibitors targeting both the IR and IGF1R more effectively reduce NSCLC cell proliferation in a manner independent of the IR:IGF1R expression ratio, providing a therapeutic rationale for the treatment of this disease.

  20. Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

    Directory of Open Access Journals (Sweden)

    Sujatha P Koduvayur

    Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

  1. Decreased expression of serum and microvascular vascular endothelial growth factor receptor-2 in meningococcal sepsis*.

    NARCIS (Netherlands)

    Flier, M. van der; Baerveldt, E.M.; Miedema, A.; Hartwig, N.G.; Hazelzet, J.A.; Emonts, M.; Groot, R. de; Prens, E.P.; Vught, A.J. van; Jansen, N.J.

    2013-01-01

    OBJECTIVES: To determine the skin microvessel expression of vascular endothelial growth factor receptor 2 and serum-soluble vascular endothelial growth factor receptor 2 levels in children with meningococcal sepsis. DESIGN: Observational study. SETTING: Two tertiary academic children hospital PICUs.

  2. Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients

    DEFF Research Database (Denmark)

    Meyer, C N; Svenson, M; Larsen, Carsten Schade

    2000-01-01

    Neutralizing cytokine antibodies are found in healthy and diseased individuals, including patients treated with recombinant cytokines. Identification of CCR-5 as co-receptor for HIV has focused interest on CC chemokines and their potential therapeutic use. Chemokine-binding components in plasma...

  3. Late manifestation of subclinical hyperthyroidism after goitrogenesis in an index patient with a N670S TSH receptor germline mutation masquerading as TSH receptor antibody negative Graves' disease.

    Science.gov (United States)

    Schaarschmidt, J; Paschke, S; Özerden, M; Jäschke, H; Huth, S; Eszlinger, M; Meller, J; Paschke, R

    2012-12-01

    In 27 families with familial non-autoimmune hyperthyroidism (FNAH) reported up to date, the onset of hyperthyroidism varies from 18 months to 60 years. Also the manifestation of goitres is variable in these families. A 74-year-old woman first presented at the age of 69 years with tachyarrhythmia and hypertension. After initial treatment of her hypertension and oral anticoagulation for her intermittent atrial fibrillation, a thyroid workup revealed a suppressed TSH and normal fT3 and fT4. TPO, TSH receptor (TSHR), and thyroglobulin antibodies were negative. Thyroid ultrasound revealed a thyroid volume of 102 ml with several nodules with diameters of up to 2.6 cm right and up to 1.8 cm left. Scintigraphy showed a homogeneous Technetium-99 m ((99 m)Tc) uptake of 1.27%. She was subsequently treated with 1 GBq radioiodine ((131)I). At the age of 74, her thyroid function was normal and her thyroid volume decreased to 90 ml. Because of the diffuse (99 m)Tc uptake and the negative TPO, TSHR, and thyroglobulin antibodies, genetic analysis of her TSHR gene was performed, in spite of her negative family history for hyperthyroidism. Sequencing revealed a N670S TSHR germline mutation. Previous in vitro characterisation of this TSHR mutation suggests a weak constitutive activity, yet the experimental data are ambiguous. This case illustrates the necessity to analyse patients with hyperthyroidism accompanied by diffuse (99 m)Tc uptake and negative TPO, TSHR, and thyroglobulin antibodies for TSHR germline mutations. Moreover, it demonstrates that TSHR germline mutations may first lead to longstanding nodular goitrogenesis before the late manifestation of subclinical hyperthyroidism. © Georg Thieme Verlag KG Stuttgart · New York.

  4. Therapies based on inhibitors of the epidermal growth factor receptor: enclosing the future

    International Nuclear Information System (INIS)

    Diaz, Arlhee; Lage, Agustin

    2007-01-01

    The Epidermal Growth Factor Receptor (EGFR) is considered an important target for rational drug design due to its key role in numerous tumors. Potential contribution of EGFR-related signaling pathways to promote tumorigenic processes, including cell proliferation, angiogenesis, and resistance to apoptosis has been well established. Two classes of anti-EGFR agents in late-stage clinical testing include monoclonal antibodies against extracellular EGFR domain (Cetuximab, Nimotuzumab) and small molecules tyrosine kinase inhibitors, which inhibit the receptor enzyme activity (Gefitinib, Erlotinib). A considerable body of evidence has emerged since its introduction in the treatment of cancer patients. However, important questions such as reliable surrogate markers to predict response to the treatment, or optimal sequence and combination of these agents with conventional therapies remain to be addressed. Identify and validate predictive factors to select patients likely to respond to EGFR inhibitors, such as mutations that confer resistance versus those associated with sensitivity is required. A better understanding of molecular mechanisms associated with antitumor activity will useful to predict the interaction of these agents with other therapies in order to avoid antagonisms or overlapping effects resulting in no adding effects. Finally, the benefits derived from EGFR inhibitors as first-line therapy in selected populations, and the optimal doses and ways to delivery to the tumor site resulting in optimal target modulation should be established by the ongoing investigation. (Author)

  5. Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation

    International Nuclear Information System (INIS)

    Martinez-Garcia, Eva; Irigoyen, Marta; Anso, Elena; Martinez-Irujo, Juan Jose; Rouzaut, Ana

    2008-01-01

    Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 μM triggered cell differentiation towards a neuronal-like phenotype: cells emitted filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure

  6. Stepwise Progress in Epidermal Growth Factor Receptor/Radiation Studies for Head and Neck Cancer

    International Nuclear Information System (INIS)

    Harari, Paul M.

    2007-01-01

    The U.S. Food and Drug Administration approval of four new epidermal growth factor receptor (EGFR) inhibitors for cancer therapy (cetuximab, panitumumab, gefitinib, and erlotinib) over the last 3 years is a remarkable milestone in oncology. Indeed, molecular inhibition of EGFR signaling represents one of the most promising current arenas for the development of molecular-targeted cancer therapies. Epidermal growth factor receptor inhibitors from both the monoclonal antibody and tyrosine kinase inhibitor class have demonstrated clinical activity in the treatment of a broad spectrum of common human malignancies. For the discipline of radiation oncology, the 2006 report of a phase III trial demonstrating a survival advantage for advanced head and neck cancer patients with the addition of weekly cetuximab during a 7-week course of radiation is particularly gratifying. Indeed, this is the first phase III trial to confirm a survival advantage with the addition of a molecular-targeted agent to radiation. Furthermore, this result seems to have been achieved with only a modest increment in overall treatment toxicity and with very high compliance to the prescribed treatment regimen. Nevertheless, much remains to be learned regarding the rational integration of EGFR inhibitors into cancer treatment regimens, as well as methods to optimize the selection of patients most likely to benefit from EGFR inhibitor strategies

  7. Epidermal Growth Factor Receptor (EGFR) Crosstalks in Liver Cancer

    International Nuclear Information System (INIS)

    Berasain, Carmen; Latasa, María Ujue; Urtasun, Raquel; Goñi, Saioa; Elizalde, María; Garcia-Irigoyen, Oihane; Azcona, María; Prieto, Jesús; Ávila, Matías A.

    2011-01-01

    Hepatocarcinogenesis is a complex multistep process in which many different molecular pathways have been implicated. Hepatocellular carcinoma (HCC) is refractory to conventional chemotherapeutic agents, and the new targeted therapies are meeting with limited success. Interreceptor crosstalk and the positive feedback between different signaling systems are emerging as mechanisms of targeted therapy resistance. The identification of such interactions is therefore of particular relevance to improve therapeutic efficacy. Among the different signaling pathways activated in hepatocarcinogenesis the epidermal growth factor receptor (EGFR) system plays a prominent role, being recognized as a “signaling hub” where different extracellular growth and survival signals converge. EGFR can be transactivated in response to multiple heterologous ligands through the physical interaction with multiple receptors, the activity of intracellular kinases or the shedding of EGFR-ligands. In this article we review the crosstalk between the EGFR and other signaling pathways that could be relevant to liver cancer development and treatment

  8. Epidermal growth factor receptor in primary human lung cancer

    International Nuclear Information System (INIS)

    Yu Xueyan; Hu Guoqiang; Tian Keli; Wang Mingyun

    1996-01-01

    Cell membranes were prepared from 12 human lung cancers for the study of the expression of epidermal growth factor receptors (EGFR). EGFR concentration was estimated by ligand binding studies using 125 I-radiolabeled EGF. The dissociation constants of the high affinity sites were identical, 1.48 nmol and 1.1 nmol in cancer and normal lung tissues, the EGFR contents were higher in lung cancer tissues (range: 2.25 to 19.39 pmol·g -1 membrane protein) than that in normal tissues from the same patients (range: 0.72 to 7.43 pmol·g -1 membrane protein). These results suggest that EGF and its receptor may play a role in the regulatory mechanisms in the control of lung cellular growth and tumor promotion

  9. Determination of anti-acetylcholine receptor antibodies in myasthenic patients by use of time-resolved fluorescence

    Czech Academy of Sciences Publication Activity Database

    Říčný, Jan; Šimková, L.; Vincent, A.

    2002-01-01

    Roč. 48, č. 3 (2002), s. 549-554 ISSN 0009-9147 R&D Projects: GA MZd NF4646 Institutional research plan: CEZ:AV0Z5011922 Keywords : nicotinic acetylcholine receptor * time-resolved fluorescence method * myasthenia gravis Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 4.788, year: 2002

  10. Factors determining anti-poliovirus type 3 antibodies among orally immunised Indian infants.

    Science.gov (United States)

    Kaliappan, Saravanakumar Puthupalayam; Venugopal, Srinivasan; Giri, Sidhartha; Praharaj, Ira; Karthikeyan, Arun S; Babji, Sudhir; John, Jacob; Muliyil, Jayaprakash; Grassly, Nicholas; Kang, Gagandeep

    2016-09-22

    Among the three poliovirus serotypes, the lowest responses after vaccination with trivalent oral polio vaccine (tOPV) are to serotype 3. Although improvements in routine immunisation and supplementary immunisation activities have greatly increased vaccine coverage, there are limited data on antibody prevalence in Indian infants. Children aged 5-11months with a history of not having received inactivated polio vaccine were screened for serum antibodies to poliovirus serotype 3 (PV3) by a micro-neutralisation assay according to a modified World Health Organization (WHO) protocol. Limited demographic information was collected to assess risk-factors for a lack of protective antibodies. Student's t-test, logistic regression and multilevel logistic regression (MLR) model were used to estimate model parameters. Of 8454 children screened at a mean age of 8.3 (standard deviation [SD]-1.8) months, 88.1% (95% confidence interval (CI): 87.4-88.8) had protective antibodies to PV3. The number of tOPV doses received was the main determinant of seroprevalence; the maximum likelihood estimate yields a 37.7% (95% CI: 36.2-38.3) increase in seroprevalence per dose of tOPV. In multivariable logistic regression analysis increasing age, male sex, and urban residence were also independently associated with seropositivity (Odds Ratios (OR): 1.17 (95% CI: 1.12-1.23) per month of age, 1.27 (1.11-1.46) and 1.24 (1.05-1.45) respectively). Seroprevalence of antibodies to PV3 is associated with age, gender and place of residence, in addition to the number of tOPV doses received. Ensuring high coverage and monitoring of response are essential as long as oral vaccines are used in polio eradication. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  11. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

    International Nuclear Information System (INIS)

    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen

    2015-01-01

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice

  12. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen, E-mail: srrshurology@163.com

    2015-08-14

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

  13. Safety and efficacy of eculizumab in anti-acetylcholine receptor antibody-positive refractory generalised myasthenia gravis (REGAIN)

    DEFF Research Database (Denmark)

    Howard, James F; Utsugisawa, Kimiaki; Benatar, Michael

    2017-01-01

    BACKGROUND: Complement is likely to have a role in refractory generalised myasthenia gravis, but no approved therapies specifically target this system. Results from a phase 2 study suggested that eculizumab, a terminal complement inhibitor, produced clinically meaningful improvements in patients...... with anti-acetylcholine receptor antibody-positive refractory generalised myasthenia gravis. We further assessed the efficacy and safety of eculizumab in this patient population in a phase 3 trial. METHODS: We did a phase 3, randomised, double-blind, placebo-controlled, multicentre study (REGAIN) in 76...... hospitals and specialised clinics in 17 countries across North America, Latin America, Europe, and Asia. Eligible patients were aged at least 18 years, with a Myasthenia Gravis-Activities of Daily Living (MG-ADL) score of 6 or more, Myasthenia Gravis Foundation of America (MGFA) class II-IV disease...

  14. Acetylcholine receptor binding antibody-associated myasthenia gravis and rhabdomyolysis induced by nivolumab in a patient with melanoma.

    Science.gov (United States)

    Shirai, Takushi; Sano, Tasuku; Kamijo, Fuminao; Saito, Nana; Miyake, Tomomi; Kodaira, Minori; Katoh, Nagaaki; Nishie, Kenichi; Okuyama, Ryuhei; Uhara, Hisashi

    2016-01-01

    We reported an 81-year-old woman with metastatic melanoma, in whom myasthenia gravis and rhabdomyolysis developed after nivolumab monotherapy. The first symptom of myasthenia gravis was dyspnea. Ultrasonography detected hypokinesis of the bilateral diaphragm suggesting myasthenia gravis, although there was no abnormal finding of the lungs in computed tomography images. Acetylcholine receptor binding antibodies were low-titer positive in the preserved serum before administration of nivolumab, strongly suggesting that the myasthenia gravis was a nivolumab-related immune adverse event. Despite the remarkable clinical benefits of immune checkpoint inhibitors for patients with advanced melanoma, it is important to recognize unexpected immune-related adverse events. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. Validation of antibodies for neuroanatomical localization of the P2Y receptor in macaque brain

    DEFF Research Database (Denmark)

    Dreisig, Karin; Degn, Matilda; Sund, Louise

    2016-01-01

    Focus on the purinergic receptor P2Y11 has increased following the finding of an association between the sleep disorder narcolepsy and a genetic variant in P2RY11 causing decreased gene expression. Narcolepsy is believed to arise from an autoimmune destruction of the hypothalamic neurons that pro......Focus on the purinergic receptor P2Y11 has increased following the finding of an association between the sleep disorder narcolepsy and a genetic variant in P2RY11 causing decreased gene expression. Narcolepsy is believed to arise from an autoimmune destruction of the hypothalamic neurons...

  16. The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

    Science.gov (United States)

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai

    2013-01-01

    Abstract Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

  17. Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis

    International Nuclear Information System (INIS)

    Ceridono, Mara; Belleudi, Francesca; Ceccarelli, Simona; Torrisi, Maria Rosaria

    2005-01-01

    Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCγ binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCγ as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR

  18. Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma.

    Science.gov (United States)

    Li, Nan; Fu, Haiying; Hewitt, Stephen M; Dimitrov, Dimiter S; Ho, Mitchell

    2017-08-08

    Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.

  19. Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein

    DEFF Research Database (Denmark)

    Järås, Marcus; Johnels, Petra; Hansen, Nils Gunder

    2010-01-01

    stem cells could be prospectively separated. In addition, by generating an anti-IL1RAP antibody, we provide proof of concept that IL1RAP can be used as a target on CML CD34(+)CD38(-) cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell...... will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test...... whether IL1RAP expression distinguishes normal (Ph(-)) and leukemic (Ph(+)) cells within the CML CD34(+)CD38(-) cell compartment, we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method, we sorted cells directly into drops on slides to investigate...

  20. Structural basis for pH-insensitive inhibition of immunoglobulin G recycling by an anti-neonatal Fc receptor antibody

    Energy Technology Data Exchange (ETDEWEB)

    Kenniston, Jon A.; Taylor, Brandy M.; Conley, Gregory P.; Cosic, Janja; Kopacz, Kris J.; Lindberg, Allison P.; Comeau, Stephen R.; Atkins, Kateri; Bullen, Jameson; TenHoor, Christopher; Adelman, Burt A.; Sexton, Daniel J.; Edwards, Thomas E.; Nixon, Andrew E. (Beryllium); (Dyax)

    2017-09-06

    The neonatal Fc receptor FcRn plays a critical role in the trafficking of IgGs across tissue barriers and in retaining high circulating concentrations of both IgG and albumin. Although generally beneficial from an immunological perspective in maintaining IgG populations, FcRn can contribute to the pathogenesis of autoimmune disorders when an abnormal immune response targets normal biological components. We previously described a monoclonal antibody (DX-2507) that binds to FcRn with high affinity at both neutral and acidic pH, prevents the simultaneous binding of IgG, and reduces circulating IgG levels in preclinical animal models. Here, we report a 2.5 Å resolution X-ray crystal structure of an FcRn–DX-2507 Fab complex, revealing a nearly complete overlap of the IgG–Fc binding site in FcRn by complementarity-determining regions in DX-2507. This overlap explains how DX-2507 blocks IgG binding to FcRn and thereby shortens IgG half-life by preventing IgGs from recycling back into circulation. Moreover, the complex structure explains how the DX-2507 interaction is pH-insensitive unlike normal Fc interactions and how serum albumin levels are unaffected by DX-2507 binding. These structural studies could inform antibody-based therapeutic approaches for limiting the effects of IgG-mediated autoimmune disease.

  1. Can anti-vascular endothelial growth factor antibody reverse radiation necrosis? A preclinical investigation.

    Science.gov (United States)

    Duan, Chong; Perez-Torres, Carlos J; Yuan, Liya; Engelbach, John A; Beeman, Scott C; Tsien, Christina I; Rich, Keith M; Schmidt, Robert E; Ackerman, Joseph J H; Garbow, Joel R

    2017-05-01

    Anti-vascular endothelial growth factor (anti-VEGF) antibodies are a promising new treatment for late time-to-onset radiation-induced necrosis (RN). We sought to evaluate and validate the response to anti-VEGF antibody in a mouse model of RN. Mice were irradiated with the Leksell Gamma Knife Perfexion™ and then treated with anti-VEGF antibody, beginning at post-irradiation (PIR) week 8. RN progression was monitored via anatomic and diffusion MRI from weeks 4-12 PIR. Standard histology, using haematoxylin and eosin (H&E), and immunohistochemistry staining were used to validate the response to treatment. After treatment, both post-contrast T1-weighted and T2-weighted image-derived lesion volumes decreased (P < 0.001), while the lesion volumes for the control group increased. The abnormally high apparent diffusion coefficient (ADC) for RN also returned to the ADC range for normal brain following treatment (P < 0.001). However, typical RN pathology was still present histologically. Large areas of focal calcification were observed in ~50% of treated mouse brains. Additionally, VEGF and hypoxia-inducible factor 1-alpha (HIF-1α) were continually upregulated in both the anti-VEGF and control groups. Despite improvements observed radiographically following anti-VEGF treatment, lesions were not completely resolved histologically. The subsequent calcification and the continued upregulation of VEGF and HIF-1α merit further preclinical/clinical investigation.

  2. Modulation of the virus-receptor interaction by mutations in the V5 loop of feline immunodeficiency virus (FIV following in vivo escape from neutralising antibody

    Directory of Open Access Journals (Sweden)

    Samman Ayman

    2010-04-01

    Full Text Available Abstract Background In the acute phase of infection with feline immunodeficiency virus (FIV, the virus targets activated CD4+ T cells by utilising CD134 (OX40 as a primary attachment receptor and CXCR4 as a co-receptor. The nature of the virus-receptor interaction varies between isolates; strains such as GL8 and CPGammer recognise a "complex" determinant on CD134 formed by cysteine-rich domains (CRDs 1 and 2 of the molecule while strains such as PPR and B2542 require a more "simple" determinant comprising CRD1 only for infection. These differences in receptor recognition manifest as variations in sensitivity to receptor antagonists. In this study, we ask whether the nature of the virus-receptor interaction evolves in vivo. Results Following infection with a homogeneous viral population derived from a pathogenic molecular clone, a quasispecies emerged comprising variants with distinct sensitivities to neutralising antibody and displaying evidence of conversion from a "complex" to a "simple" interaction with CD134. Escape from neutralising antibody was mediated primarily by length and sequence polymorphisms in the V5 region of Env, and these alterations in V5 modulated the virus-receptor interaction as indicated by altered sensitivities to antagonism by both anti-CD134 antibody and soluble CD134. Conclusions The FIV-receptor interaction evolves under the selective pressure of the host humoral immune response, and the V5 loop contributes to the virus-receptor interaction. Our data are consistent with a model whereby viruses with distinct biological properties are present in early versus late infection and with a shift from a "complex" to a "simple" interaction with CD134 with time post-infection.

  3. Modulation of the virus-receptor interaction by mutations in the V5 loop of feline immunodeficiency virus (FIV) following in vivo escape from neutralising antibody.

    Science.gov (United States)

    Willett, Brian J; Kraase, Martin; Logan, Nicola; McMonagle, Elizabeth L; Samman, Ayman; Hosie, Margaret J

    2010-04-26

    In the acute phase of infection with feline immunodeficiency virus (FIV), the virus targets activated CD4+ T cells by utilising CD134 (OX40) as a primary attachment receptor and CXCR4 as a co-receptor. The nature of the virus-receptor interaction varies between isolates; strains such as GL8 and CPGammer recognise a "complex" determinant on CD134 formed by cysteine-rich domains (CRDs) 1 and 2 of the molecule while strains such as PPR and B2542 require a more "simple" determinant comprising CRD1 only for infection. These differences in receptor recognition manifest as variations in sensitivity to receptor antagonists. In this study, we ask whether the nature of the virus-receptor interaction evolves in vivo. Following infection with a homogeneous viral population derived from a pathogenic molecular clone, a quasispecies emerged comprising variants with distinct sensitivities to neutralising antibody and displaying evidence of conversion from a "complex" to a "simple" interaction with CD134. Escape from neutralising antibody was mediated primarily by length and sequence polymorphisms in the V5 region of Env, and these alterations in V5 modulated the virus-receptor interaction as indicated by altered sensitivities to antagonism by both anti-CD134 antibody and soluble CD134. The FIV-receptor interaction evolves under the selective pressure of the host humoral immune response, and the V5 loop contributes to the virus-receptor interaction. Our data are consistent with a model whereby viruses with distinct biological properties are present in early versus late infection and with a shift from a "complex" to a "simple" interaction with CD134 with time post-infection.

  4. Anti-phospholipase A receptor antibodies correlate with clinical status in idiopathic membranous nephropathy

    NARCIS (Netherlands)

    Hofstra, J.M.; Beck Jr., L.H.; Beck, D.M.; Wetzels, J.F.M.; Salant, D.J.

    2011-01-01

    BACKGROUND AND OBJECTIVES: Circulating autoantibodies against the M-type phospholipase A(2) receptor (anti-PLA(2)R) were recently identified in the majority of patients in the United States with idiopathic membranous nephropathy (iMN). The objectives of this study were to assess the prevalence of

  5. An assessment of radioimmunoassay procedures for determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis

    International Nuclear Information System (INIS)

    Carter, B.; Harrison, R.; Lunt, G.G.; Morris, H.; Savage-Marengo, T.; Stephenson, F.A.

    1981-01-01

    A reproducible radioimmunoassay procedure for the determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis is described and examined in detail. The assay combines features of a number of methods previously outlined and allows repeat determinations of antibody titre in a given myasthenic serum sample with coefficient of variation 6%. The mean +- standard deviation for normal human serum anti-acetylcholine receptor antibodies was found by this procedure to be 0.024 +- 0.033 nmol/l α-bungarotoxin binding sites whereas the range for myasthenic patients was 0-139.14 nmol/l with a mean value of 7.55 nmol/l α-bungarotoxin binding sites. (author)

  6. Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor

    Science.gov (United States)

    Schreiner, Jens; Thommen, Daniela S.; Herzig, Petra; Bacac, Marina; Klein, Christian; Roller, Andreas; Belousov, Anton; Levitsky, Victor; Savic, Spasenija; Moersig, Wolfgang; Uhlenbrock, Franziska; Heinzelmann-Schwarz, Viola A.; Umana, Pablo; Pisa, Pavel; von Bergwelt-Baildon, M.; Lardinois, Didier; Müller, Philipp; Karanikas, Vaios; Zippelius, Alfred

    2016-01-01

    ABSTRACT T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction. PMID:27057429

  7. Did hypocretin receptor 2 autoantibodies cause narcolepsy with hypocretin deficiency in Pandemrix-vaccinated children? Comment on “Antibodies to influenza nucleoprotein cross-react with human hypocretin receptor 2”

    OpenAIRE

    Vassalli Anne

    2015-01-01

    Abstract Did hypocretin receptor 2 auto antibodies cause narcolepsy with hypocretin deficiency in Pandemrix vaccinated children as suggested by Ahmed et al.? Using newly developed mouse models to report and inactivate hypocretin receptor expression Vassalli et al. now show that hypocretin neurons (whose loss causes narcolepsy) do not express hypocretin autoreceptors raising questions to the interpretation of Ahmed et al.’s findings. Mouse Genome Informatics: www.informatics.jax.org/reference/...

  8. Epidermal growth factor and its receptors in human pancreatic carcinoma

    International Nuclear Information System (INIS)

    Chen, Y.F.; Pan, G.Z.; Hou, X.; Liu, T.H.; Chen, J.; Yanaihara, C.; Yanaihara, N.

    1990-01-01

    The role of epidermal growth factor (EGF) in oncogenesis and progression of malignant tumors is a subject of vast interest. In this study, radioimmunoassay and radioreceptor assay of EGF were established. EGF contents in malignant and benign pancreatic tumors, in normal pancreas tissue, and in culture media of a human pancreatic carcinoma cell line were determined. EGF receptor binding studies were performed. It was shown that EGF contents in pancreatic carcinomas were significantly higher than those in normal pancreas or benign pancreatic tumors. EGF was also detected in the culture medium of a pancreatic carcinoma cell line. The binding of 125I-EGF to the pancreatic carcinoma cells was time and temperature dependent, reversible, competitive, and specific. Scatchard analysis showed that the dissociation constant of EGF receptor was 2.1 X 10(-9) M, number of binding sites was 1.3 X 10(5) cell. These results indicate that there is an over-expression of EGF/EGF receptors in pancreatic carcinomas, and that an autocrine regulatory mechanism may exist in the growth-promoting effect of EGF on tumor cells

  9. Insulin/insulin like growth factors in cancer: new roles for the aryl hydrocarbon receptor, tumor resistance mechanisms and new blocking strategies

    Directory of Open Access Journals (Sweden)

    Travis B Salisbury

    2015-02-01

    Full Text Available The insulin-like growth factor 1 receptor (IGF1R and the insulin receptor (IR are receptor tyrosine kinases (RTKs that are expressed in cancer cells. The results of different studies indicate that tumor proliferation and survival is dependent on the IGF1R and IR, and that their inhibition leads to reductions in proliferation and increases in cell death. Molecular targeting therapies that have been used in solid tumors include: anti-IGF1R antibodies, anti-IGF1/IGF2 antibodies and small molecule inhibitors that suppress IGF1R and IR kinase activity. New advances in the molecular basis of anti-IGF1R blocking antibodies reveal they are biased agonists and promote the binding of IGF1 to integrin β3 receptors in some cancer cells. Our recent reports indicate that pharmacological aryl hydrocarbon receptor (AHR ligands inhibit breast cancer cell responses to IGFs, suggesting that targeting AHR may have benefit in cancers whose proliferation and survival are dependent on insulin/IGF signaling. Novel aspects of IGF1R/IR in cancer, such as biased agonism, integrin β3 signaling, AHR and new therapeutic targeting strategies will be discussed.

  10. 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.

  11. Expression of insulin-like growth factor-1 and insulin-like growth factor-1 receptors in EL4 lymphoma cells overexpressing growth hormone.

    Science.gov (United States)

    Weigent, Douglas A; Arnold, Robyn E

    2005-03-01

    Almost all of the previous studies with growth hormone (GH) have been done with exogenously supplied GH and, therefore, involve actions of the hormone through its receptor. However, the actions of endogenous or lymphocyte GH are still unclear. In a previous study, we showed that overexpression of GH (GHo) in a lymphoid cell line resulted in protection of the cells to apoptosis mediated by nitric oxide (NO). In the present study, we show that the protection from apoptosis could be transferred to control cells with culture fluids obtained from GHo cells and blocked by antibodies to the insulin-like growth factor-1 (IGF-1) or antibodies to the IGF-1-receptor (IGF-1R). Northern and Western blot analysis detected significantly higher levels of IGF-1 in cells overexpressing GH. An increase in the expression of the IGF-1R in GHo cells was also detected by Western blot analysis, (125)I-IGF-1 binding and analysis of IGF-1R promoter luciferase constructs. Transfection of GHo cells with a dominant negative IGF-1R mutant construct blocked the generation of NO and activation of Akt seen in GHo cells compared to vector alone control EL4 cells. The results suggest that one of the consequences of the overexpression of GH, in cells lacking the GH receptor, is an increase in the expression of IGF-1 and the IGF-1R which mediate the protection of EL4 lymphoma cells from apoptosis.

  12. Metal oxide nanosensors using polymeric membranes, enzymes and antibody receptors as ion and molecular recognition elements.

    Science.gov (United States)

    Willander, Magnus; Khun, Kimleang; Ibupoto, Zafar Hussain

    2014-05-16

    The concept of recognition and biofunctionality has attracted increasing interest in the fields of chemistry and material sciences. Advances in the field of nanotechnology for the synthesis of desired metal oxide nanostructures have provided a solid platform for the integration of nanoelectronic devices. These nanoelectronics-based devices have the ability to recognize molecular species of living organisms, and they have created the possibility for advanced chemical sensing functionalities with low limits of detection in the nanomolar range. In this review, various metal oxides, such as ZnO-, CuO-, and NiO-based nanosensors, are described using different methods (receptors) of functionalization for molecular and ion recognition. These functionalized metal oxide surfaces with a specific receptor involve either a complex formation between the receptor and the analyte or an electrostatic interaction during the chemical sensing of analytes. Metal oxide nanostructures are considered revolutionary nanomaterials that have a specific surface for the immobilization of biomolecules with much needed orientation, good conformation and enhanced biological activity which further improve the sensing properties of nanosensors. Metal oxide nanostructures are associated with certain unique optical, electrical and molecular characteristics in addition to unique functionalities and surface charge features which shows attractive platforms for interfacing biorecognition elements with effective transducing properties for signal amplification. There is a great opportunity in the near future for metal oxide nanostructure-based miniaturization and the development of engineering sensor devices.

  13. Metal Oxide Nanosensors Using Polymeric Membranes, Enzymes and Antibody Receptors as Ion and Molecular Recognition Elements

    Directory of Open Access Journals (Sweden)

    Magnus Willander

    2014-05-01

    Full Text Available The concept of recognition and biofunctionality has attracted increasing interest in the fields of chemistry and material sciences. Advances in the field of nanotechnology for the synthesis of desired metal oxide nanostructures have provided a solid platform for the integration of nanoelectronic devices. These nanoelectronics-based devices have the ability to recognize molecular species of living organisms, and they have created the possibility for advanced chemical sensing functionalities with low limits of detection in the nanomolar range. In this review, various metal oxides, such as ZnO-, CuO-, and NiO-based nanosensors, are described using different methods (receptors of functionalization for molecular and ion recognition. These functionalized metal oxide surfaces with a specific receptor involve either a complex formation between the receptor and the analyte or an electrostatic interaction during the chemical sensing of analytes. Metal oxide nanostructures are considered revolutionary nanomaterials that have a specific surface for the immobilization of biomolecules with much needed orientation, good conformation and enhanced biological activity which further improve the sensing properties of nanosensors. Metal oxide nanostructures are associated with certain unique optical, electrical and molecular characteristics in addition to unique functionalities and surface charge features which shows attractive platforms for interfacing biorecognition elements with effective transducing properties for signal amplification. There is a great opportunity in the near future for metal oxide nanostructure-based miniaturization and the development of engineering sensor devices.

  14. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials

    DEFF Research Database (Denmark)

    Voldborg, B R; Damstrup, L; Spang-Thomsen, M

    1997-01-01

    The epidermal growth factor receptor (EGFR) is a growth factor receptor that induces cell differentiation and proliferation upon activation through the binding of one of its ligands. The receptor is located at the cell surface, where the binding of a ligand activates a tyrosine kinase in the intr...... aspects of therapeutic targeting of EGFR....

  15. Engineering chimeric human and mouse major histocompatibility complex (MHC) class I tetramers for the production of T-cell receptor (TCR) mimic antibodies

    Science.gov (United States)

    Bentley, Carol; Yates, Jenna; Salimi, Maryam; Greig, Jenny; Wiblin, Sarah; Hassanali, Tasneem; Banham, Alison H.

    2017-01-01

    Therapeutic monoclonal antibodies targeting cell surface or secreted antigens are among the most effective classes of novel immunotherapies. However, the majority of human proteins and established cancer biomarkers are intracellular. Peptides derived from these intracellular proteins are presented on the cell surface by major histocompatibility complex class I (MHC-I) and can be targeted by a novel class of T-cell receptor mimic (TCRm) antibodies that recognise similar epitopes to T-cell receptors. Humoural immune responses to MHC-I tetramers rarely generate TCRm antibodies and many antibodies recognise the α3 domain of MHC-I and β2 microglobulin (β2m) that are not directly involved in presenting the target peptide. Here we describe the production of functional chimeric human-murine HLA-A2-H2Dd tetramers and modifications that increase their bacterial expression and refolding efficiency. These chimeric tetramers were successfully used to generate TCRm antibodies against two epitopes derived from wild type tumour suppressor p53 (RMPEAAPPV and GLAPPQHLIRV) that have been used in vaccination studies. Immunisation with chimeric tetramers yielded no antibodies recognising the human α3 domain and β2m and generated TCRm antibodies capable of specifically recognising the target peptide/MHC-I complex in fully human tetramers and on the cell surface of peptide pulsed T2 cells. Chimeric tetramers represent novel immunogens for TCRm antibody production and may also improve the yield of tetramers for groups using these reagents to monitor CD8 T-cell immune responses in HLA-A2 transgenic mouse models of immunotherapy. PMID:28448627

  16. alpha-MSH and its receptors in regulation of tumor necrosis factor-alpha production by human monocyte/macrophages.

    Science.gov (United States)

    Taherzadeh, S; Sharma, S; Chhajlani, V; Gantz, I; Rajora, N; Demitri, M T; Kelly, L; Zhao, H; Ichiyama, T; Catania, A; Lipton, J M

    1999-05-01

    The hypothesis that macrophages contain an autocrine circuit based on melanocortin [ACTH and alpha-melanocyte-stimulating hormone (alpha-MSH)] peptides has major implications for neuroimmunomodulation research and inflammation therapy. To test this hypothesis, cells of the THP-1 human monocyte/macrophage line were stimulated with lipopolysaccharide (LPS) in the presence and absence of alpha-MSH. The inflammatory cytokine tumor necrosis factor (TNF)-alpha was inhibited in relation to alpha-MSH concentration. Similar inhibitory effects on TNF-alpha were observed with ACTH peptides that contain the alpha-MSH amino acid sequence and act on melanocortin receptors. Nuclease protection assays indicated that expression of the human melanocortin-1 receptor subtype (hMC-1R) occurs in THP-1 cells; Southern blots of RT-PCR product revealed that additional subtypes, hMC-3R and hMC-5R, also occur. Incubation of resting macrophages with antibody to hMC-1R increased TNF-alpha concentration; the antibody also markedly reduced the inhibitory influence of alpha-MSH on TNF-alpha in macrophages treated with LPS. These results in cells known to produce alpha-MSH at rest and to increase secretion of the peptide when challenged are consistent with an endogenous regulatory circuit based on melanocortin peptides and their receptors. Targeting of this neuroimmunomodulatory circuit in inflammatory diseases in which myelomonocytic cells are prominent should be beneficial.

  17. Production and properties of monoclonal antibodies to human blood coagulation factor VII and factor VIIa

    International Nuclear Information System (INIS)

    Mann, P.; Nesbitt, J.A.; Ge, M.; Kisiel, W.

    1986-01-01

    Human factor VII is a trace vitamin K-dependent protein that circulates in blood as a single-chain precursor to a serine protease. Upon activation, two-chain factor VIIa activates factor x in the presence of tissue factor and calcium. Purified preparations of single-chain (SC) human factor VII and two-chain (TC) factor VIIa were utilized to immunize Balb/c mice. Spleen cells from these immunized mice were fused to a non-secreting NS-1 derivative of X63-Ag8 myeloma cells and grown in selective medium. Analysis of culture supernatants by EIA revealed several hybridomas that were secreting IgG specific for Sc-factor VII and TC-factor VIIa. In addition, several hybridomas secreted IgG that reacted equally well with factor VII and factor VIIa. One of the latter McAb (A-29) reacted with the heavy chain of factor VIIa and the intact factor VII molecule equally as judged by Western blotting. A-29 was produced in ascites fluid, purified and coupled to activated CH-Sepharose. Application of one liter of normal human plasma to 10 ml of this immunoadsorbent column, elution of factor VII and subsequent Western blot using 125 I-rabbit anti-human factor VII indicated a single species of factor VII(M/sub r/ = 50 KDa) in normal plasma. These specific factor VII/VIIa McAbs may prove useful in the analysis of these factors, and in the separation of SC-factor VII from TC-factor VIIa

  18. Fibroblast growth factor receptor 4 (FGFR4) and fibroblast growth factor 19 (FGF19) autocrine enhance breast cancer cells survival.

    Science.gov (United States)

    Tiong, Kai Hung; Tan, Boon Shing; Choo, Heng Lungh; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Tan, Si Hoey; Khor, Nelson Tze Woei; Wong, Shew Fung; See, Sze-Jia; Tan, Yuen-Fen; Rosli, Rozita; Cheong, Soon-Keng; Leong, Chee-Onn

    2016-09-06

    Basal-like breast cancer is an aggressive tumor subtype with poor prognosis. The discovery of underlying mechanisms mediating tumor cell survival, and the development of novel agents to target these pathways, is a priority for patients with basal-like breast cancer. From a functional screen to identify key drivers of basal-like breast cancer cell growth, we identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of cell survival. We found that FGFR4 mediates cancer cell survival predominantly via activation of PI3K/AKT. Importantly, a subset of basal-like breast cancer cells also secrete fibroblast growth factor 19 (FGF19), a canonical ligand specific for FGFR4. siRNA-mediated silencing of FGF19 or neutralization of extracellular FGF19 by anti-FGF19 antibody (1A6) decreases AKT phosphorylation, suppresses cancer cell growth and enhances doxorubicin sensitivity only in the FGFR4+/FGF19+ breast cancer cells. Consistently, FGFR4/FGF19 co-expression was also observed in 82 out of 287 (28.6%) primary breast tumors, and their expression is strongly associated with AKT phosphorylation, Ki-67 staining, higher tumor stage and basal-like phenotype. In summary, our results demonstrated the presence of an FGFR4/FGF19 autocrine signaling that mediates the survival of a subset of basal-like breast cancer cells and suggest that inactivation of this autocrine loop may potentially serve as a novel therapeutic intervention for future treatment of breast cancers.

  19. Comparison between sensitivity of autologous skin serum test and autologous plasma skin test in patients with Chronic Idiopathic Urticaria for detection of antibody against IgE or IgE receptor (FcεRIα).

    Science.gov (United States)

    Sajedi, Vahid; Movahedi, Masoud; Aghamohammadi, Asghar; Aghamohamadi, Asghar; Gharagozlou, Mohammad; Ghareguzlou, Mohammad; Shafiei, Alireza; Soheili, Habib; Sanajian, Nahal

    2011-06-01

    Intradermal injection of autologous serum and plasma elicit a cutaneous reactivity in almost 45-60% of patients with Chronic Idiopathic Urticaria (CIU). This reactivity is associated with the presence of auto antibodies against IgE or IgE receptors. This study was carried out to compare the cutaneous reactivity of autologous serum and plasma skin tests in a series of patients with CIU for diagnosis of auto antibodies against IgE or IgE receptor. Fifty eight patients with CIU were injected intradermally with autologous serum and plasma (anticoagulated by citrate). Histamine was used as positive control and normal saline as negative control. The study group was checked by routine laboratory tests (CBC, U/A etc), allergens with skin prick tests, and serum IgE level, and auto antibodies against thyroid as well. Duration of urticaria was another factor which was assessed.There was no significant difference between positive ASST and positive APST patients for the above mentioned tests. 77.6% of the patients were Positive for APST and 65.5% were ASST positive. Duration of urticaria was longer in patients with positive ASST and APST than ASST and APST negative patients, although the difference was not statistically significant.Autologus serum skin test (ASST) and autologous plasma skin test (APST) could be used for estimation of duration and severity of urticaria and planning for the treatment.

  20. Heterophilic antibody interference affecting multiple hormone assays: Is it due to rheumatoid factor?

    Science.gov (United States)

    Mongolu, Shiva; Armston, Annie E; Mozley, Erin; Nasruddin, Azraai

    2016-01-01

    Assay interference with heterophilic antibodies has been well described in literature. Rheumatoid factor is known to cause similar interference leading to falsely elevated hormone levels when measured by immunometric methods like enzyme-linked immunosorbent assay (ELISA) or multiplex immunoasays (MIA). We report a case of a 60-year-old male patient with a history of rheumatoid arthritis referred to our endocrine clinic for investigation of hypogonadism and was found to have high serum levels of LH, FSH, SHBG, Prolactin, HCG and TSH. We suspected assay interference and further tests were performed. We used Heteroblock tubes and PEG precipitation to eliminate the interference and the hormone levels post treatment were in the normal range. We believe the interference was caused by high serum levels of rheumatoid factor. Although he was treated with thyroxine for 3 years, we believe he may have been treated inappropriately as his Free T4 level was always normal despite high TSH due to assay interference. Our case illustrates the phenomenon of heterophilic antibody interference likely due to high levels of rheumatoid factor. It is essential for clinicians and endocrinologists in particular to be aware of this possibility when making treatment decisions in these groups of patients.

  1. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

    International Nuclear Information System (INIS)

    Andersen, A.S.; Kjeldsen, T.; Wiberg, F.C.; Christensen, P.M.; Rasmussen, J.S.; Norris, K.; Moeller, K.B.; Moeller, N.P.H.

    1990-01-01

    To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the α-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor

  2. Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation

    International Nuclear Information System (INIS)

    Diermeier, Simone; Horvath, Gabor; Knuechel-Clarke, Ruth; Hofstaedter, Ferdinand; Szoellosi, Janos; Brockhoff, Gero

    2005-01-01

    heterointeractions, as well as the presence or absence of growth factors. C-erbB2 overexpression alone is insufficient to predict the impact of growth factors and antibodies on cell proliferation. The optimization and specification of therapeutic approaches based on erbB-receptor targeting requires to account for EGFR coexpression as well as the potential presence of erbB-receptor relevant growth factors

  3. The epidermal growth factor receptor as a target for gastrointestinal cancer therapy.

    Science.gov (United States)

    Tedesco, Karen L; Lockhart, A Craig; Berlin, Jordan D

    2004-10-01

    The epidermal growth factor receptor (EGFR) is a member of the family of transmembrane protein kinase receptors known as the erbB or HER receptor family. When activated, EGFR phosphorylates and activates other intracellular proteins that affect cell signaling pathways, cellular proliferation, control of apoptosis and angiogenesis. EGFR signaling is best thought of as a network of activating and inactivating proteins with EGFR as the entry point into the network. EGFR overexpression occurs in most GI malignancies and while data are not entirely consistent, EGFR overexpression often confers a poor prognosis in those GI malignancies that have been studied. It often correlates with poorly differentiated histology, more advanced stage and other known poor prognostic markers. The EGFR is a tempting target because of its presence and overexpression on so many tumor types. However, downstream of the EGFR are several proteins that may be activated without EGFR thus allowing blockade to be overcome. Therefore, while blocking the activity of the EGFR protein appears to be a promising anticancer strategy, a simplistic strategy of blocking only EGFR is likely to only impact a minority of patients. It is time for the laboratory and clinical researchers to work closely together to develop this treatment strategy, moving back and forth from clinical to laboratory to best understand how to block this network effectively enough to produce a broader antitumor effect. While multiple methods of targeting the EGFR pathway are under development, including the inhibition of downstream proteins, only two modalities have entered clinical trials in GI malignancies: small molecule inhibitors of the intracellular kinase domain of EGFR and antibodies designed to block the extracellular ligand-binding domain of EGFR. EGFR inhibitors are still experimental in every GI malignancy with the notable exception of cetuximab that is approved for second or third-line therapy of metastatic colorectal

  4. Targeting the epidermal growth factor receptor in radiotherapy: radiobiological mechanisms, preclinical and clinical results

    International Nuclear Information System (INIS)

    Baumann, Michael; Krause, Mechthild

    2004-01-01

    Background and purpose: Inhibition of the epidermal growth factor receptor (EGFR) is a fastly developing field in preclinical and clinical cancer research. This review presents the current status of knowledge and discusses radiobiological mechanisms which may underly the efficacy of EGFR inhibitors combined with irradiation. Materials and methods: Preclinical and clinical results on combined targeting of the EGFR and irradiation from the literature and from this laboratory are reviewed. Focus is given to the radiobiological rationale of this approach and to endpoints of experimental radiotherapy. Results: Overexpression of the EGFR is associated with decreased local tumour control after radiotherapy, especially when the overall treatment time is long. Inhibition of the EGFR either alone or in combination with irradiation decreases the growth rate of tumours expressing this receptor. Preclinical data provide proof-of-principle that local tumour control may be improved by combining irradiation with C225 mAb. In a randomised phase III clinical trial, simultaneous irradiation and treatment with the EGFR antibody Cetuximab (Erbitux[reg]; C225) in head and neck cancer patients resulted in significantly improved locoregional tumour control and survival compared to curative irradiation alone. Acute skin reactions increased in the experimental arm. The underlying mechanisms of enhanced radiation effects of combined EGFR inhibition with irradiation and of the partly conflicting results in different studies are poorly understood. There is increasing evidence, that important intertumoral heterogeneity in the response to EGFR inhibition alone and combined with irradiation exists, which appears to be at least partly dependent on specific mutations of the receptor as well as of molecules that are involved in the intracellular signal transduction pathway. Conclusions and outlook: Further investigations at all levels of the translational research chain exploring the mechanisms of

  5. Compensatory insulin receptor (IR) activation on inhibition of insulin-like growth factor-1 receptor (IGF-1R): rationale for cotargeting IGF-1R and IR in cancer.

    Science.gov (United States)

    Buck, Elizabeth; Gokhale, Prafulla C; Koujak, Susan; Brown, Eric; Eyzaguirre, Alexandra; Tao, Nianjun; Rosenfeld-Franklin, Maryland; Lerner, Lorena; Chiu, M Isabel; Wild, Robert; Epstein, David; Pachter, Jonathan A; Miglarese, Mark R

    2010-10-01

    Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase-AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti-IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti-IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone.

  6. Exposure to Folate Receptor Alpha Antibodies during Gestation and Weaning Leads to Severe Behavioral Deficits in Rats: A Pilot Study.

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    Jeffrey M Sequeira

    Full Text Available The central nervous system continues to develop during gestation and after birth, and folate is an essential nutrient in this process. Folate deficiency and folate receptor alpha autoantibodies (FRα-AuAb have been associated with pregnancy-related complications and neurodevelopmental disorders. In this pilot study, we investigated the effect of exposure to FRα antibodies (Ab during gestation (GST, the pre-weaning (PRW, and the post weaning (POW periods on learning and behavior in adulthood in a rat model. In the open field test and novel object recognition task, which examine locomotor activity and anxiety-like behavior, deficits in rats exposed to Ab during gestation and pre-weaning (GST+PRW included more time spent in the periphery or corner areas, less time in the central area, frequent self-grooming akin to stereotypy, and longer time to explore a novel object compared to a control group; these are all indicative of increased levels of anxiety. In the place avoidance tasks that assess learning and spatial memory formation, only 30% of GST+PRW rats were able to learn the passive place avoidance task. None of these rats learned the active place avoidance task indicating severe learning deficits and cognitive impairment. Similar but less severe deficits were observed in rats exposed to Ab during GST alone or only during the PRW period, suggesting the extreme sensitivity of the fetal as well as the neonatal rat brain to the deleterious effects of exposure to Ab during this period. Behavioral deficits were not seen in rats exposed to antibody post weaning. These observations have implications in the pathology of FRα-AuAb associated with neural tube defect pregnancy, preterm birth and neurodevelopmental disorders including autism.

  7. Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

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    Bipulendu Jena

    Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  8. Characterisation of anifrolumab, a fully human anti-interferon receptor antagonist antibody for the treatment of systemic lupus erythematosus

    Science.gov (United States)

    Rajan, Bhargavi; Zerrouki, Kamelia; Karnell, Jodi L; Sagar, Divya; Vainshtein, Inna; Farmer, Erika; Rosenthal, Kimberly; Morehouse, Chris; de los Reyes, Melissa; Schifferli, Kevin; Liang, Meina; Sanjuan, Miguel A; Sims, Gary P; Kolbeck, Roland

    2018-01-01

    Objective We investigated the mechanistic and pharmacological properties of anifrolumab, a fully human, effector-null, anti-type I interferon (IFN) alpha receptor 1 (IFNAR1) monoclonal antibody in development for SLE. Methods IFNAR1 surface expression and internalisation on human monocytes before and after exposure to anifrolumab were assessed using confocal microscopy and flow cytometry. The effects of anifrolumab on type I IFN pathway activation were assessed using signal transducer and activator of transcription 1 (STAT1) phosphorylation, IFN-stimulated response element–luciferase reporter cell assays and type I IFN gene signature induction. The ability of anifrolumab to inhibit plasmacytoid dendritic cell (pDC) function and plasma cell differentiation was assessed by flow cytometry and ELISA. Effector-null properties of anifrolumab were assessed in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays with B cells. Results Anifrolumab reduced cell surface IFNAR1 by eliciting IFNAR1 internalisation. Anifrolumab blocked type I IFN-dependent STAT1 phosphorylation and IFN-dependent signalling induced by recombinant and pDC-derived type I IFNs and serum of patients with SLE. Anifrolumab suppressed type I IFN production by blocking the type I IFN autoamplification loop and inhibited proinflammatory cytokine induction and the upregulation of costimulatory molecules on stimulated pDCs. Blockade of IFNAR1 suppressed plasma cell differentiation in pDC/B cell co-cultures. Anifrolumab did not exhibit CDC or ADCC activity. Conclusions Anifrolumab potently inhibits type I IFN-dependent signalling, including the type I IFN autoamplification loop, and is a promising therapeutic for patients with SLE and other diseases that exhibit chronic dysfunctional type I IFN signalling. PMID:29644082

  9. Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis.

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    Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Ootsubo, Michiko; Izawa, Ken-ichi; Kohroki, Junya; Masuho, Yasuhiko

    2016-01-01

    The Fc domain of human IgG1 binds to Fcγ receptors (FcγRs) to induce effector functions such as phagocytosis. There are four interchain disulfide bonds between the H and L chains. In this study, the disulfide bonds within the IgG1 trastuzumab (TRA), which is specific for HER2, were cleaved by mild S-sulfonation or by mild reduction followed by S-alkylation with three different reagents. The cleavage did not change the binding activities of TRA to HER2-bearing SK-BR-3 cells. The binding activities of TRA to FcγRIIA and FcγRIIB were greatly enhanced by modification with mild reduction and S-alkylation with ICH2CONH2 or N-(4-aminophenyl) maleimide, while the binding activities of TRA to FcγRI and FcγRIIIA were decreased by any of the four modifications. However, the interchain disulfide bond cleavage by the different modifications did not change the antibody-dependent cell-mediated phagocytosis (ADCP) of SK-BR-3 cells by activated THP-1 cells. The order of FcγR expression levels on the THP-1 cells was FcγRII > FcγRI > FcγRIII and ADCP was inhibited by blocking antibodies against FcγRI and FcγRII. These results imply that the effect of the interchain disulfide bond cleavage on FcγRs binding and ADCP is dependent on modifications of the cysteine residues and the FcγR isotypes. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  10. Seroprevalence of Ehrlichia ruminantium antibodies and its associated risk factors in indigenous goats of South Africa.

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    Mdladla, Khanyisile; Dzomba, Edgar F; Muchadeyi, Farai C

    2016-03-01

    The present study investigated the seroprevalence of antibodies to Ehrlichia ruminantium and the associated risk factors in goats from five different farming provinces of South Africa. Sera collected from 686 goats of the commercial meat type (n=179), mohair type (n=9), non-descript indigenous goats from Eastern Cape (n=56), KwaZulu-Natal (n=209), Limpopo (n=111), North West (n=61) and Northern Cape (n=11) provinces and a feral Tankwa goat (n=50) were tested for the presence of immunoglobulin G (IgG) antibodies to antigens of E. ruminantium using the indirect fluorescent-antibody test (IFAT). Fifty two percent of these goats had ticks. The overall seroprevalence of antibodies to E. ruminantium was 64.87% (445/686) with the highest seroprevalence reported for Limpopo (95.50%) and lowest for Northern Cape (20.29%). Highest seroprevalence for antibodies to E. ruminantium was observed in goats from endemic regions (76.09%), and from smallholder production systems (89.54%). High seroprevalence was also observed in non-descript indigenous goats (85.04%), adult goat (69.62%), in does (67.46%) and goats infested with ticks (85.79%). The logistic model showed a gradient of increasing risk for commercial meat type Savanna (OR=3.681; CI=1.335-10.149) and non-descript indigenous (OR=3.466; CI=1.57-7.645) compared to Boer goats and for goats from the smallholder production system (OR=2.582; CI=1.182-5.639) and those with ticks (OR=3.587; CI=2.105-6.112). Results from this study showed that E. ruminantium infections were prevalent but were widely and unevenly distributed throughout South Africa. Findings from the study facilitate identification and mapping of risk areas for heartwater and its endeminicity in South Africa and should be taken into consideration for future disease control strategies and local goat improvement programs. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. TITERS OF ANTIBODIES TO Β1-ADRENOCEPTOR AND M2 CHOLINERGIC RECEPTORS IN PATIENTS WITH VENTRICULAR ARRHYTHMIAS WITHOUT AN ORGANIC CARDIOVASCULAR DISEASE AND THEIR POSSIBLE CLINICAL SIGNIFICANCE

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    M. M. Rogova

    2012-01-01

    Full Text Available Aim. To identify the most promising epitopes that simulate various sites β1-adrenergic and M2-cholinergic receptors, and to evaluate their possible contribution to the development and maintenance of cardiac arrhythmias, particularly idiopathic ventricular arrhythmia. Material and methods. Patients with ventricular arrhythmias without organic cardiovascular disease (the study group; n=70 were included in the study. The control group consisted of 20 healthy volunteers. Evaluation of levels of antibodies to antigenic determinants, modeling various sites β1-adrenergic and M2-cholinergic performed in all patients. Causal treatment with clarithromycin and valacyclovir performed in part of patients. Results. Antibodies to different peptide sequences of β1-adrenergic and M2-cholinergic receptors have been identified in 25% of main group patients. A direct correlation between the frequency of episodes of ventricular tachycardia and IgG levels to MRI-MRIV (p=0.02 revealed. Increase in titre of antibodies to β1-adrenoceptors, to a peptide sequence β8 (p=0.02, and lower titers of antibodies to the M2 acetylcholine receptor — chimera MRI-MRIV IgM (p=0.06 and ARI-MRIV IgM (p=0.07 were observed when assessing the efficacy of the therapy in the causal dynamics in the group of "untreated" patients. IgG titer reduction of ARI-MRIV (p=0.02, which is 4 times out of 10 with reduction of ventricular ectopic activity , recorded after valacyclovir therapy. Clarithromycin therapy on the level of antibodies exerted no significant effect. Conclusion. Possible involvement of antibodies to β1-adrenoceptor and M2-cholinergic receptors in the development of idiopathic ventricular arrhythmias demonstrated. The relationship between the frequency of episodes of ventricular tachycardia and levels of antibody titers to M2-cholinergic receptors found. Attempt of causal treatment, depending on the possible mechanisms of the autoimmune process is executed. Further studies to

  12. [Analysis of epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome].

    Science.gov (United States)

    Tsuboi, Hiroto; Matsuo, Naomi; Iizuka, Mana; Nakamura, Yumi; Matsumoto, Isao; Sumida, Takayuki

    2010-01-01

    Sjögren's syndrome (SS) is an autoimmune disease that affects exocrine glands including salivary and lacrimal glands. It is characterized by lymphocytic infiltration into exocrine glands, leading to dry mouth and eyes. A number of auto-antibodies, such as anti-SS-A and SS-B antibodies, are detected in patients with SS. However, no SS-specific pathologic auto-antibodies have yet been found in this condition. M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with SS carried inhibitory auto-antibodies against M3R. To clarify the epitopes and function of anti-M3R antibodies in SS, we examined antibodies to the extracellular domains (N terminal region, the first, second, and third extracellular loop) of M3R by ELISA using synthesized peptide antigens encoding these domains in 42 SS and 42 healthy controls (HC). Titers and positivity of anti-M3R antibodies to every extracellular domain of M3R were significantly higher in SS than in HC. For functional analysis, human salivary gland (HSG) cells were pre-cultured with IgG from anti-M3R antibodies positive SS, negative SS, and HC. HSG cells were stimulated with cevimeline hydrochloride and intracellular calcium concentration ([Ca(2+)](i)) was measured. IgG from anti-M3R antibodies to the second loop positive SS inhibited the increase of [Ca(2+)](i), but IgG from antibodies to the N terminal or the first loop positive SS enhanced it, while IgG from antibodies to the third loop positive SS showed no effect on [Ca(2+)](i) as well as IgG from anti-M3R antibodies negative SS and HC. These findings indicated the presence of several B cell epitopes on M3R in SS and effect of anti-M3R antibodies on the salivary secretion might differ with these epitopes.

  13. Ex-vivo expanded human NK cells express activating receptors that mediate cytotoxicity of allogeneic and autologous cancer cell lines by direct recognition and antibody directed cellular cytotoxicity

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    Campana Dario

    2010-10-01

    Full Text Available Abstract Background The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i the small number of NK cells in peripheral blood, (ii the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii the need to activate the NK cells in order to induce NK cell mediated killing and (iv the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii their ability to kill allogeneic and autologous tumor targets by direct cytotoxitiy and by antibody-mediated cellular cytotoxicity and (iii defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing. Methods Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated. Results Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors. Conclusion These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical

  14. Preparation and functional studies of hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody

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    Yang J

    2014-05-01

    Full Text Available Jingjing Yang,1,3,* Xiaoping Huang,1,3,* Fanghong Luo,1 Xiaofeng Cheng,3 Lianna Cheng,3 Bin Liu,4 Lihong Chen,2 Ruyi Hu,1,3 Chunyan Shi,1,3 Guohong Zhuang,1,3 Ping Yin2 1Anti-Cancer Research Center, Medical College, Xiamen University, Fujian, People's Republic of China, 2The Department of Pathology, Zhongshan Hospital, Xiamen University, Xiamen, People's Republic of China, 3Organ transplantation institution, Xiamen University, Xiamen, People's Republic of China, 4Jilin Vocational College of Industry and Technology, Jilin, People's Republic of China  *These authors contributed equally to this work Objective: To prepare hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody, and study their characteristics, functions, and mechanisms of action. Materials and methods: The anti-human death receptor 5 single-chain antibody was constructed and expressed. Protein-loaded hydroxyethyl chitosan nanoparticles were prepared, and their size, morphology, particle-size distribution and surface zeta potential were measured by scanning electron microscopy and laser particle-size analysis. Mouse H22 hepatocellular carcinoma cells were cultured, and growth inhibition was examined using the CellTiter-Blue cell-viability assay. Flow cytometry and Hoechst 33342 were employed to measure cell apoptosis. Kunming mice with H22 tumor models were treated with protein-loaded hydroxyethyl chitosan nanoparticles, and their body weight and tumor size were measured, while hematoxylin and eosin staining was used to detect antitumor effects in vivo and side effects from tumors. Results: The protein-loaded hydroxyethyl chitosan nanoparticles had good stability; the zeta potential was -24.2±0.205, and the dispersion index was 0.203. The inhibition of the protein-loaded hydroxyethyl chitosan nanoparticles on H22 growth was both time- and dose-dependent. Increased expressions of active caspase 8, active caspase 3, and BAX were detected

  15. Development and Characterization of a Camelid Single Domain Antibody–Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2

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    Baomin Tian

    2017-08-01

    Full Text Available Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor, which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFRs. In this study, we describe the development and characterization of V21-DOS47, an immunoconjugate that targets VEGFR2. V21-DOS47 is composed of a camelid single domain anti-VEGFR2 antibody (V21 and the enzyme urease. The conjugate specifically binds to VEGFR2 and urease converts endogenous urea into ammonia, which is toxic to tumor cells. Previously, we developed a similar antibody–urease conjugate, L-DOS47, which is currently in clinical trials for non-small cell lung cancer. Although V21-DOS47 was designed from parameters learned from the generation of L-DOS47, additional optimization was required to produce V21-DOS47. In this study, we describe the expression and purification of two versions of the V21 antibody: V21H1 and V21H4. Each was conjugated to urease using a different chemical cross-linker. The conjugates were characterized by a panel of analytical techniques, including SDS-PAGE, size exclusion chromatography, Western blotting, and LC-MSE peptide mapping. Binding characteristics were determined by ELISA and flow cytometry assays. To improve the stability of the conjugates at physiologic pH, the pIs of the V21 antibodies were adjusted by adding several amino acid residues to the C-terminus. For V21H4, a terminal cysteine was also added for use in the conjugation chemistry. The modified V21 antibodies were expressed in the E. coli BL21 (DE3 pT7 system. V21H1 was conjugated to urease using the heterobifunctional cross-linker succinimidyl-[(N-maleimidopropionamido-diethyleneglycol] ester (SM(PEG2, which targets lysine resides in the antibody. V21H4 was conjugated to urease using the homobifunctional cross-linker, 1,8-bis

  16. Kinetics of platelets in dogs with thrombocytopenia induced by antiglycoprotein IIb/IIIa receptor monoclonal antibody

    International Nuclear Information System (INIS)

    Hosono, Makoto; Sone, Naoaki; Endo, Keigo; Saga, Tsuneo; Kobayashi, Hisataka; Hosono, Masako N.; Sakahara, Harumi; Yasunaga, Kojiro; Konishi, Junji

    1995-01-01

    To experimentally assess the kinetics of platelets in thrombocytopenia, we constructed a canine model using 111 In-oxine labeled autologous platelets and an intact antiplatelet monoclonal antibody (MAb) NNKY2-11 (IgG2a). With the infusion of radiolabeled autologous platelets into dogs, the peripheral platelet count and blood radioactivity level were examined, and the radioactivity in the liver, spleen and heart was determined with scintigraphic analysis. Thereafter, i.v. injection of 100 μg/kg of NNKY2-11 had no effect on platelet counts or the biodistribution of radiolabeled platelets. However, 200 and 300 μg/kg of MAb reduced the platelets, and the radioactivity of the liver and spleen augmented clearly after injection of MAb. Platelet radioactivity in serum, which had decreased after MAb infusion, did not recover, even when peripheral platelet counts returned to the normal levels, indicating that these new platelets might be derived from the platelet-storage pool or new thrombocytogenesis. This model of antiplatelet MAb induced thrombocytopenia seems to be useful for analyzing the kinetics of platelets in thrombocytopenia

  17. Placental growth factor neutralising antibodies give limited anti-angiogenic effects in an in vitro organotypic angiogenesis model.

    Science.gov (United States)

    Brave, Sandra R; Eberlein, Cath; Shibuya, Masabumi; Wedge, Stephen R; Barry, Simon T

    2010-12-01

    Vascular Endothelial Growth Factor Receptor (VEGFR) mediated signalling drives angiogenesis. This is predominantly attributed to the activity of VEGFR-2 following binding of VEGF-A. Whether other members of the VEGFR and ligand families such as VEGFR-1 and its ligand Placental Growth Factor (PlGF) can also contribute to developmental and pathological angiogenesis is less clear. We explored the function of PlGF in VEGF-A dependent angiogenesis using an in vitro co-culture assay in which endothelial cells are cultured on a fibroblast feeder layer. In the presence of 2% FS MCDB media (containing limited growth factors) in vitro endothelial tube formation is driven by endogenous angiogenic stimuli which are produced by the fibroblast and endothelial cells. Under these conditions independent sequestration of either free VEGF-A or PlGF with polyclonal and monoclonal antibodies inhibited tube formation suggesting that both ligands are required to drive an angiogenic response. Endothelial tube formation could only be driven within this assay by the addition of exogenous VEGF-A, VEGF-E or VEGF-A/PlGF heterodimer, but not by PlGF alone, implying that activation of either VEGFR-2/VEGFR-1 heterodimers or VEGFR-2 homodimers were responsible for eliciting an angiogenic response directly, but not VEGFR-1 homodimers. In contrast to results obtained with an endogenous angiogenic drive, sequestration of PlGF did not affect endothelial tube formation when the assay was driven by 1 ng/ml exogenous VEGF-A. These data suggest that although neutralising PlGF can be shown to reduce endothelial tube formation in vitro, this effect is only observed under restricted culture conditions and is influenced by VEGF-A. Such data questions whether neutralising PlGF would have a therapeutic benefit in vivo in the presence of pathological concentrations of VEGF-A.

  18. Antibodies to watch in 2014.

    Science.gov (United States)

    Reichert, Janice M

    2014-01-01

    Since 2010, mAbs has documented the biopharmaceutical industry's progress in transitioning antibody therapeutics to first Phase 3 clinical studies and regulatory review, and its success at gaining first marketing approvals for antibody-based products. This installment of the "Antibodies to watch" series outlines events anticipated to occur between December 2013 and the end of 2014, including first regulatory actions on marketing applications for vedolizumab, siltuximab, and ramucirumab, as well as the Fc fusion proteins Factor IX-Fc and Factor VIII-Fc; and the submission of first marketing applications for up to five therapeutics (secukinumab, ch14.18, onartuzumab, necitumumab, gevokizumab). Antibody therapeutics in Phase 3 studies are described, with an emphasis on those with study completion dates in 2014, including antibodies targeting interleukin-17a or the interleukin-17a receptor (secukinumab, ixekizumab, brodalumab), proprotein convertase subtilisin/kexin type 9 (alirocumab, evolocumab, bococizumab), and programmed death 1 receptor (lambrolizumab, nivolumab). Five antibodies with US Food and Drug Administration's Breakthrough Therapy designation (obinutuzumab, ofatumumab, lambrolizumab, bimagrumab, daratumumab) are also discussed.

  19. Expression of nerve growth factor and its receptor, tyrosine kinase receptor A, in rooster testes.

    Science.gov (United States)

    Ma, Wei; Wang, Chunqiang; Su, Yuhong; Tian, Yumin; Zhu, Hongyan

    2015-10-01

    Nerve growth factor (NGF), which is required for the survival and differentiation of the nervous system, is also thought to play an important role in the development of mammalian reproductive tissues. To explore the function of NGF in the male reproductive system of non-mammalian animals, we determined the presence of NGF and its receptor, tyrosine kinase receptor A (TrkA), in rooster testes and investigated the regulation of NGF and TrkA expression by follicle-stimulating hormone (FSH). The mRNA and protein levels of NGF and TrkA in 6-week-old rooster testes were lower than those in 12-, 16- or 20-week age groups; levels were highest in the 16-week group. Immunohistochemistry showed that NGF and TrkA were both detected in spermatogonia, spermatocytes and spermatids. NGF immunoreactivity was observed in Leydig cells and strong TrkA signals were present in Sertoli cells. Meanwhile, FSH increased TrkA transcript levels in rooster testes in a dose-dependent manner. We present novel evidence for the developmental and FSH-regulated expression of the NGF/TrkA system, and our findings suggest that the NGF/TrkA system may play a prominent role in chicken spermatogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. The herpes virus Fc receptor gE-gI mediates antibody bipolar bridging to clear viral antigens from the cell surface.

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    Blaise Ndjamen

    2014-03-01

    Full Text Available The Herpes Simplex Virus 1 (HSV-1 glycoprotein gE-gI is a transmembrane Fc receptor found on the surface of infected cells and virions that binds human immunoglobulin G (hIgG. gE-gI can also participate in antibody bipolar bridging (ABB, a process by which the antigen-binding fragments (Fabs of the IgG bind a viral antigen while the Fc binds to gE-gI. IgG Fc binds gE-gI at basic, but not acidic, pH, suggesting that IgG bound at extracellular pH by cell surface gE-gI would dissociate and be degraded in acidic endosomes/lysosomes if endocytosed. The fate of viral antigens associated with gE-gI-bound IgG had been unknown: they could remain at the cell surface or be endocytosed with IgG. Here, we developed an in vitro model system for ABB and investigated the trafficking of ABB complexes using 4-D confocal fluorescence imaging of ABB complexes with transferrin or epidermal growth factor, well-characterized intracellular trafficking markers. Our data showed that cells expressing gE-gI and the viral antigen HSV-1 gD endocytosed anti-gD IgG and gD in a gE-gI-dependent process, resulting in lysosomal localization. These results suggest that gE-gI can mediate clearance of infected cell surfaces of anti-viral host IgG and viral antigens to evade IgG-mediated responses, representing a general mechanism for viral Fc receptors in immune evasion and viral pathogenesis.

  1. Incidence of radiation-induced Graves' disease in patients treated with radioiodine for thyroid autonomy before and after introduction of a high-sensitivity TSH receptor antibody assay

    International Nuclear Information System (INIS)

    Dunkelmann, Simone; Wolf, Ricarda; Koch, Annedore; Kittner, Christian; Groth, Peter; Schuemichen, Carl

    2004-01-01

    Autoimmune hyperthyroidism may occur several months after radioiodine therapy (RIT) for functional thyroid autonomy. Exacerbation of pre-existing subclinical Graves' disease (GD) has been held responsible for this phenomenon. Determination of TSH receptor antibody using solubilised porcine epithelial cell membranes is insensitive and may have failed to diagnose GD in these patients before RIT. Following the introduction of a more sensitive assay, using the human TSH receptor as an antigen, it has been expected that the incidence of radiation-induced GD after RIT for functional thyroid autonomy will be reduced. In a first group of 1,428 patients treated between November 1993 and March 1997 (group I) we used the porcine TRAb assay to exclude GD, while in a second group comprising 1,408 patients treated between January 2000 and December 2001 (group II), GD was excluded using the human TRAb assay. A matched control group of 231 patients was derived from group II. In group I a total of 15 (1.05%) patients developed obvious or suspected radiation-induced GD, while in group II 17 (1.2%) did so; the interval until development of GD was 8.4 and 8.8 months, respectively, after RIT. Serum anti-thyroid peroxidase levels before RIT were elevated in 36.4% of group I patients and 47.1% of group II patients, but in only 5.6% of the control group. Other non-specific signs of mild immunopathy of the thyroid were seen retrospectively in 73.3%, 64.7% and 16.0% of the patients in these three groups, respectively. In conclusion, the introduction of a high-sensitivity TRAb assay did not reduce the incidence of autoimmune hyperthyroidism occurring late after RIT for functional thyroid autonomy, but mild immunopathy of the thyroid is seen more frequently in these patients and seems to be a predisposing factor in the development of radiation-induced GD. (orig.)

  2. Nonneutralizing antibodies against factor VIII and risk of inhibitor development in severe hemophilia A.

    Science.gov (United States)

    Cannavò, Antonino; Valsecchi, Carla; Garagiola, Isabella; Palla, Roberta; Mannucci, Pier Mannuccio; Rosendaal, Frits R; Peyvandi, Flora

    2017-03-09

    The development of anti-factor VIII (FVIII) neutralizing antibodies (inhibitors) is the major complication in hemophilia A. Nonneutralizing antibodies (NNAs) have been detected in hemophilia patients and also in unaffected individuals. The aim of this study was to assess the prevalence of NNAs and to evaluate whether their presence is associated with the development of inhibitors in a cohort of previously untreated or minimally treated patients with hemophilia A; plasma samples of 237 patients with severe hemophilia A enrolled in the SIPPET trial were collected before any exposure to FVIII concentrates and analyzed for the presence of anti-FVIII NNAs. Patients were observed for the development of neutralizing antibodies. NNAs were found in 18 (7.6%) of 237 patients at screening, and there was a clear age gradient. Of those with NNAs, 7 patients subsequently developed an inhibitor for a cumulative incidence of 45.4% (95% confidence interval [CI], 19.5% to 71.3%); among the 219 patients without NNAs, 64 (29%) developed an inhibitor (cumulative incidence, 34.0%; 95% CI, 27.1%-40.9%). In Cox regression analyses, patients with NNAs at screening had an 83% higher incidence of inhibitor development than patients without NNAs (hazard ratio [HR], 1.83; 95% CI, 0.84-3.99). For high-titer inhibitors, the incidence rate had an almost threefold increase (HR, 2.74; 95% CI, 1.23-6.12). These associations did not materially change after adjustment. The presence of anti-FVIII NNAs in patients with severe hemophilia A who were not previously exposed to FVIII concentrates is associated with an increased incidence of inhibitors. © 2017 by The American Society of Hematology.

  3. Nerve growth factor receptor immunostaining suggests an extrinsic origin for hypertrophic nerves in Hirschsprung's disease.

    OpenAIRE

    Kobayashi, H; O'Briain, D S; Puri, P

    1994-01-01

    The expression of nerve growth factor receptor in colon from 20 patients with Hirshsprung's disease and 10 controls was studied immunohistochemically. The myenteric and submucous plexuses in the ganglionic bowel and hypertrophic nerve trunks in the aganglionic bowel displayed strong expression of nerve growth factor receptor. The most important finding was the identical localisation of nerve growth factor receptor immunoreactivity on the perineurium of both hypertrophic nerve trunks in Hirshs...

  4. The negative correlation between thyrotropin receptor-stimulating antibodies and bone mineral density in postmenopausal patients with Graves' disease.

    Science.gov (United States)

    Amashukeli, Medea; Korinteli, Maka; Zerekidze, Tamar; Jikurauli, Nino; Shanava, Shorena; Tsagareli, Marina; Giorgadze, Elen

    2013-06-01

    Graves' disease is an autoimmune disorder with various clinical manifestations. Thyrotropin receptor antibodies (TRAbs), the circulating autoantibodies specific to Graves' disease, are the cause for hyperthyroidism, the most prevalent abnormality. Hyperthyroidism leads to increased bone turnover and a negative bone balance. The aims of the present study were to determine the relationship between TRAbs and bone mineral density (BMD), to assess the extent of BMD change in patients with Graves' disease, and to determine the impact of conservative and surgical therapy on BMD. Fifty female postmenopausal patients with Graves' disease were chosen for this study. Twenty women had a recent diagnosis of Graves' disease, 30 women presented with a compensated disease state after either conservative or surgical treatment, and 30 healthy postmenopausal women served as controls. Thyroid parameters were measured, and BMD values were obtained by dual energy x-ray absorptiometry scan.Femoral neck and lumbar spine BMD and T-scores were significantly lower in newly diagnosed patients compared with the control group, but a difference was not observed between the treated and control groups. Statistical analysis revealed a strong and significant negative correlation between femoral neck and lumbar spine BMD and TRAb values.Both surgical and conservative therapies are effective for restoring BMD in postmenopausal patients with Graves' disease, and the increased level of TRAb can be a useful marker of bone density impairment.

  5. A patient with Graves’ disease showing only psychiatric symptoms and negativity for both TSH receptor autoantibody and thyroid stimulating antibody

    Directory of Open Access Journals (Sweden)

    Hamasaki Hidetaka

    2012-12-01

    Full Text Available Abstract Background Both thyroid stimulating hormone (TSH and thyroid stimulating antibody (TSAb negative Graves’s disease (GD is extremely rare. Here we present such a patient. Case presentation The patient was a 76-year-old woman who was diagnosed as having schizophrenia forty years ago. She did not show characteristic symptoms for hyperthyroidism, such as swelling of thyroid, exophthalmos, tachycardia and tremor, however, she showed only psychomotor agitation. Serum free triiodothyronine and free thyroxine levels were elevated and TSH level was suppressed, suggesting the existence of hyperthyroidism. However, both the first generation TSH receptor autoantibody (TRAb1 and the thyroid stimulating autoantibody (TSAb were negative. Slightly increased blood flow and swelling was detected by thyroid echography. Thyroid scintigraphy demonstrated diffuse and remarkably elevated uptake of 123I uptake. Finally, we diagnosed her as having GD. She was treated by using methimazole, and hyperthyroidism and her psychiatric symptoms were promptly ameliorated. Discussion We experienced a patient with GD who did not show characteristic symptoms except for psychiatric symptoms, and also showed negativity for both TRAb1 and TSAb. Thyroid autoantibody-negative GD is extremely rare. Thyroid scintigraphy was useful to diagnose such a patient.

  6. Detection of thyroid stimulating hormone receptor antibodies (TRAb) by radioreceptor assay (RRA) and enzyme-linked immunosorbent assay (ELISA)

    International Nuclear Information System (INIS)

    Dumrongpisutikul, S.; Tuchinda, S.

    1990-01-01

    Thyroid stimulating hormone receptor antibodies (TRAb) were determined in 100 patients using radioreceptor assay (RRA) and enzyme-linked immunosorbent assay (ELISA). The sensitivity of RRA and ELISA were found to be 70.6% and 88.2% respectively (n=51). The specificity of both assays were 100% (n=16). With RRA as the standard test the sensitivity and specificity of ELISA were 75.8% and 86.8%. In the untreated hyperthyroid the RRA result which expressed as % specific 125 I-TSH inhibition was 33.6% (n=51), decline to 26.9% in the treated hyperthyroid (n=33) and 14.1% in the euthyroid (n=16). The mean 0.D 492nm of TRAb-ELISA were 0.861 in untreated hyperthyroid, 0.437 in treated hyperthyroid and 0.135 in euthyroid Phi coefficient analysis show that the RRA was 60.4% correlated to hyperthyroidism where as TRAb-ELISA was 80.1%

  7. Arctigenin induced gallbladder cancer senescence through modulating epidermal growth factor receptor pathway.

    Science.gov (United States)

    Zhang, Mingdi; Cai, Shizhong; Zuo, Bin; Gong, Wei; Tang, Zhaohui; Zhou, Di; Weng, Mingzhe; Qin, Yiyu; Wang, Shouhua; Liu, Jun; Ma, Fei; Quan, Zhiwei

    2017-05-01

    Gallbladder cancer has poor prognosis and limited therapeutic options. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms involved in the antitumor effect of arctigenin on gallbladder cancer have not been fully elucidated. The expression levels of epidermal growth factor receptor were examined in 100 matched pairs of gallbladder cancer tissues. A positive correlation between high epidermal growth factor receptor expression levels and poor prognosis was observed in gallbladder cancer tissues. Pharmacological inhibition or inhibition via RNA interference of epidermal growth factor receptor induced cellular senescence in gallbladder cancer cells. The antitumor effect of arctigenin on gallbladder cancer cells was primarily achieved by inducing cellular senescence. In gallbladder cancer cells treated with arctigenin, the expression level of epidermal growth factor receptor significantly decreased. The analysis of the activity of the kinases downstream of epidermal growth factor receptor revealed that the RAF-MEK-ERK signaling pathway was significantly inhibited. Furthermore, the cellular senescence induced by arctigenin could be reverted by pcDNA-epidermal growth factor receptor. Arctigenin also potently inhibited the growth of tumor xenografts, which was accompanied by the downregulation of epidermal growth factor receptor and induction of senescence. This study demonstrates arctigenin could induce cellular senescence in gallbladder cancer through the modulation of epidermal growth factor receptor pathway. These data identify epidermal growth factor receptor as a key regulator in arctigenin-induced gallbladder cancer senescence.

  8. Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor

    DEFF Research Database (Denmark)

    Rønne, E; Behrendt, N; Ellis, V

    1991-01-01

    We have raised four monoclonal antibodies recognizing different epitopes within the human cell-surface receptor for urokinase-type plasminogen activator (u-PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro......-u-PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP-inactivated [125I]-u-PA to U937 cells, is directed against the u-PA binding NH2-terminal domain of u-PAR, a well-defined fragment formed by limited chymotrypsin digestion of purified u......-PAR, demonstrating the functional independence of the u-PA binding domain as well as the critical role of u-PAR in the assembly of the cell-surface plasminogen activation system....

  9. [Antibodies against TSH receptors (TRAb) as indicators in prognosing the effectiveness of Tiamazol therapy for Grave's Disease].

    Science.gov (United States)

    Bojarska-Szmygin, Anna; Ciechanek, Roman

    2003-01-01

    The aim of the study was to evaluate the usefulness of TRAb determinations in prognosing and monitoring the efficacy of conservative treatment in Graves' disease. The examinations were performed in 54 patients. During the 18-month observation all the patients were treated with Tiamazol. The control group consisted of 20 healthy volunteers. The TRAb levels were determined before as well as 12 and 18 months after thyrostatic treatment. Simultaneously, the levels of TSH and FT4 were analysed. Moreover, all the patients underwent ultrasound examinations to assess the size of the thyroid gland. The findings of the 18-month follow up showed that in 31 patients (57%) the thyroid function became normal (group I--euthyreosis), in 23 patients (43%) hyperactivity persisted (group II--hyperthyreosis). The TRAb levels were analysed in both groups of patients. An increased initial level of TRAb was found in the hyperactivity group mean -54.39 + 31.21 U/l which was statistically significantly different from the TRAb levels in the euthyreosis group mean -29.13 +/- 19.14 U/l and in controls mean -2.75 +/- 2.06 U/l (p Graves' disease. High initial levels of antibodies are the poor prognostic factors. The TRAb determinations are of some prognostic value not only before but also 12 months since the onset of therapy. The lack of antibody level normalization during treatment is connected with persisting hyperactivity. The TRAb concentration correlates with the thyroid size.

  10. A monoclonal antibody to an early pregnancy factor-induced suppressor factor (EPF-S1) disrupts implantation in mice.

    Science.gov (United States)

    Athanasas-Platsis, S; Hoskin, M J; Rolfe, B E; Cavanagh, A C; Morton, H

    1995-03-01

    The importance of EPF during pregnancy has been established previously but the importance of the EPF-induced suppressor factor EPF-S1 in pregnancy has to date been unaddressed. Investigations were therefore conducted in order to study this. Monoclonal antibodies to EPF-S1 were produced, and one antibody, designated R2T gamma, was characterized. Mated mice were passively immunized with R2T gamma and the effect on implantation determined. Characterization of anti-EPF-S1 R2T gamma revealed that it cross-reacted with EPF-S1 of different MHC restriction but not with EPF or EPF-S2. When injected into mated mice on days 1 to 4, R2T gamma had no effect on pregnancy but when injections continued to day 5, pregnancy was affected; the number of embryos implanted on day 7 were significantly less than the number of corpora lutea counted, signifying embryonic loss. These studies show that anti-EPF-S1 R2T gamma disrupts implantation in mice when injected on days 1 to 5 of pregnancy but not when injected on days 1 to 4, demonstrating that EPF-S1 exerts its effects around the time of implantation.

  11. Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

    Science.gov (United States)

    Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

    1994-05-13

    Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

  12. Targeting Fibroblast Growth Factor 23 Signaling with Antibodies and Inhibitors, Is There a Rationale?

    Directory of Open Access Journals (Sweden)

    Seiji Fukumoto

    2018-02-01

    Full Text Available Fibroblast growth factor 23 (FGF23 is a phosphotropic hormone mainly produced by bone. FGF23 reduces serum phosphate by suppressing intestinal phosphate absorption through reducing 1,25-dihydroxyvitamin D and proximal tubular phosphate reabsorption. Excessive actions of FG23 result in several kinds of hypophosphatemic rickets/osteomalacia including X-linked hypophosphatemic rickets (XLH and tumor-induced osteomalacia. While neutral phosphate and active vitamin D are standard therapies for child patients with XLH, these medications have several limitations both in their effects and adverse events. Several approaches that inhibit FGF23 actions including anti-FGF23 antibodies and inhibitors of FGF signaling have been shown to improve phenotypes of model mice for FG23-related hypophosphatemic diseases. In addition, clinical trials indicated that a humanized anti-FGF23 antibody increased serum phosphate and improved quality of life in patients with XLH. Furthermore, circulatory FGF23 is high in patients with chronic kidney disease (CKD. Many epidemiological studies indicated the association between high FGF23 levels and various adverse events especially in patients with CKD. However, it is not known whether the inhibition of FGF23 activities in patients with CKD is beneficial for these patients. In this review, recent findings concerning the modulation of FGF23 activities are discussed.

  13. Chimeric monoclonal antibody to tumor necrosis factor alpha (infliximab in psoriasis

    Directory of Open Access Journals (Sweden)

    Sridhar J

    2006-01-01

    Full Text Available Background: Insights into the pathogenesis of psoriasis have provided opportunities to target key steps in the disease process. Tumor necrosis factor-alpha (TNF-a being crucial to the pathogenesis of psoriasis, monoclonal antibodies against this cytokine have proved useful in its treatment. Aim: To study the efficacy of chimeric monoclonal antibody to TNF-a (infliximab in Indian patients with recalcitrant psoriasis vulgaris. Materials and Methods: Three patients with recalcitrant psoriasis vulgaris were studied. Baseline haemogram, biochemical parameters, chest radiograph and Mantoux skin test were performed. A loading dose regimen of 5 mg/kg infliximab was administered at weeks 0, 2 and 6. PASI assessment, adverse drug event monitoring and laboratory assessments were carried out at 2-week intervals until week 10. Patients were followed up until week 22 for relapse. Results: Infliximab was well tolerated. The mean PASI was 25.4 at presentation and declined to 5.5 at 10 weeks. PASI 75 was attained at a mean of 9.6 weeks. Relapse occurred at a mean of 18.6 weeks after the first infusion. Conclusions: This study on Indian patients brings out the importance of cytokine-based therapies in psoriasis. Indigenous production could make these therapies a viable therapeutic option for psoriasis patients in the near future.

  14. Human epidermal growth factor receptor 2 (HER2) immunoreactivity

    DEFF Research Database (Denmark)

    Rasmussen, Anne-Sofie Schrohl; Pedersen, Hans Christian; Jensen, Sussie Steen

    2011-01-01

    The availability of specific antibody-based test systems is essential to testing of HER2 protein expression. Here, we mapped epitopes recognized by three pharmacodiagnostic HER2 antibodies and investigated their specificity towards peptides and fusion proteins homologous to the intracellular doma...... domains of HER1, HER2, HER3 and HER4. The investigated antibodies were PATHWAY(®) HER2 (clone 4B5; Ventana Medical Systems Inc., Tucson, AZ, USA), HercepTest™ (Dako Denmark A/S, Glostrup, Denmark), and Oracle(®) HER2 (clone CB11; Leica Microsystems GmbH, Wetzlar, Germany)....

  15. Antibodies to Chlamydia trachomatis heat shock proteins in women with tubal factor infertility are associated with prior infection by C. trachomatis but not by C. pneumoniae

    DEFF Research Database (Denmark)

    Persson, K; Osser, S; Birkelund, Svend

    1999-01-01

    The antibody response to heat shock proteins 60 and 10 were studied in 163 patients with tubal factor infertility and in 163 age-matched pregnant women. The associations of these antibodies with specific antibodies to Chlamydia trachomatis and to Chlamydia pneumoniae as well as with antibodies...... proteins and to C. trachomatis but no independent influence of antibodies to C. pneumoniae. No interaction between C. trachomatis and C. pneumoniae suggesting a synergistic effect was found although the heat shock proteins from these two organisms are immunologically similar. Antibodies to the chlamydial...

  16. Radiotherapy and receptor of epidermal growth factor; Radiotherapie et recepteur de l'Epidermal Growth Factor

    Energy Technology Data Exchange (ETDEWEB)

    Deberne, M. [Institut Gustave-Roussy, 94 - Villejuif (France)

    2009-10-15

    The expression level of the receptor of the epidermal growth factor is in correlation with the tumor cells radiosensitivity. An overexpression of the E.G.F.R. is often present in the bronchi cancer, epidermoid carcinomas of the O.R.L. sphere, esophagus, uterine cervix, and anal duct but also in the rectum cancers and glioblastomas. At the clinical level, the E.G.F.R. expression is in correlation with an unfavourable prognosis after radiotherapy in numerous tumoral localizations. In the rectum cancers it is an independent prognosis factor found in multifactorial analysis: increase of the rate of nodes and local recurrence when the E.G.F.R. is over expressed. In the uterine cervix cancers, the survival is is negatively affected in multifactorial analysis by the E.G.F.R. membranes expression level. At the therapy level, the development of anti E.G.F.R. targeted therapies (tyrosine kinase inhibitors and monoclonal antibodies) opens a new therapy field at radio-sensitivity potentiality. The irradiation makes an activation of the E.G.F.R. way that would be partially responsible of the post irradiation tumoral repopulation. This activation leads the phosphorylation of the PI3 kinase ways and M.A.P. kinase ones, then the Akt protein one that acts an apoptotic modulator part. It has been shown that blocking the E.G.F.R. way acts on three levels: accumulation of ells in phase G1, reduction of the cell repair and increasing of apoptosis. he inhibition of post irradiation action of the E.G.F.R. signal way is a factor explaining the ionizing radiation - anti E.G.F.R. synergy. The preclinical data suggest that the E.G.F.R. blocking by the monoclonal antibodies is more important than the use of tyrosine kinase inhibitors. A first positive randomized study with the cetuximab, published in 2006 in the epidermoid carcinomas of the O.R.L. sphere lead to its authorization on the market with the radiotherapy for this localization. The use of cetuximab in other indication with or in

  17. Epidermal growth factor receptor inhibition by anti-CD147 therapy in cutaneous squamous cell carcinoma.

    Science.gov (United States)

    Frederick, John W; Sweeny, Larissa; Hartman, Yolanda; Zhou, Tong; Rosenthal, Eben L

    2016-02-01

    Advanced cutaneous squamous cell carcinoma (SCC) is an uncommon and aggressive malignancy. As a result, there is limited understanding of its biology and pathogenesis. CD147 and epidermal growth factor receptor (EGFR) have been identified as oncologically important targets, but their relationship remains undefined in cutaneous SCC. Multiple cutaneous SCC cell lines (Colo-16, SRB-1, and SRB-12), were treated in vitro with a range of chimeric anti-CD147 monoclonal antibody (mAb) (0, 50, 100, and 200 µg/mL) or transfected with a small interfering RNA against CD147 (SiCD147). Cell proliferation, migration (scratch wound healing assay), and protein expression was then assessed. In vivo, Colo-16 flank xenografts were treated anti-CD147 mAb (150 µg i.p. triweekly). After treatment with anti-CD147 (200 µg/mL), there was a significant decrease in proliferation for all cell lines relative to controls (p CD147 (200 µg/mL) resulted in decreased cell migration for all cell lines, with an average of 43% reduction in closure compared to controls (p CD147 antibody therapy and siRNA mediated reduction in CD147 expression were both found to decrease protein expression of EGFR, which correlated with a reduction in downstream total and phosphorylated protein kinase B (pAKT). Tumor growth in vivo was reduced for both the anti-CD147 treatment group and the SiCD147 group relative to controls. Inhibition and downregulation of CD147 in cutaneous SCC resulted in suppression of the malignant phenotype in vitro and in vivo, which may be mediated in part by an alteration in EGFR expression. As a result, CD147 may serve as a potential therapeutic target for advanced cutaneous SCC. © 2014 Wiley Periodicals, Inc.

  18. Fcγ-receptor IIa-mediated Src Signaling Pathway Is Essential for the Antibody-Dependent Enhancement of Ebola Virus Infection.

    Directory of Open Access Journals (Sweden)

    Wakako Furuyama

    2016-12-01

    Full Text Available Antibody-dependent enhancement (ADE of Ebola virus (EBOV infection has been demonstrated in vitro, raising concerns about the detrimental potential of some anti-EBOV antibodies. ADE has been described for many viruses and mostly depends on the cross-linking of virus-antibody complexes to cell surface Fc receptors, leading to enhanced infection. However, little is known about the molecular mechanisms underlying this phenomenon. Here we show that Fcγ-receptor IIa (FcγRIIa-mediated intracellular signaling through Src family protein tyrosine kinases (PTKs is required for ADE of EBOV infection. We found that deletion of the FcγRIIa cytoplasmic tail abolished EBOV ADE due to decreased virus uptake into cellular endosomes. Furthermore, EBOV ADE, but not non-ADE infection, was significantly reduced by inhibition of the Src family protein PTK pathway, which was also found to be important to promote phagocytosis/macropinocytosis for viral uptake into endosomes. We further confirmed a significant increase of the Src phosphorylation mediated by ADE. These data suggest that antibody-EBOV complexes bound to the cell surface FcγRIIa activate the Src signaling pathway that leads to enhanced viral entry into cells, providing a novel perspective for the general understanding of ADE of virus infection.

  19. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways

    DEFF Research Database (Denmark)

    Pandey, A; Fernandez, M M; Steen, H

    2000-01-01

    In an effort to clone novel tyrosine-phosphorylated substrates of the epidermal growth factor receptor, we have initiated an approach coupling affinity purification using anti-phosphotyrosine antibodies to mass spectrometry-based identification. Here, we report the identification of a signaling m...

  20. Emerging role of epidermal growth factor receptor inhibition in therapy for advanced malignancy: focus on NSCLC

    International Nuclear Information System (INIS)

    Langer, Corey J.

    2004-01-01

    Combination chemotherapy regimens have emerged as the standard approach in advanced non-small-cell lung cancer. Meta-analyses have demonstrated a 2-month increase in median survival after platinum-based therapy vs. best supportive care, and an absolute 10% improvement in the 1-year survival rate. Just as importantly, cytotoxic therapy has produced benefits in symptom control and quality of life. Newer agents, including the taxanes, vinorelbine, gemcitabine, and irinotecan, have expanded our therapeutic options in the treatment of advanced non-small-cell lung cancer. Despite their contributions, we have reached a therapeutic plateau, with response rates seldom exceeding 30-40% in cooperative group studies and 1-year survival rates stable between 30% and 40%. It is doubtful that substituting one agent for another in various combinations will lead to any further improvement in these rates. The thrust of current research has focused on targeted therapy, and epidermal growth factor receptor inhibition is one of the most promising clinical strategies. Epidermal growth factor receptor inhibitors currently under investigation include the small molecules gefitinib (Iressa, ZD1839) and erlotinib (Tarceva, OSI-774), as well as monoclonal antibodies such as cetuximab (IMC-225, Erbitux). Agents that have only begun to undergo clinical evaluation include CI-1033, an irreversible pan-erbB tyrosine kinase inhibitor, and PKI166 and GW572016, both examples of dual kinase inhibitors (inhibiting epidermal growth factor receptor and Her2). Preclinical models have demonstrated synergy for all these agents in combination with either chemotherapy or radiotherapy, leading to great enthusiasm regarding their ultimate contribution to lung cancer therapy. However, serious clinical challenges persist. These include the identification of the optimal dose(s); the proper integration of these agents into popular, established cytotoxic regimens; and the selection of the optimal setting(s) in which

  1. Therapeutic efficacy of antibodies lacking Fcγ receptor binding against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies [corrected].

    Directory of Open Access Journals (Sweden)

    Katherine L Williams

    2013-02-01

    Full Text Available Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS are life-threatening complications following infection with one of the four serotypes of dengue virus (DENV. At present, no vaccine or antiviral therapies are available against dengue. Here, we characterized a panel of eight human or mouse-human chimeric monoclonal antibodies (MAbs and their modified variants lacking effector function and dissected the mechanism by which some protect against antibody-enhanced lethal DENV infection. We found that neutralizing modified MAbs that recognize the fusion loop or the A strand epitopes on domains II and III of the envelope protein, respectively, act therapeutically by competing with and/or displacing enhancing antibodies. By analyzing these relationships, we developed a novel in vitro suppression-of-enhancement assay that predicts the ability of modified MAbs to act therapeutically against antibody-enhanced disease in vivo. These studies provide new insight into the biology of DENV pathogenesis and the requirements for antibodies to treat lethal DENV disease.

  2. Higher serum levels of rheumatoid factor and anti-nuclear antibodies in helicobacter pylori-infected peptic ulcer patients.

    Science.gov (United States)

    Jafarzadeh, Abdollah; Nemati, Maryam; Rezayati, Mohammad Taghi; Nabizadeh, Mansooreh; Ebrahimi, Medhi

    2013-07-01

    H. pylori infection has been associated with some autoimmune disorders. The aim of this study was to evaluate the serum concentrations of rheumatoid factor and anti-nuclear antibodies in H. pylori-infected peptic ulcer patients, H. pylori-infected asymptomatic carriers and a healthy control group. A Total of 100 H. pylori-infected peptic ulcer patients, 65 asymptomatic carriers and 30 healthy H. pylori-negative subjects (as a control group) were enrolled into study. Serum samples of participants tested for the levels of rheumatoid factor and anti-nuclear antibodies by use of ELISA. The mean serum levels of rheumatoid factor and anti-nuclear antibodies in peptic ulcer group was significantly higher in comparison to the control group (ppeptic ulcer patients and asymptomatic carriers groups regarding the mean serum levels of rheumatoid factor and anti-nuclear antibodies. The mean serum levels of rheumatoid factor in men with peptic ulcer was significantly higher compared to the group of healthy men (ppeptic ulcer patients or asymptomatic carriers groups, the mean serum levels of rheumatoid factor was higher than that in healthy women, but the differences were not statistically significant. Also, no significant differences were observed between men and women with peptic ulcer, asymptomatic carriers control groups based on the serum levels of anti-nuclear antibodies. The results showed higher serum levels of rheumatoid factor and anti-nuclear antibodies in H. pylori-infected patients with peptic ulcer disease which represent the H. pylori-related immune disturbance in these patients. Additional follow-up studies are necessary to clarify the clinical significance of these autoantibodies in patients with H. pylori infection.

  3. Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib-resistance of EML4-ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors.

    Science.gov (United States)

    Taniguchi, Hirokazu; Takeuchi, Shinji; Fukuda, Koji; Nakagawa, Takayuki; Arai, Sachiko; Nanjo, Shigeki; Yamada, Tadaaki; Yamaguchi, Hiroyuki; Mukae, Hiroshi; Yano, Seiji

    2017-01-01

    Crizotinib, a first-generation anaplastic lymphoma kinase (ALK) tyrosine-kinase inhibitor, is known to be effective against echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive non-small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib-resistant cells (A925LPE3-CR) via long-term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4-ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib-resistant cells, and these cells were cross-resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3-CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine-kinase inhibitor (erlotinib) or an anti-EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4-ALK lung cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  4. Comparison of Levels of Antibodies against Chlamydia Trachomatis in Infertile Women Due to Tubal Factors and Fertile Women

    Directory of Open Access Journals (Sweden)

    F Ghalmbor

    2009-01-01

    Full Text Available Introduction: Chlamydia trachomatis infection is a common pathogen in sexual transmitted disease, but most of female patients with this infection are asymptomatic. Sequealae include pelvic inflammatory disease, infertility and ectopic pregnancy. The aim of the study was to determine the association between Chlamydia trachomatis and tubal factor infertility, if significant. Methods: This prospective, case -control study was done in April 2005-April2006. The study group consisted of 125 patients with tubal factor infertility and the control group included 125 fertile women. The level of antibodies to Chlamydia trachomatis was determined in both groups by ELIZA method. Results: Antibody to Chlamydia trachomatis was present in 29 women in the study group (23.2% and in15 women in the control group ( 12%, respectively, (P< 0.005. The mean level of antibody in both groups was 0.76 and 0.49, respectively (P<0.0005. Conclusion: The study showed that the level of antibody against Chlamydia is significantly more in tubal factor infertile women. We therefore suggest the screening of Chlamydia antibody testing is necessary for tubal factor infertility workup.

  5. The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection.

    Science.gov (United States)

    Rajalingam, Raja

    2016-01-01

    Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.

  6. The impact of HLA class I-specific killer cell immunoglobulin-like receptors on antibody-dependent natural killer cell-mediated cytotoxicity and organ allograft rejection

    Directory of Open Access Journals (Sweden)

    Raja Rajalingam

    2016-12-01

    Full Text Available Natural killer (NK cells of the innate immune system are cytotoxic lymphocytes that play important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self HLA class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIR is involved in the calibration of NK cell effector capacities during a developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self HLA class I (due to virus infection or tumor transformation or HLA class I disparities (in the setting of allogeneic transplantation. NK cells expressing an inhibitory KIR binding self HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC, triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.

  7. Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

    DEFF Research Database (Denmark)

    Pandey, A; Podtelejnikov, A V; Blagoev, B

    2000-01-01

    Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2...

  8. Clinical Association of Thyroid Stimulating Hormone Receptor Antibody Levels with Disease Severity in the Chronic Inactive Stage of Graves' Orbitopathy.

    Science.gov (United States)

    Woo, Young Jae; Jang, Sun Young; Lim, Tyler Hyung Taek; Yoon, Jin Sook

    2015-08-01

    To investigate associations between serum thyroid stimulating hormone (TSH) receptor antibody (TRAb) levels and Graves' orbitopathy (GO) activity/severity in chronic-stage GO and compare the performance of two newly-developed TRAb assays (third-generation TSH-binding inhibition immunoglobulin [TBII] assay versus Mc4 thyroid-stimulating immunoglobulin [TSI] bioassay). This study is a retrospective review of medical charts and blood tests from Korean GO patients who first visited the departments of ophthalmology and endocrinology, Yonsei University College of Medicine from January 2008 to December 2011, were diagnosed with GO and Graves' hyperthyroidism, and were followed up for ≥18 months. Third-generation M22-TBII and Mc4-TSI assays were performed in the chronic-inactive GO patients in whom euthyroidism status was restored. Patients' GO activity/severity clinical activity scores (CAS), and modified NOSPECS scores were examined for a correlation with TRAb assays. Fifty patients (mean age, 41.3 years; 41 females) were analyzed. The mean duration of Graves' hyperthyroidism symptom was 63 months (range, 18 to 401 months) and that of GO was 46 months (range, 18 to 240 months). All patients had been treated previously with anti-thyroid drugs for a median period of 52.3 months, and two patients underwent either radioiodine therapy or total thyroidectomy. Mean CAS and NOSPECS scores were 0.5 ± 0.9 (standard deviation) and 4.8 ± 3.1, respectively. Mean M22-TBII and Mc4-TSI values were 7.5 ± 10.2 IL/L and 325.9 ± 210.1 specimen-to-reference control ratio. TSI was significantly correlated with NOSPECS score (R = 0.479, p 0.05), because GO inflammatory activity subsided in the chronic stages of GO. In chronic-inactive GO after euthyroid restoration, GO activity score did not associate with serum levels of TRAb or TBII. However, levels of the functional antibody Mc4-TSI did correlate with GO severity. Therefore, the TSI bioassay is a clinically relevant measure of disease

  9. Development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor receptor and insulin receptor expression in equine tissue

    Directory of Open Access Journals (Sweden)

    Stephen B. Hughes

    2013-08-01

    Full Text Available The insulin-like growth factor system (insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor and six insulin-like growth factor-binding proteins and insulin are essential to muscle metabolism and most aspects of male and female reproduction. Insulin-like growth factor and insulin play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. In order to better understand the local factors that regulate equine physiology, such as muscle metabolism and reproduction (e.g., germ cell development and fertilisation, real-time reverse transcription polymerase chain reaction assays for quantification of equine insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were developed. The assays were sensitive: 192 copies/µLand 891 copies/µL for insulin-like growth factor 1 receptor, messenger ribonucleic acid and insulin receptor respectively (95%limit of detection, and efficient: 1.01 for the insulin-like growth factor 1 receptor assay and 0.95 for the insulin receptor assay. The assays had a broad linear range of detection (seven logs for insulin-like growth factor 1 receptor and six logs for insulin receptor. This allowed for analysis of very small amounts of messenger ribonucleic acid. Low concentrations of both insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were detected in endometrium, lung and spleen samples, whilst high concentrations were detected in heart, muscle and kidney samples, this was most likely due to the high level of glucose metabolism and glucose utilisation by these tissues. The assays developed for insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid expression have been shown to work on equine tissue and will contribute to the understanding of insulin and insulin-like growth factor 1

  10. Seroprevalence and risk factors of anti-brucella antibodies in cattle in Khartoum State, the Sudan

    Directory of Open Access Journals (Sweden)

    Wegdan Osman Mohamed-Khair

    2016-06-01

    Material and methods: A total of 300 serum samples were taken from jugular veins of cattle and screened by Rose Bengal Plate Test (RBPT and Serum Agglutination Test (SAT. The RBPT-positive samples were all tested using c-ELISA. Results: Antibodies were detected with an overall seroprevalence of 25.7% using RBPT and 22.7% using SAT while slightly less than two thirds of the RBPT-positive samples were cELISA-positive. The herd and within-herd seroprevalences were 76.7% (n=23 and from 10.0-80.0%. Moreover, significant statistical dissimilarities were not observed between the seroprevalence of the different categories of the investigated risk factors by RBPT. Only milking method ( and #967;2=3.976; P=0.046 was found to have an influence on the RBPT-positive status for brucella infection in the univariate analysis. Additionally, natural breeding (OR=3.61; 95% CI 1.192 and ndash;10.96; P=0.023 was the only observed risk factor with an increased odd of being RBPT positive. The Kappa analysis showed an almost perfect agreement between the results of the RBPT and the SAT tests. Conclusion: The prevalence of anti-brucella antibodies in Khartoum state was relatively higher; therefore, brucellosis in cattle is, perhaps, a significant public health problem. It is recommended to raise awareness of cattle owners and/or herders on the routes of transmission of brucellosis. [J Adv Vet Anim Res 2016; 3(2.000: 134-144

  11. Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses*

    Science.gov (United States)

    Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A.; Manna, Sunil K.

    2010-01-01

    The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy. PMID:20018848

  12. Azadirachtin interacts with the tumor necrosis factor (TNF) binding domain of its receptors and inhibits TNF-induced biological responses.

    Science.gov (United States)

    Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A; Manna, Sunil K

    2010-02-19

    The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

  13. The clinical significance and risk factors of anti-platelet factor 4/heparin antibody on maintenance hemodialysis patients: a two-year prospective follow-up.

    Directory of Open Access Journals (Sweden)

    Delong Zhao

    Full Text Available BACKGROUND: Heparin-induced thrombocytopenia is an immune response mediated by anti-PF4/heparin antibody, which is clinically characterized by thrombocytopenia and thromboembolic events. In this study, a prospective and multi-center clinical investigation 1 determined the positive rate of anti-PF4/heparin antibody in maintenance hemodialysis patients in China, 2 identified the related risk factors, and 3 further explored the effect of the anti-PF4/heparin antibody on bleeding, thromboembolic events, and risk of death in the patients. METHODS: The serum anti-PF4/heparin antibody was measured in 661 patients from nine hemodialysis centers, detected by IgG-specific ELISA and followed by confirmation with excess heparin. Risk factors of these patients were analyzed. Based on a two-year follow-up, the association between the anti-PF4/heparin antibody and bleeding, thromboembolic events, and risk of death in the patients was investigated. RESULTS: 1 The positivity rate of the anti-PF4/heparin antibody in maintenance hemodialysis patients was 5.6%. With diabetes as an independent risk factor, the positivity rate of the anti-PF4/heparin antibody decreased in the patients undergoing weekly dialyses ≥3 times. 2 The positivity rate of the anti-PF4/heparin antibody was not related to the occurrence of clinical thromboembolic events and was not a risk factor for death within two years in maintenance hemodialysis patients. 3 Negativity for the anti-PF4/heparin antibody combined with a reduction of the platelet count or combined with the administration of antiplatelet drugs yielded a significant increase in bleeding events. However, the composite determination of the anti-PF4/heparin antibody and thrombocytopenia, as well as the administration of antiplatelet drugs, was not predictive for the risk of thromboembolic events in the maintenance hemodialysis patients. CONCLUSIONS: A single detection of the anti-PF4/heparin antibody did not predict the occurrence

  14. Glycosylation as a Main Regulator of Growth and Death Factor Receptors Signaling

    Directory of Open Access Journals (Sweden)

    Inês Gomes Ferreira

    2018-02-01

    Full Text Available Glycosylation is a very frequent and functionally important post-translational protein modification that undergoes profound changes in cancer. Growth and death factor receptors and plasma membrane glycoproteins, which upon activation by extracellular ligands trigger a signal transduction cascade, are targets of several molecular anti-cancer drugs. In this review, we provide a thorough picture of the mechanisms bywhich glycosylation affects the activity of growth and death factor receptors in normal and pathological conditions. Glycosylation affects receptor activity through three non-mutually exclusive basic mechanisms: (1 by directly regulating intracellular transport, ligand binding, oligomerization and signaling of receptors; (2 through the binding of receptor carbohydrate structures to galectins, forming a lattice thatregulates receptor turnover on the plasma membrane; and (3 by receptor interaction with gangliosides inside membrane microdomains. Some carbohydrate chains, for example core fucose and β1,6-branching, exert a stimulatory effect on all receptors, while other structures exert opposite effects on different receptors or in different cellular contexts. In light of the crucial role played by glycosylation in the regulation of receptor activity, the development of next-generation drugs targeting glyco-epitopes of growth factor receptors should be considered a therapeutically interesting goal.

  15. Brain metastasis in human epidermal growth factor receptor 2-positive breast cancer: from biology to treatment

    Energy Technology Data Exchange (ETDEWEB)

    Koo, Tae Ryool [Dept. of Radiation Oncology, Hallym University Chuncheon Sacred Heart Hospital, Chuncheon (Korea, Republic of); Kim, In Ah [Dept. of Radiation Oncology, Seoul National University Bundang Hospital, Seongnam (Korea, Republic of)

    2016-03-15

    Overexpression of human epidermal growth factor receptor 2 (HER2) is found in about 20% of breast cancer patients. With treatment using trastuzumab, an anti-HER2 monoclonal antibody, systemic control is improved. Nonetheless, the incidence of brain metastasis does not be improved, rather seems to be increased in HER2-positive breast cancer. The mainstay treatment for brain metastases is radiotherapy. According to the number of metastatic lesions and performance status of patients, radiosurgery or whole brain radiotherapy can be performed. The concurrent use of a radiosensitizer further improves intracranial control. Due to its large molecular weight, trastuzumab has a limited ability to cross the blood-brain barrier. However, small tyrosine kinase inhibitors such as lapatinib, has been noted to be a promising agent that can be used as a radiosensitizer to affect HER2-positive breast cancer. This review will outline general management of brain metastases and will focus on preclinical findings regarding the radiosensitizing effect of small molecule HER2 targeting agents.

  16. Biologic effects of platelet-derived growth factor receptor α blockade in uterine cancer.

    Science.gov (United States)

    Roh, Ju-Won; Huang, Jie; Hu, Wei; Yang, XiaoYun; Jennings, Nicholas B; Sehgal, Vasudha; Sohn, Bo Hwa; Han, Hee Dong; Lee, Sun Joo; Thanapprapasr, Duangmani; Bottsford-Miller, Justin; Zand, Behrouz; Dalton, Heather J; Previs, Rebecca A; Davis, Ashley N; Matsuo, Koji; Lee, Ju-Seog; Ram, Prahlad; Coleman, Robert L; Sood, Anil K

    2014-05-15

    Platelet-derived growth factor receptor α (PDGFRα) expression is frequently observed in many kinds of cancer and is a candidate for therapeutic targeting. This preclinical study evaluated the biologic significance of PDGFRα and PDGFRα blockade (using a fully humanized monoclonal antibody, 3G3) in uterine cancer. Expression of PDGFRα was examined in uterine cancer clinical samples and cell lines, and biologic effects of PDGFRα inhibition were evaluated using in vitro (cell viability, apoptosis, and invasion) and in vivo (orthotopic) models of uterine cancer. PDGFRα was highly expressed and activated in uterine cancer samples and cell lines. Treatment with 3G3 resulted in substantial inhibition of PDGFRα phosphorylation and of downstream signaling molecules AKT and mitogen-activated protein kinase (MAPK). Cell viability and invasive potential of uterine cancer cells were also inhibited by 3G3 treatment. In orthotopic mouse models of uterine cancer, 3G3 monotherapy had significant antitumor effects in the PDGFRα-positive models (Hec-1A, Ishikawa, Spec-2) but not in the PDGFRα-negative model (OVCA432). Greater therapeutic effects were observed for 3G3 in combination with chemotherapy than for either drug alone in the PDGFRα-positive models. The antitumor effects of therapy were related to increased apoptosis and decreased proliferation and angiogenesis. These findings identify PDGFRα as an attractive target for therapeutic development in uterine cancer. ©2014 American Association for Cancer Research.

  17. New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome.

    Science.gov (United States)

    Tsuboi, H; Matsumoto, I; Wakamatsu, E; Nakamura, Y; Iizuka, M; Hayashi, T; Goto, D; Ito, S; Sumida, T

    2010-10-01

    M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögren's syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti-M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human-M3R including the N-terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme-linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti-M3R antibody-positive SS, -negative SS and controls for 12 h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca(2+) concentrations [(Ca(2+) )i] were measured. Antibodies to the N-terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS-IgG inhibited the increase of (Ca(2+) )i induced by cevimeline hydrochloride. Antibodies to the N-terminal positive SS-IgG and antibodies to the first loop positive SS-IgG enhanced it, while antibodies to the third loop positive SS-IgG showed no effect on (Ca(2+) )i as well as anti-M3R antibody-negative SS-IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes. © 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.

  18. Diagnostic values of vascular endothelial growth factor and epidermal growth factor receptor for benign and malignant hydrothorax.

    Science.gov (United States)

    Gu, Yan; Zhang, Min; Li, Guo-Hua; Gao, Jun-Zhen; Guo, Liping; Qiao, Xiao-Juan; Wang, Li-Hong; He, Lan; Wang, Mei-Ling; Yan, Li; Fu, Xiu-Hua

    2015-02-05

    Hydrothorax, as one of the common complications of malignant tumors, still cannot be sensitively detected in clinical practice, thus requiring a sensitive, specific method for diagnosis. The aim of this study was to analyze the correlation between levels of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) in patients with benign and malignant hydrothorax. The contents of VEGF in the pleural effusion and serum of the patients with malignant pleural effusion (n = 35) and benign pleural effusion (n = 30) were detected by double antibody sandwich enzyme linked immunosorbent assay. The gene copy number level of EGFR in pleural effusion was detected by fluorescence in situ hybridization (FISH). The points with the highest sensitivity and specificity were selected as the critical values to calculate the diagnostic value of the VEGF in pleural effusion and serum, and EGFR gene copy number in pleural effusion. The contents of VEGF in pleural effusion and serum of patients with malignant hydrothorax were (384.91 ± 120.18), and (129.62 ± 46.35) ng/L, respectively, which were significantly higher than those of the patients with benign hydrothorax (207.97 ± 64.04), (63.49 ± 24.58) ng/L (P benign and malignant hydrothorax. The sensitivity and specificity of serum were 74.3% and 96.7%, respectively (the boundary value was 99.21 ng/L) for diagnosing benign and malignant hydrothorax. The diagnostic efficiencies of EGFR and VEGF in hydrothorax were similar. There was a significant correlation between EGFR and VEGF in hydrothorax (P benign and malignant pleural effusions, which contributed to differential diagnosis results of benign and malignant pleural effusions. It is feasible to detect the gene copy number of the pleural effusion cell mass EGFR by FISH technique. Joint detection can improve the diagnostic sensitivity.

  19. Anti-thyrotropin receptor antibody levels after radioiodine therapy in patients of childbearing age with Graves' disease

    International Nuclear Information System (INIS)

    Takeuchi, Mizuho; Tojo, Katsuyoshi; Tajima, Naoko; Yoshimura, Hiroshi; Ito, Koichi

    2006-01-01

    Following radioiodine therapy for Graves' disease, transient elevation of anti-thyrotropin receptor antibody (TRAb) is observed. Elevation of TRAb causes neonatal hyperthyroidism. Serum TRAb levels before radioiodine therapy, 2 months to 1 year, 1 to 2 years, 2 to 3 years, and 3 to 4 years after radioiodine therapy were retrospectively analyzed in 25 women of childbearing age with Graves' disease. The normal range for TRAb is ≤15%. The one patient with serum TRAb levels <10% before radioiodine therapy did not have TRAb levels ≥50% after radioiodine therapy. However, in patients with serum TRAb levels of 10% to 30% before radioiodine therapy (n=8), TRAb were ≥50% in 75.0% 2 months to 1 year after radioiodine therapy, in 25.0% 1 to 2 years after, and in 37.5% 2 to 4 years after. In patients with serum TRAb levels of 30% to 50% before radioiodine therapy (n=3), TRAb levels were ≥50% in 33.3% 2 months to 1 year after radioiodine therapy and in 0.0% 1 to 4 years after. In patients with serum TRAb levels of 50% to 70% before radioiodine therapy (n=6), TRAb were ≥50% in 83.3% 2 months to 1 year after radioiodine therapy, in 66.6% 1 to 2 years after, and in 33.3% 2 to 4 years after. In patients with serum TRAb levels ≥70% before radioiodine therapy (n=7), TRAb levels were ≥50% in 100% 2 months to 1 year after radioiodine therapy, in 85.7% 1 to 2 years after, in 71.4% 2 to 3 years after, and in 57.1% 3 to 4 years after. Serum TRAb levels are more likely to be ≥50% after radioiodine therapy in patients with high serum TRAb levels before radioiodine therapy. (author)

  20. Population PK and IgE pharmacodynamic analysis of a fully human monoclonal antibody against IL4 receptor.

    Science.gov (United States)

    Kakkar, Tarundeep; Sung, Cynthia; Gibiansky, Leonid; Vu, Thuy; Narayanan, Adimoolam; Lin, Shao-Lee; Vincent, Michael; Banfield, Christopher; Colbert, Alex; Hoofring, Sarah; Starcevic, Marta; Ma, Peiming

    2011-10-01

    For AMG 317, a fully human monoclonal antibody to interleukin receptor IL-4Rα, we developed a population pharmacokinetic (PK) model by fitting data from four early phase clinical trials of intravenous and subcutaneous (SC) routes simultaneously, investigated important PK covariates, and explored the relationship between exposure and IgE response. Data for 294 subjects and 2183 AMG 317 plasma concentrations from three Phase 1 and 1 Phase 2 studies were analyzed by nonlinear mixed effects modeling using first-order conditional estimation with interaction. The relationship of IgE response with post hoc estimates of exposure generated from the final PK model was explored based on data from asthmatic patients. The best structural model was a two-compartment quasi-steady-state target-mediated drug disposition model with linear and non-linear clearances. For a typical 80-kg, 40-year subject, linear clearance was 35.0 mL/hr, central and peripheral volumes of distribution were 1.78 and 5.03 L, respectively, and SC bioavailability was 24.3%. Body weight was an important covariate on linear clearance and central volume; age influenced absorption rate. A significant treatment effect was observable between the cumulative AUC and IgE response measured. The population PK model adequately described AMG 317 PK from IV and SC routes over a 60-fold range of doses with two dosing strengths across multiple studies covering healthy volunteers and patients with mild to severe asthma. IgE response across a range of doses and over the sampling time points was found to be related to cumulative AMG 317 exposure.

  1. Restoration of pharyngeal dilator muscle force in dystrophin-deficient (mdx) mice following co-treatment with neutralizing interleukin-6 receptor antibodies and urocortin 2.

    Science.gov (United States)

    Burns, David P; Rowland, Jane; Canavan, Leonie; Murphy, Kevin H; Brannock, Molly; O'Malley, Dervla; O'Halloran, Ken D; Edge, Deirdre

    2017-09-01

    What is the central question of this study? We previously reported impaired upper airway dilator muscle function in the mdx mouse model of Duchenne muscular dystrophy (DMD). Our aim was to assess the effect of blocking interleukin-6 receptor signalling and stimulating corticotrophin-releasing factor receptor 2 signalling on mdx sternohyoid muscle structure and function. What is the main finding and its importance? The interventional treatment had a positive inotropic effect on sternohyoid muscle force, restoring mechanical work and power to wild-type values, reduced myofibre central nucleation and preserved the myosin heavy chain type IIb fibre complement of mdx sternohyoid muscle. These data might have implications for development of pharmacotherapies for DMD with relevance to respiratory muscle performance. The mdx mouse model of Duchenne muscular dystrophy shows evidence of impaired pharyngeal dilator muscle function. We hypothesized that inflammatory and stress-related factors are implicated in airway dilator muscle dysfunction. Six-week-old mdx (n = 26) and wild-type (WT; n = 26) mice received either saline (0.9% w/v) or a co-administration of neutralizing interleukin-6 receptor antibodies (0.2 mg kg -1 ) and corticotrophin-releasing factor receptor 2 agonist (urocortin 2; 30 μg kg -1 ) over 2 weeks. Sternohyoid muscle isometric and isotonic contractile function was examined ex vivo. Muscle fibre centronucleation and muscle cellular infiltration, collagen content, fibre-type distribution and fibre cross-sectional area were determined by histology and immunofluorescence. Muscle chemokine content was examined by use of a multiplex assay. Sternohyoid peak specific force at 100 Hz was significantly reduced in mdx compared with WT. Drug treatment completely restored force in mdx sternohyoid to WT levels. The percentage of centrally nucleated muscle fibres was significantly increased in mdx, and this was partly ameliorated after drug treatment. The areal

  2. Epidermal growth factor receptor mutation in gastric cancer.

    Science.gov (United States)

    Liu, Zhimin; Liu, Lina; Li, Mei; Wang, Zhaohui; Feng, Lu; Zhang, Qiuping; Cheng, Shihua; Lu, Shen

    2011-04-01

    Epidermal growth factor receptor (EGFR) and Kirsten-RAS (KRAS) mutations have been identified as predictors of response to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer. We aimed to screen the mutations of both genes in gastric carcinoma to detect the suitability of EGFR TKIs for patients with gastric carcinoma. We screened EGFR mutation in exons 19-21 and KRAS mutation in exon 2 in 58 gastric adenocarcinomas from China using high resolution melting analysis (HRMA). Positive samples were confirmed by DNA sequencing. Three EGFR missense mutations (5.2%) and 22 single nucleotide polymorphisms (SNP, Q787Q, 37.9%) were identified. To our knowledge, we report for the first time three mutation patterns of EGFR, Y801C, L858R and G863D, in gastric carcinoma. Two samples with EGFR mutation were mucinous adenocarcinoma. These three samples were collected from male patients aged over 75 years old. The frequency of KRAS mutation was 10.3% (6/58). The exclusiveness of EGFR and KRAS mutations was proven for the first time in gastric cancer. Gastric carcinoma of the mucinous adenocarcinoma type collected from older male patients may harbour EGFR mutations. The small subset of gastric adenocarcinoma patients may respond to EGFR TKIs.

  3. Association of coatomer proteins with the beta-receptor for platelet-derived growth factor

    DEFF Research Database (Denmark)

    Hansen, Klaus; Rönnstrand, L; Rorsman, C

    1997-01-01

    The nonreceptor tyrosine kinase Src binds to and is activated by the beta-receptor for platelet-derived growth factor (PDGF). The interaction leads to Src phosphorylation of Tyr934 in the kinase domain of the receptor. In the course of the functional characterization of this phosphorylation, we...... of intracellular vesicle transport. In order to explore the functional significance of the interaction between alpha- and beta'-COP and the PDGF receptor, a receptor mutant was made in which the conserved histidine residue 928 was mutated to an alanine residue. The mutant receptor, which was unable to bind alpha...

  4. 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.

  5. Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against α-transforming growth factor

    International Nuclear Information System (INIS)

    Hazarika, P.; Pardue, R.L.; Earls, R.; Dedman, J.R.

    1987-01-01

    Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human α-transforming growth factor (α-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting α-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native α-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of α-TCF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of α-TGF has a cellular role beyond that of an autocrine growth factor

  6. Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against. cap alpha. -transforming growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Hazarika, P.; Pardue, R.L.; Earls, R.; Dedman, J.R.

    1987-04-07

    Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human ..cap alpha..-transforming growth factor (..cap alpha..-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting ..cap alpha..-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native ..cap alpha..-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of ..cap alpha..-TCF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of ..cap alpha..-TGF has a cellular role beyond that of an autocrine growth factor.

  7. Soluble CD30 and HLA antibodies as potential risk factors for kidney transplant rejection.

    Science.gov (United States)

    Slavcev, Antonij; Lácha, Jiri; Honsová, Eva; Sajdlová, Helena; Lodererová, Alena; Vitko, Stefan; Skibová, Jelena; Striz, Ilja

    2005-06-01

    Recent literary data suggest that high pre- and post-transplant serum levels of the soluble CD30 (sCD30) molecule may be a risk factor for acute rejection and worse prognosis of the transplanted kidney. The aim of our study was to correlate the concentrations of sCD30 and the presence of HLA antibodies as defined by flow cytometry and ELISA with the clinical course and graft prognosis after transplantation. One hundred and seventeen kidney transplant patients were included into the study. The incidence of rejection episodes, graft function and graft survival for up to 1 year post-transplant were evaluated. Soluble CD30 levels before transplantation were virtually the same in patients who experienced rejection and in non-rejecting patients. In both patient groups, a significant decrease of sCD30 was detected 2 weeks after transplantation (104.4 U/ml before vs. 37.0 U/ml post-transplant, P sCD30 between rejecting and non-rejecting patients. Patients without rejection had lower sCD30 values (31.2 U/ml post-transplant) compared to patients who experienced rejection episodes (62.9 U/ml), P antigens and elevated concentrations of sCD30 shortly after transplantation were associated with increased risk for acute rejection in the first post-transplant year. Measurement of soluble CD30 after transplantation, taken into consideration with the presence of HLA class II antibodies, might be helpful for evaluating the potential risk for acute rejection.

  8. The Pseudo signal peptide of the corticotropin-releasing factor receptor type 2A prevents receptor oligomerization.

    Science.gov (United States)

    Teichmann, Anke; Rutz, Claudia; Kreuchwig, Annika; Krause, Gerd; Wiesner, Burkhard; Schülein, Ralf

    2012-08-03

    N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF(2(a))R and the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF(2(a))R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general.

  9. A transcription factor active on the epidermal growth factor receptor gene

    International Nuclear Information System (INIS)

    Kageyama, R.; Merlino, G.T.; Pastan, I.

    1988-01-01

    The authors have developed an in vitro transcription system for the epidermal growth factor receptor (EGFR) oncogene by using nuclear extracts of A431 human epidermoid carcinoma cells, which overproduce EGFR. They found that a nuclear factor, termed EGFR-specific transcription factor (ETF), specifically stimulated EGFR transcription by 5- to 10-fold. In this report, ETF, purified by using sequence-specific oligonucleotide affinity chromatography, is shown by renaturing material eluted from a NaDodSO 4 /polyacrylamide gel to be a protein with a molecular mass of 120 kDa. ETF binds to the promoter region, as measured by DNase I footprinting and gel-mobility-shift assays, and specifically stimulates the transcription of the EGFR gene in a reconstituted in vitro transcription system. These results suggest that ETF could play a role in the overexpression of the cellular oncogene EGFR

  10. The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

    Energy Technology Data Exchange (ETDEWEB)

    Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi; Gittis, Apostolos G.; Su, Hua-Poo; Mikami, Bunzo; Okazaki, Taku; Honjo, Tasuku; Minato, Nagahiro; Garboczi, David N. (NIH); (Kyoto)

    2008-07-29

    Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

  11. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

    Science.gov (United States)

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2013-05-07

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

  12. IgA antibodies against β2 glycoprotein I in hemodialysis patients are an independent risk factor for mortality.

    Science.gov (United States)

    Serrano, Antonio; García, Florencio; Serrano, Manuel; Ramírez, Elisa; Alfaro, F Javier; Lora, David; de la Cámara, Agustín Gómez; Paz-Artal, Estela; Praga, Manuel; Morales, Jose M

    2012-06-01

    Cardiovascular complications are the most important cause of death in patients on dialysis with end-stage renal disease. Antibodies reacting with β-glycoprotein I seem to play a pathogenic role in antiphospholipid syndrome and stroke and are involved in the origin of atherosclerosis. Here we evaluated the presence of anticardiolipin and anti-β-glycoprotein I antibodies together with other vascular risk factors and their relationship with mortality and cardiovascular morbidity in a cohort of 124 hemodialysis patients prospectively followed for 2 years. Of these, 41 patients were significantly positive for IgA anti-β-glycoprotein I, and the remaining had normal values. At 24 months, overall and cardiovascular mortality and thrombotic events were all significantly higher in patients with high anti-β-glycoprotein I antibodies. Multivariate analysis using Cox regression modeling found that age, hypoalbuminemia, use of dialysis catheters, and IgA β-glycoprotein I antibodies were independent risk factors for death. Thus, IgA antibodies to β-glycoprotein I are detrimental to the clinical outcome of hemodialysis patients.

  13. Association Between Serum Antibodies to Periodontal Bacteria and Rheumatoid Factor in the Third National Health and Nutrition Examination Survey.

    Science.gov (United States)

    Goh, Charlene E; Kopp, Jacob; Papapanou, Panos N; Molitor, Jerry A; Demmer, Ryan T

    2016-10-01

    Alterations in the microbiome, including the periodontal microbiome, may be a risk factor for rheumatoid arthritis (RA). Most studies that have analyzed this association are relatively small, focus primarily on a single periodontal pathogen (Porphyromonas gingivalis), and are not population based. This study was undertaken to investigate the association between elevated serum levels of IgG antibodies to 19 periodontal species and the prevalence of rheumatoid factor (RF) in a large nationally representative sample of adults. The Third National Health and Nutrition Examination Survey (NHANES-III) is a cross-sectional sample of the noninstitutionalized US population (n = 33,994). Our study population included all dentate participants who were 60 years and older, did not have RA as defined by a modified version of the American College of Rheumatology 1987 criteria, and had complete data for both serum IgG antibodies against periodontal bacteria and serum RF antibody titer (n = 2,461). Adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) summarizing the relationship between the 19 periodontal serum IgG antibodies and RF seropositivity ranged from 0.53 (95% CI 0.29-0.97) to 1.27 (95% CI 0.79-2.06), and 17 of the 19 observed ORs were periodontal bacteria are mostly unassociated with RF seropositivity in the nationally representative NHANES-III. Elevated levels of antibodies to P intermedia and C ochracea are associated with lower odds of RF seropositivity. © 2016, American College of Rheumatology.

  14. Host factors that modify Plasmodium falciparum adhesion to endothelial receptors.

    Science.gov (United States)

    Mahamar, Almahamoudou; Attaher, Oumar; Swihart, Bruce; Barry, Amadou; Diarra, Bacary S; Kanoute, Moussa B; Cisse, Kadidia B; Dembele, Adama B; Keita, Sekouba; Gamain, Benoît; Gaoussou, Santara; Issiaka, Djibrilla; Dicko, Alassane; Duffy, Patrick E; Fried, Michal

    2017-10-24

    P. falciparum virulence is related to adhesion and sequestration of infected erythrocytes (IE) in deep vascular beds, but the endothelial receptors involved in severe malaria remain unclear. In the largest ever study of clinical isolates, we surveyed adhesion of freshly collected IE from children under 5 years of age in Mali to identify novel vascular receptors, and examined the effects of host age, hemoglobin type, blood group and severe malaria on levels of IE adhesion to a panel of endothelial receptors. Several novel molecules, including integrin α3β1, VE-cadherin, ICAM-2, junctional adhesion molecule-B (JAM-B), laminin, and cellular fibronectin, supported binding of IE from children. Severe malaria was not significantly associated with levels of IE adhesion to any of the 19 receptors. Hemoglobin AC, which reduces severe malaria risk, reduced IE binding to the receptors CD36 and integrin α5β1, while hemoglobin AS did not modify IE adhesion to any receptors. Blood groups A, AB and B significantly reduced IE binding to ICAM-1. Severe malaria risk varies with age, but age significantly impacted the level of IE binding to only a few receptors: IE binding to JAM-B decreased with age, while binding to CD36 and integrin α5β1 significantly increased with age.

  15. Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

    International Nuclear Information System (INIS)

    He Yuxian; Zhou Yusen; Liu Shuwen; Kou Zhihua; Li Wenhui; Farzan, Michael; Jiang Shibo

    2004-01-01

    The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS

  16. Bevacizumab, an anti-vascular endothelial growth factor antibody, inhibits osteoarthritis.

    Science.gov (United States)

    Nagai, Toshihiro; Sato, Masato; Kobayashi, Miyuki; Yokoyama, Munetaka; Tani, Yoshiki; Mochida, Joji

    2014-09-18

    Angiogenesis is an important factor in the development of osteoarthritis (OA). We investigated the efficacy of bevacizumab, an antibody against vascular endothelial growth factor and an inhibitor of angiogenesis, in the treatment of OA using a rabbit model of anterior cruciate ligament transection. First, we evaluated the response of gene expression and histology of the normal joint to bevacizumab treatment. Next, in a rabbit model of OA induced by anterior cruciate ligament transection, we used macroscopic and histological evaluations and real-time polymerase chain reaction (PCR) to examine the responses to intravenous (systemic) administration of bevacizumab (OAB IV group). We also investigated the efficacy of intra-articular (local) administration of bevacizumab in OA-induced rabbits (OAB IA group). Histologically, bevacizumab had no negative effect in normal joints. Bevacizumab did not increase the expression of genes for catabolic factors in the synovium, subchondral bone, or articular cartilage, but it increased the expression of collagen type 2 in the articular cartilage. Macroscopically and histologically, the OAB IV group exhibited a reduction in articular cartilage degeneration and less osteophyte formation and synovitis compared with the control group (no bevacizumab; OA group). Real-time PCR showed significantly lower expression of catabolic factors in the synovium in the OAB IV group compared with the OA group. In articular cartilage, expression levels of aggrecan, collagen type 2, and chondromodulin-1 were higher in the OAB IV group than in the OA group. Histological evaluation and assessment of pain behaviour showed a superior effect in the OAB IA group compared with the OAB IV group 12 weeks after administration of bevacizumab, even though the total dosage given to the OAB IA group was half that received by the OAB IV group. Considering the dosage and potential adverse effects of bevacizumab, the local administration of bevacizumab is a more

  17. Effect of baseline rheumatoid factor and anticitrullinated peptide antibody serotype on rituximab clinical response: a meta-analysis

    NARCIS (Netherlands)

    Isaacs, John D.; Cohen, Stanley B.; Emery, Paul; Tak, Paul P.; Wang, Jianmei; Lei, Guiyuan; Williams, Sarah; Lal, Preeti; Read, Simon J.

    2013-01-01

    Studies examining the relationship between serological status (rheumatoid factor and/or anticitrullinated antibody) and rituximab treatment outcome in rheumatoid arthritis (RA) have been hampered by limited numbers of seronegative patients. To carry out a meta-analysis of trials from the rituximab

  18. Aging-dependent decline of IL-10 producing B cells coincides with production of antinuclear antibodies but not rheumatoid factors

    NARCIS (Netherlands)

    van der Geest, Kornelis S. M.; Lorencetti, Pedro G.; Abdulahad, Wayel H.; Horst, Gerda; Huitema, Minke; Roozendaal, Caroline; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M. H.

    Aging is associated with development of autoimmunity. Loss of B cell tolerance in the elderly is suggested by an increased prevalence of anti-nuclear antibodies (ANAs) and rheumatoid factors (RFs). Accumulating evidence indicates that B cells also impact autoimmunity via secretion of cytokines. So

  19. Association of the leukemia inhibitory factor gene mutation and the antiphospholipid antibodies in the peripheral blood of infertile women

    Czech Academy of Sciences Publication Activity Database

    Králíčková, M.; Ulčová-Gallová, Z.; Šíma, R.; Vaněček, T.; Šíma, Petr; Křižan, Jiří; Suchá, J.; Uher, P.; Hes, O.; Novotný, Z.; Rokyta, Z.; Větvička, E.

    2007-01-01

    Roč. 52, č. 5 (2007), s. 543-548 ISSN 0015-5632 R&D Projects: GA ČR GA301/05/0078 Institutional research plan: CEZ:AV0Z50200510 Keywords : leukemia inhibitory faktor * lif gene mutation * antiphospholipid antibodies Subject RIV: EE - Microbiology, Virology Impact factor: 0.989, year: 2007

  20. Targeting fibroblast growth factor receptor signaling inhibits prostate cancer progression.

    Science.gov (United States)

    Feng, Shu; Shao, Longjiang; Yu, Wendong; Gavine, Paul; Ittmann, Michael

    2012-07-15

    Extensive correlative studies in human prostate cancer as well as studies in vitro and in mouse models indicate that fibroblast growth factor receptor (FGFR) signaling plays an important role in prostate cancer progression. In this study, we used a probe compound for an FGFR inhibitor, which potently inhibits FGFR-1-3 and significantly inhibits FGFR-4. The purpose of this study is to determine whether targeting FGFR signaling from all four FGFRs will have in vitro activities consistent with inhibition of tumor progression and will inhibit tumor progression in vivo. Effects of AZ8010 on FGFR signaling and invasion were analyzed using immortalized normal prostate epithelial (PNT1a) cells and PNT1a overexpressing FGFR-1 or FGFR-4. The effect of AZ8010 on invasion and proliferation in vitro was also evaluated in prostate cancer cell lines. Finally, the impact of AZ8010 on tumor progression in vivo was evaluated using a VCaP xenograft model. AZ8010 completely inhibits FGFR-1 and significantly inhibits FGFR-4 signaling at 100 nmol/L, which is an achievable in vivo concentration. This results in marked inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and invasion in PNT1a cells expressing FGFR-1 and FGFR-4 and all prostate cancer cell lines tested. Treatment in vivo completely inhibited VCaP tumor growth and significantly inhibited angiogenesis and proliferation and increased cell death in treated tumors. This was associated with marked inhibition of ERK phosphorylation in treated tumors. Targeting FGFR signaling is a promising new approach to treating aggressive prostate cancer.

  1. Antiprothrombin Antibodies

    Directory of Open Access Journals (Sweden)

    Polona Žigon

    2015-05-01

    Full Text Available In patients with the antiphospholipid syndrome (APS, the presence of a group of pathogenic autoantibodies called antiphospholipid antibodies causes thrombosis and pregnancy complications. The most frequent antigenic target of antiphospholipid antibodies are phospholipid bound β2-glycoprotein 1 (β2GPI and prothrombin. The international classification criteria for APS connect the occurrence of thrombosis and/or obstetric complications together with the persistence of lupus anticoagulant, anti-cardiolipin antibodies (aCL and antibodies against β2GPI (anti-β2GPI into APS. Current trends for the diagnostic evaluation of APS patients propose determination of multiple antiphospholipid antibodies, among them also anti-prothrombin antibodies, to gain a common score which estimates the risk for thrombosis in APS patients. Antiprothrombin antibodies are common in APS patients and are sometimes the only antiphospholipid antibodies being elevated. Methods for their determination differ and have not yet been standardized. Many novel studies confirmed method using phosphatidylserine/prothrombin (aPS/PT ELISA as an antigen on solid phase encompass higher diagnostic accuracy compared to method using prothrombin alone (aPT ELISA. Our research group developed an in-house aPS/PT ELISA with increased analytical sensitivity which enables the determination of all clinically relevant antiprothrombin antibodies. aPS/PT exhibited the highest percentage of lupus anticoagulant activity compared to aCL and anti-β2GPI. aPS/PT antibodies measured with the in-house method associated with venous thrombosis and presented the strongest independent risk factor for the presence of obstetric complications among all tested antiphospholipid antibodies

  2. Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity.

    Science.gov (United States)

    Giuntini, Serena; Beernink, Peter T; Reason, Donald C; Granoff, Dan M

    2012-01-01

    Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity. Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity. The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.

  3. Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics

    International Nuclear Information System (INIS)

    Ayyagari, R.R.; Khan-Dawood, F.S.

    1987-01-01

    Epidermal growth factor receptors are present in many reproductive tissues but have not been demonstrated in the human corpus luteum. To determine the presence of epidermal growth factor receptors and its binding characteristics, we carried out studies on the plasma cell membrane fraction of seven human corpora lutea (days 16 to 25) of the menstrual cycle. Specific epidermal growth factor receptors were present in human corpus luteum. Insulin, nerve growth factor, and human chorionic gonadotropin did not competitively displace epidermal growth factor binding. The optimal conditions for corpus luteum-epidermal growth factor receptor binding were found to be incubation for 2 hours at 4 degrees C with 500 micrograms plasma membrane protein and 140 femtomol 125 I-epidermal growth factor per incubate. The number (mean +/- SEM) of epidermal growth factor binding sites was 12.34 +/- 2.99 X 10(-19) mol/micrograms protein; the dissociation constant was 2.26 +/- 0.56 X 10(-9) mol/L; the association constant was 0.59 +/- 0.12 X 10(9) L/mol. In two regressing corpora lutea obtained on days 2 and 3 of the menstrual cycle, there was no detectable specific epidermal growth factor receptor binding activity. Similarly no epidermal growth factor receptor binding activity could be detected in ovarian stromal tissue. Our findings demonstrate that specific receptors for epidermal growth factor are present in the human corpus luteum. The physiologic significance of epidermal growth factor receptors in human corpus luteum is unknown, but epidermal growth factor may be involved in intragonadal regulation of luteal function

  4. An experimental test of stroke recovery by implanting a hyaluronic acid hydrogel carrying a Nogo receptor antibody in a rat model

    International Nuclear Information System (INIS)

    Ma Jun; Tian Weiming; Hou Shaoping; Xu Qunyuan; Spector, Myron; Cui Fuzhai

    2007-01-01

    The objective of the study was to determine the effects of a hyaluronic-acid-based (HA-based) hydrogel implant, carrying a polyclonal antibody to the Nogo-66 receptor (NgR), on adult rats that underwent middle cerebral artery occlusion (MCAO). Behavioral tests of a forelimb-reaching task suggested that the disabled function of the impaired forelimb in this stroke model was ameliorated by the implant to a certain extent. These behavioral findings were correlated with immunohistochemical results of investigating the distribution of NgR antibody, neurofilaments (NF) and neuron-specific class III β-tubulin (TuJ1) in the brain sections. The porous hydrogel functioned as a scaffold to deliver the NgR antibody, support cell migration and development. In addition, it was found NF-positive and TuJ1-positive expressions were distributed in the implanted hydrogel. Collectively, the results demonstrate the promise of the HA hydrogel as a scaffold material and the delivery vehicle of the NgR antibody for the repair of defects and the support of neural regeneration in the brain

  5. Monoclonal antibody 1.6.1 against human MPL receptor allows HSC enrichment of CB and BM CD34(+)CD38(-) populations.

    Science.gov (United States)

    Petit Cocault, Laurence; Fleury, Maud; Clay, Denis; Larghero, Jérôme; Vanneaux, Valérie; Souyri, Michèle

    2016-04-01

    Thrombopoietin (TPO) and its receptor Mpl (CD110) play a crucial role in the regulation of hematopoietic stem cells (HSCs). Functional study of Mpl-expressing HSCs has, however, been hampered by the lack of efficient monoclonal antibodies, explaining the very few data available on Mpl(+) HSCs during human embryonic development and after birth. Investigating the main monoclonal antibodies used so far to sort CD110(+) cells from cord blood (CB) and adult bone marrow (BM), we found that only the recent monoclonal antibody 1.6.1 engineered by Immunex Corporation was specific. Using in vitro functional assays, we found that this antibody can be used to sort a CD34(+)CD38(-)CD110(+) population enriched in hematopoietic progenitor stem cells, both in CB and in adult BM. In vivo injection into NSG mice further indicated that the CB CD34(+)CD38(-)CD110(+) population is highly enriched in HSCs compared with both CD34(+)CD38(-)CD110(-) and CD34(+)CD38(-) populations. Together our results validate MAb1.6.1 as an important tool, which has so far been lacking, in the HSC field. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  6. An experimental test of stroke recovery by implanting a hyaluronic acid hydrogel carrying a Nogo receptor antibody in a rat model

    Energy Technology Data Exchange (ETDEWEB)

    Ma Jun [Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Tian Weiming [Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Hou Shaoping [Beijing Institute of Neuroscience, Capital University of Medical Sciences, Beijing 100054 (China); Xu Qunyuan [Beijing Institute of Neuroscience, Capital University of Medical Sciences, Beijing 100054 (China); Spector, Myron [Tissue Engineering, VA Boston Healthcare System, Harvard Medical School, Boston, MA (United States); Cui Fuzhai [Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China)

    2007-12-15

    The objective of the study was to determine the effects of a hyaluronic-acid-based (HA-based) hydrogel implant, carrying a polyclonal antibody to the Nogo-66 receptor (NgR), on adult rats that underwent middle cerebral artery occlusion (MCAO). Behavioral tests of a forelimb-reaching task suggested that the disabled function of the impaired forelimb in this stroke model was ameliorated by the implant to a certain extent. These behavioral findings were correlated with immunohistochemical results of investigating the distribution of NgR antibody, neurofilaments (NF) and neuron-specific class III {beta}-tubulin (TuJ1) in the brain sections. The porous hydrogel functioned as a scaffold to deliver the NgR antibody, support cell migration and development. In addition, it was found NF-positive and TuJ1-positive expressions were distributed in the implanted hydrogel. Collectively, the results demonstrate the promise of the HA hydrogel as a scaffold material and the delivery vehicle of the NgR antibody for the repair of defects and the support of neural regeneration in the brain.

  7. Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

    International Nuclear Information System (INIS)

    Nagai, Kentaro; Nakahata, Shingo; Shimosaki, Shunsuke; Tamura, Tomohiro; Kondo, Yuudai; Baba, Takashi; Taki, Tomohiko; Taniwaki, Masafumi; Kurosawa, Gene; Sudo, Yukio; Okada, Seiji; Sakoda, Sumio; Morishita, Kazuhiro

    2014-01-01

    Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Up to 20% of oral dysplasia cases have been suggested to undergo malignant transformation to OSCC; however, there are no methods to predict OSCC development. In this study, to identify the genes associated with oral dysplasia progression, we performed genomic copy number analyses of genomic DNA samples isolated from primary oral dysplasia and OSCC via the microdissection method and found elevated expression of transferrin receptor C (TfR1/TFRC) with genomic amplification in oral dysplasia and OSCC. The expression rate of TFRC in OSCC was significantly higher than that in dysplasia, suggesting that OSCC disease progression might be related to TFRC expression. Additionally, we investigated the in vitro and in vivo impacts of a newly established anti-human TFRC monoclonal antibody, which was isolated from a human cDNA library using the phage-display method, on cell proliferation and survival. The anti-TFRC antibody blocked the interaction between transferrin and TFRC and consequently inhibited iron uptake, leading to the iron deprivation-mediated suppression of cell growth and induction of apoptosis. Moreover, we demonstrated that the anti-TFRC antibody efficiently inhibited tumor growth in a murine xenograft OSCC model. Therefore, we suggest our developed complete human anti-human TFRC antibody as a useful, novel treatment for oral dysplasia and OSCC

  8. Use of antibodies against the variable regions of the T-cell receptor alpha/beta heterodimer for the study of cutaneous T-cell lymphomas.

    Science.gov (United States)

    Ralfkiaer, E; Wollf-Sneedorff, A; Vejlsgaard, G L

    1991-11-01

    Recent studies have suggested that antibodies against the variable (V) regions of the T-cell antigen receptor (TCR) may be used as markers for clonality and malignancy in T-cell infiltrates. We have investigated this by examining biopsy samples from 45 patients with cutaneous T-cell lymphomas (CTCL) for reactivity with seven antibodies against different V-gene families on the TCR alpha/beta heterodimer, i.e. ICI (V beta 5a), W112 (V beta 5b), OT145 (V beta 6a), 16G8 (V beta 8a), S511 (V beta 12a), F1 (V alpha 2a) and LC4 (alpha beta Va). Serial biopsies were available in 13 patients and a total of 62 samples were studied. The neoplastic cells in five cases were positive for either V beta 5 (one case), V beta 6 (one case), V beta 8 (two cases) or V beta 12 (one case). In the remaining 40 cases, no staining was seen of the neoplastic cells. These findings indicate that while antibodies against the TCR V-regions may be used as clonotypic markers for certain T-cell neoplasms, there is as yet not a sufficient number of anti-TCR V-region antibodies available for the routine diagnosis of these conditions.

  9. A novel monoclonal antibody of human stem cell factor inhibits umbilical cord blood stem cell ex vivo expansion

    Directory of Open Access Journals (Sweden)

    Fan Jie

    2012-12-01

    Full Text Available Abstract Stem cell factor (SCF activates hematopoietic stem cell (HSC self-renewal and is being used to stimulate the ex vivo expansion of HSCs. The mechanism by which SCF supports expansion of HSCs remains poorly understood. In cord blood ex vivo expansion assays, a newly produced anti-SCF monoclonal antibody (clone 23C8 was found to significantly inhibit the expansion of CD34+ cells. This antibody appears to bind directly to a part of SCF that is critical for biological activity toward expansion of CD34+ cells, which is located in the first 104 amino acids from the NH2-terminus.

  10. Napsin A and Thyroid Transcription Factor-1-Positive Cerebellar Tumor with Epidermal Growth Factor Receptor Mutation

    Directory of Open Access Journals (Sweden)

    Taiji Kuwata

    2011-12-01

    Full Text Available We present a very rare case of cerebellar metastasis of unknown origin, in which a primary lung adenocarcinoma was diagnosed by pathological examination of a cerebellar metastatic tumor, using immunohistochemical markers and epidermal growth factor receptor (EGFR mutation of primary lung cancer. A 69-year-old woman was admitted to our hospital because of a hemorrhagic cerebellar tumor and multiple small brain tumors. She underwent cerebellar tumor resection. On pathological examination, the tumor was diagnosed as adenocarcinoma. However, the primary tumor site was unidentifiable even with several imaging inspections. On immunohistochemical analysis, the resected tumor was positive for napsin A and thyroid transcription factor-1. In addition, an EGFR mutation was detected in the tumor. Therefore, primary lung cancer was diagnosed and the patient was started on gefitinib (250 mg/day therapy.

  11. Production of thyrotropin receptor antibodies in acute phase of infectious mononucleosis due to Epstein-Barr virus primary infection: a case report of a child.

    Science.gov (United States)

    Nagata, Keiko; Okuno, Keisuke; Ochi, Marika; Kumata, Keisuke; Sano, Hitoshi; Yoneda, Naohiro; Ueyama, Jun-Ichi; Matsushita, Michiko; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Kanzaki, Susumu; Hayashi, Kazuhiko

    2015-01-01

    Various autoantibodies have been reported to be detected during the progression of infectious mononucleosis. We observed a case of infectious mononucleosis due to Epstein-Barr virus primary infection for 2 months, and noticed the transiently increased titer of thyrotropin receptor autoantibodies detected at the acute phase on the 3rd day after admission. At that time, real-time quantitative PCR also revealed the mRNA expressions of an immediate early lytic gene, BZLF1, and a latent gene, EBNA2. The expression of BZLF1 mRNA means that Epstein-Barr virus infects lytically, and EBNA2 protein has an important role in antibody production as well as the establishment of Epstein-Barr virus latency. These results suggest that Epstein-Barr virus lytic infection is relevant to thyrotropin receptor autoantibody production. Thyrotropin receptor autoantibodies stimulate thyroid follicular cells to produce excessive thyroid hormones and cause Graves' disease. Recently, we reported the thyrotropin receptor autoantibody production from thyrotropin receptor autoantibody-predisposed Epstein-Barr virus-infected B cells by the induction of Epstein-Barr virus lytic infection in vitro. This case showed in vivo findings consistent with our previous reports, and is important to consider the pathophysiology of Graves' disease and one of the mechanisms of autoimmunity.

  12. An assay for serum vitamin-B12 and for intrinsic factor antibody type I by means of hog intrinsic factor

    International Nuclear Information System (INIS)

    Hudak, J.; Berger, Z.; Varga, L.

    1980-01-01

    A new radioassay method was elaborated for the determination of the plasma level of vitamin B 12 and of the intrinsic factor antibody type I. The assay applies vitamin-B 12 labelled with 58 Co, but replaces human intrinsic factor by hog intrinsic factor. 124 cases were investigated by both the original and this modified method, and the results were in very good agreement. (L.E.)

  13. Characterization of the glucagon-like peptide-1 receptor in male mouse brain using a novel antibody and in situ hybridization

    DEFF Research Database (Denmark)

    Jensen, Casper Bo; Pyke, Charles; Rasch, Morten Grønbech

    2017-01-01

    was abundantly expressed in numerous regions including the septal nucleus, the hypothalamus and the brain stem. GLP-1R protein expression was also observed on neuronal projections in brain regions devoid of any mRNA which has not been observed in earlier reports. Taken together, these findings provide new......Glucagon-like peptide-1 (GLP-1) is a physiological regulator of appetite and long-acting GLP-1 receptor agonists (GLP-1RA) lower food intake and bodyweight in both human and animal studies. The effects are mediated through brain GLP-1Rs, and several brain nuclei expressing the GLP-1R may...... be involved. To date, mapping the complete location of GLP-1R protein in the brain has been challenged by lack of good antibodies and the discrepancy between mRNA and protein especially relevant in neuronal axonal processes. Here, we present a novel and specific monoclonal GLP-1R antibody...

  14. Effects of anti-vascular endothelial growth factor monoclonal antibody (bevacizumab on lens epithelial cells

    Directory of Open Access Journals (Sweden)

    Jun JH

    2016-06-01

    Full Text Available Jong Hwa Jun,1 Wern-Joo Sohn,2 Youngkyun Lee,2 Jae-Young Kim21Department of Ophthalmology, School of Medicine, Dongsan Medical Center, Keimyung University, 2Department of Oral Biochemistry, School of Dentistry, IHBR, Kyungpook National University, Daegu, South KoreaAbstract: The molecular and cellular effects of anti-vascular endothelial growth factor monoclonal antibody (bevacizumab on lens epithelial cells (LECs were examined using both an immortalized human lens epithelial cell line and a porcine capsular bag model. After treatment with various concentrations of bevacizumab, cell viability and proliferation patterns were evaluated using the water-soluble tetrazolium salt assay and 5-bromo-2'-deoxyuridine enzyme-linked immunosorbent assay, respectively. The scratch assay and Western blot analysis were employed to validate the cell migration pattern and altered expression levels of signaling molecules related to the epithelial–mesenchymal transition (EMT. Application of bevacizumab induced a range of altered cellular events in a concentration-dependent manner. A 0.1–2 mg/mL concentration demonstrated dose-dependent increase in proliferation and viability of LECs. However, 4 mg/mL decreased cell proliferation and viability. Cell migrations displayed dose-dependent retardation from 0.1 mg/mL bevacizumab treatment. Transforming growth factor-β2 expression was markedly increased in a dose-dependent manner, and α-smooth muscle actin, matrix metalloproteinase-9, and vimentin expression levels showed dose-dependent changes in a B3 cell line. Microscopic observation of porcine capsular bag revealed changes in cellular morphology and a decline in cell density compared to the control after 2 mg/mL treatment. The central aspect of posterior capsule showed delayed confluence, and the factors related to EMT revealed similar expression patterns to those identified in the cell line. Based on these results, bevacizumab modulates the proliferation

  15. Nonclinical safety of mavrilimumab, an anti-GMCSF receptor alpha monoclonal antibody, in cynomolgus monkeys: Relevance for human safety

    Energy Technology Data Exchange (ETDEWEB)

    Ryan, Patricia C., E-mail: ryanp@medimmune.com [MedImmune, LLC, Gaithersburg, MD (United States); Sleeman, Matthew A. [MedImmune, LLC, Cambridge (United Kingdom); Rebelatto, Marlon [MedImmune, LLC, Gaithersburg, MD (United States); Wang, Bing; Lu, Hong [MedImmune, LLC, Moutain View, CA (United States); Chen, Xiaomin [Novartis, East Hanover, NJ (United States); Wu, Chi-Yuan [MedImmune, LLC, Moutain View, CA (United States); Hinrichs, Mary Jane; Roskos, Lorin [MedImmune, LLC, Gaithersburg, MD (United States); Towers, Heidi [MedImmune, LLC, Cambridge (United Kingdom); McKeever, Kathleen; Dixit, Rakesh [MedImmune, LLC, Gaithersburg, MD (United States)

    2014-09-01

    Mavrilimumab (CAM-3001) is an investigational human IgG4 monoclonal antibody (MAb) targeting GM-CSF receptor alpha which is currently being developed for the treatment of RA. GM-CSF plays a central role in the pathogenesis of rheumatoid arthritis (RA) through the activation, differentiation, and survival of macrophages and neutrophils. To support clinical development, the nonclinical safety of mavrilimumab was evaluated in several studies with cynomolgus monkeys as the pharmacologically relevant species. Comprehensive toxicity parameters were assessed in each study, and treatment duration ranged from 4 to 26 weeks. Mavrilimumab has an acceptable safety profile in monkeys with no changes in any parameters other than microscopic findings in lung. In several studies, minimal accumulation of foamy alveolar macrophages was observed. This finding was only seen in studies of at least 11 weeks duration, was reversible following a dose-free recovery period and was considered non-adverse. At higher dose levels (≥ 30 mg/kg/week), in a 26-week repeat-IV dose study, the presence of lung foreign material, cholesterol clefts, and granulomatous inflammation was also observed in a few animals and was considered adverse. The dose- and time-related accumulation of foamy macrophages in lung following exposure to mavrilimumab observed in several NHP studies was expected based upon the known role of GM-CSFRα signaling in the function of alveolar macrophages. Overall, a clean no-observed-adverse-effect-level (NOAEL) without any effects in lung was established and provided adequate clinical safety margins. In clinical studies in RA patients, mavrilimumab has demonstrated good clinical activity with adequate safety to support further clinical development. A Phase 2b study of mavrilimumab in subjects with RA is in progress. - Highlights: • Mavrilimumab is a MAB targeting GM-CSFRα being developed for RA therapy. • Mavrilimumab has an acceptable safety profile in cynomolgus monkeys.

  16. Antibody-mediated immunity to the obligate intracellular bacterial pathogen Coxiella burnetii is Fc receptor- and complement-independent

    Directory of Open Access Journals (Sweden)

    Heinzen Robert A

    2009-05-01

    Full Text Available Abstract Background The obligate intracellular bacterial pathogen Coxiella burnetii causes the zoonosis Q fever. The intracellular niche of C. burnetii has led to the assumption that cell-mediated immunity is the most important immune component for protection against this pathogen. However, passive immunization with immune serum can protect naïve animals from challenge with virulent C. burnetii, indicating a role for antibody (Ab in protection. The mechanism of this Ab-mediated protection is unknown. Therefore, we conducted a study to determine whether Fc receptors (FcR or complement contribute to Ab-mediated immunity (AMI to C. burnetii. Results Virulent C. burnetii infects and replicates within human dendritic cells (DC without inducing their maturation or activation. We investigated the effects of Ab opsonized C. burnetii on human monocyte-derived and murine bone marrow-derived DC. Infection of DC with Ab-opsonized C. burnetii resulted in increased expression of maturation markers and inflammatory cytokine production. Bacteria that had been incubated with naïve serum had minimal effect on DC, similar to virulent C. burnetii alone. The effect of Ab opsonized C. burnetii on DC was FcR dependent as evidenced by a reduced response of DC from FcR knockout (FcR k/o compared to C57Bl/6 (B6 mice. To address the potential role of FcR in Ab-mediated protection in vivo, we compared the response of passively immunized FcR k/o mice to the B6 controls. Interestingly, we found that FcR are not essential for AMI to C. burnetii in vivo. We subsequently examined the role of complement in AMI by passively immunizing and challenging several different strains of complement-deficient mice and found that AMI to C. burnetii is also complement-independent. Conclusion Despite our data showing FcR-dependent stimulation of DC in vitro, Ab-mediated immunity to C. burnetii in vivo is FcR-independent. We also found that passive immunity to this pathogen is independent of

  17. Nonclinical safety of mavrilimumab, an anti-GMCSF receptor alpha monoclonal antibody, in cynomolgus monkeys: Relevance for human safety

    International Nuclear Information System (INIS)

    Ryan, Patricia C.; Sleeman, Matthew A.; Rebelatto, Marlon; Wang, Bing; Lu, Hong; Chen, Xiaomin; Wu, Chi-Yuan; Hinrichs, Mary Jane; Roskos, Lorin; Towers, Heidi; McKeever, Kathleen; Dixit, Rakesh

    2014-01-01

    Mavrilimumab (CAM-3001) is an investigational human IgG4 monoclonal antibody (MAb) targeting GM-CSF receptor alpha which is currently being developed for the treatment of RA. GM-CSF plays a central role in the pathogenesis of rheumatoid arthritis (RA) through the activation, differentiation, and survival of macrophages and neutrophils. To support clinical development, the nonclinical safety of mavrilimumab was evaluated in several studies with cynomolgus monkeys as the pharmacologically relevant species. Comprehensive toxicity parameters were assessed in each study, and treatment duration ranged from 4 to 26 weeks. Mavrilimumab has an acceptable safety profile in monkeys with no changes in any parameters other than microscopic findings in lung. In several studies, minimal accumulation of foamy alveolar macrophages was observed. This finding was only seen in studies of at least 11 weeks duration, was reversible following a dose-free recovery period and was considered non-adverse. At higher dose levels (≥ 30 mg/kg/week), in a 26-week repeat-IV dose study, the presence of lung foreign material, cholesterol clefts, and granulomatous inflammation was also observed in a few animals and was considered adverse. The dose- and time-related accumulation of foamy macrophages in lung following exposure to mavrilimumab observed in several NHP studies was expected based upon the known role of GM-CSFRα signaling in the function of alveolar macrophages. Overall, a clean no-observed-adverse-effect-level (NOAEL) without any effects in lung was established and provided adequate clinical safety margins. In clinical studies in RA patients, mavrilimumab has demonstrated good clinical activity with adequate safety to support further clinical development. A Phase 2b study of mavrilimumab in subjects with RA is in progress. - Highlights: • Mavrilimumab is a MAB targeting GM-CSFRα being developed for RA therapy. • Mavrilimumab has an acceptable safety profile in cynomolgus monkeys.

  18. Anti-citrullinated protein antibody and rheumatoid factor in patients with end-stage renal disease.

    Science.gov (United States)

    Romic, Zeljko; Unic, Adriana; Derek, Lovorka; Zivkovic, Marcela; Marijancevic, Domagoj; Kes, Petar; Pehar, Mario

    2009-01-01

    Patients with end-stage renal disease (ESRD) and on hemodialysis (HD) are at increased risk for developing rheumatoid arthritis (RA), as a result of defective immunity. Our aim was to examine if ESRD and the length of HD treatment impact the clinical utility of antibodies to cyclic citrullinated peptides (anti-CCP) and rheumatoid factor (RF) as diagnostic tools for RA. We included 94 subjects in our study: 37 healthy volunteers and 57 patients with ESRD who had been undergoing HD for 1-12 years, and without confirmed RA. In order to test our hypothesis, we measured and correlated anti-CCP and RF as laboratory markers of RA. Our study showed that there is no significant difference between values for anti-CCP (p=0.11) and RF (p=0.98) in control subjects as well as in patients undergoing HD, regardless of the length of time that patients had been undergoing HD treatment. Our study indicates that HD does not impair the specificity of anti-CCP and RF for RA in patients where the disease has not yet developed. Future prospective studies may show whether there is any use in determinating RF, and especially anti-CCP, as early predictors of RA in patients with ESRD who are at greater risk of developing this condition.

  19. 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

  20. Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

    International Nuclear Information System (INIS)

    Hirose, Tetsuro; Terajima, Hiroaki; Yamauchi, Akira

    1997-01-01

    Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125 I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3 H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125 I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125 I-insulin specific binding was not affected. The decrease in 125 I-epidermal growth factor specific binding was explained by the decrease in the number or available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/10 5 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immuno-detection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125 I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125 I-epidermal growth factor binding

  1. Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

    Directory of Open Access Journals (Sweden)

    Marchal Joëlle

    2005-10-01

    Full Text Available Abstract Background Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. Methods Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. Results Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. Conclusion The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

  2. Receptores do factor estimulante de colónias de macrófagos do robalo

    OpenAIRE

    Oliveira, Márcio Adriano Guiomar de

    2010-01-01

    O receptor do factor estimulante de colónias de macrófagos, também conhecido como receptor do factor estimulante de colónias-1 (CSF1R), é um receptor de um factor de crescimento hematopoiético que é especificamente expresso em células do sistema fagocítico-mononuclear e desempenha um papel essencial no desenvolvimento e regulação destas células. O CSF1R já foi descrito em vários mamíferos e a sua biologia tem sido exaustivamente caracterizada nestes vertebrados mas o conheci...

  3. Human Epidermal Growth Factor Receptor 2 Overexpression in Micropapillary and Other Variants of Urothelial Carcinoma.

    Science.gov (United States)

    Behzatoğlu, Kemal; Yörükoğlu, Kutsal; Demir, Hale; Bal, Nebil

    2016-06-21

    Human epidermal growth factor receptor 2 (HER2) protein overexpression or gene amplification has been shown in urothelial bladder cancer. This could be helpful when using targeted anti-HER2 therapy on these tumors. To evaluate HER2 immunohistochemical expression in conventional urothelial carcinoma (UC), in situ UC, and UC variants primarily in micropapillary urothelial carcinoma (MPUC). The study evaluated 60 MPUC cases; 25 invasive, 20 low-grade noninvasive, and 10 high-grade noninvasive UC cases; 8 in situ UC cases; and 69 UC variant cases. The immunohistochemistry staining was scored according to recommendations of the American Society of Clinical Oncology/College of American Pathologists 2013 HER2 test guideline established for breast cancer and only 3+ staining was considered HER2 overexpression. HER2 overexpression was determined by 3+ staining. 34 of 60 MPUC cases (56%) showed HER2 overexpression (3+ staining). We observed 3+ staining HER2 overexpression in nine of 25 conventional invasive UC cases (36%), four of eight in situ UC cases (50%), and three of six lipid cell variant cases (50%). 3+ staining HER2 overexpression was not seen in eight glandular, six small cell, and five sarcomatoid variant cases. HER2 overexpression was negative in the 20 low-grade noninvasive UC cases but positive in two of the 10 high-grade noninvasive UC cases (20%). We observed HER2 overexpression most commonly in MPUC cases. We also found HER2 overexpression in conventional invasive and in situ UC cases. Pure in situ UC and conventional invasive UC, especially MPUC, could be candidate tumors for treatment with anti-HER2 antibody (trastuzumab therapy). Targeted therapy has a limited place in treatment of bladder cancer. In this study, human epidermal growth factor receptor 2 (HER2) overexpression in bladder carcinomas was evaluated in a large number of cases. Anti-HER2 therapy could be used in bladder cancers, as in breast and gastric cancers. Copyright © 2016 European

  4. The transferrin receptor of Actinobacillus pleuropneumoniae: Quantitation of expression and structural characterization using a peptide-specific monoclonal antibody

    DEFF Research Database (Denmark)

    Bøg, Yang S.; Andresen, Lars Ole; Bastholm, L.

    2001-01-01

    transferrin. This complex was studied using a monoclonal antibody (Mab 1.48) raised against a synthetic peptide corresponding to a hydrophilic domain of Tbp2 common to several A. pp serotypes. The antibody reacted specifically with a 60-70 kDa Tbp2-antigen found in all serotypes of A. pp obtained from iron...... expressing Tbp2 and in wild type A. pp grown under iron restricted conditions. The subcellular location of Tbp2 in A. pp was studied by immunoelectron microscopy using the Mab 1.48. Interestingly, all antibody binding was found inside the A. pp cells, while Tbp2 expressed in recombinant E. coli was found...

  5. Anti-cyclic citrullinated peptide antibodies and rheumatoid factor in Sjögren's syndrome.

    Science.gov (United States)

    Barcelos, Filipe; Abreu, Isabel; Patto, José Vaz; Trindade, Hélder; Teixeira, Ana

    2009-01-01

    The purpose of this study was to evaluate the prevalence and clinical significance of anti-cyclic citrullinated peptide antibodies (anti-CCP-Abs), IgM and IgA rheumatoid factors (RFs) in primary Sjögren's Syndrome (pSS). We compared clinical and serological characteristics of 31 pSS and 31 Rheumatoid Arthritis (RA) patients. Both, anti-CCP-Abs and RFs (IgM, IgA) directed against Fc determinants of IgG from humans and rabbit were detected by enzyme-linked immunosorbent assay (ELISA). We included 31 blood donors as control group for the evaluation of RFs and anti-CCP-Abs. Nine (29%) pSS patients presented arthritis, and 10 (32,3%) RA patients also had secondary Sjögren's syndrome (sSS) RESULTS: IgM and IgA RFs prevalence was similar in pSS and RA, whichever the antigene (Human or Rabbit IgG) used. However, RA patients with sSS showed a tendency to present more often RF positivity, longer disease duration and higher ESR and CRP when compared with pSS patients with arthritis. Anti-CCP-Abs were detected in 64,5% of RA patients and in only 6,9% of pSS patients (p<0,0005). Anti-CCP-Abs were more often positive in RA patients with sSS (RA/sSS) (8 patients, 80%) than in RA patients without sSS (18 patients, 58,1%), and were absent in pSS patients with arthritis. RF-positive pSS patients presented more often pulmonary involvement and higher inflammatory parameters, and less often neuropathy compared to RF-negative patients. In controls, anti-CCP-Abs were absent and RFs were negligible. Anti-CCP-Abs were detected in only a few pSS patients, none of whom presented arthritis, which contrasts with the high frequency of these antibodies in RA/sSS. These results suggest that anti-CCP-Abs could be useful in the distinction between pSS and RA with sSS. Although not useful for the differential diagnosis between RA and pSS, RFs may have a prognostic role in pSS.

  6. Multistep change in epidermal growth factor receptors during spontaneous neoplastic progression in Chinese hamster embryo fibroblasts

    International Nuclear Information System (INIS)

    Wakshull, E.; Kraemer, P.M.; Wharton, W.

    1985-01-01

    Whole Chinese hamster embryo lineages have been shown to undergo multistep spontaneous neoplastic progression during serial passage in culture. The authors have studied the binding, internalization, and degradation of 125 I-labeled epidermal growth factor at four different stages of transformation. The whole Chinese hamster embryo cells lost cell surface epidermal growth factor receptors gradually during the course of neoplastic progression until only 10% of the receptor number present in the early-passage cells (precrisis) were retained in the late-passage cells (tumorigenic). No differences in internalization rates, chloroquine sensitivity, or ability to degrade hormone between the various passage levels were seen. No evidence for the presence in conditioned medium of transforming growth factors which might mask or down-regulate epidermal growth factor receptor was obtained. These results suggest that a reduction in cell surface epidermal growth factor receptor might be an early event during spontaneous transformation in whole Chinese hamster embryo cells

  7. Hierarchical classification strategy for Phenotype extraction from epidermal growth factor receptor endocytosis screening

    NARCIS (Netherlands)

    L. Cao (Lu); M. Graauw (Marjo de); K. Yan (Kuan); L.C.J. Winkel (Leah C.J.); F.J. Verbeek (Fons)

    2016-01-01

    textabstractBackground: Endocytosis is regarded as a mechanism of attenuating the epidermal growth factor receptor (EGFR) signaling and of receptor degradation. There is increasing evidence becoming available showing that breast cancer progression is associated with a defect in EGFR endocytosis. In

  8. Targeting of the tumor necrosis factor receptor superfamily for cancer immunotherapy

    NARCIS (Netherlands)

    Bremer, Edwin

    2013-01-01

    The tumor necrosis factor (TNF) ligand and cognate TNF receptor superfamilies constitute an important regulatory axis that is pivotal for immune homeostasis and correct execution of immune responses. TNF ligands and receptors are involved in diverse biological processes ranging from the selective

  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. Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    Directory of Open Access Journals (Sweden)

    Ahnen Dennis

    2005-01-01

    Full Text Available Abstract Background Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs is associated with a decreased mortality from colorectal cancer (CRC. NSAIDs induce apoptotic cell death in colon cancer cells in vitro and inhibit growth of neoplastic colonic mucosa in vivo however, the biochemical mechanisms required for these growth inhibitory effects are not well defined. We previously reported that metabolites of the NSAID sulindac downregulate extracellular-signal regulated kinase 1/2 (ERK1/2 signaling and that this effect is both necessary and sufficient for the apoptotic effects of these drugs. The goal of this project was to specifically test the hypothesis that sulindac metabolites block activation and/or expression of the epidermal growth factor (EGF receptor (EGFR. Methods HT29 human colon cancer cells were treated with EGF, alone, or in the presence of sulindac sulfide or sulindac sulfone. Cells lysates were assayed by immunoblotting for phosphorylated EGFR (pEGFR, pY1068, total EGFR, phosphorylated ERK1/2 (pERK1/2, total ERK1/2, activated caspase-3, and α-tubulin. Results EGF treatment rapidly induced phosphorylation of both EGFR and ERK1/2 in HT29 colon cancer cells. Pretreatment with sulindac metabolites for 24 h blocked EGF-induced phosphorylation of both EGFR and ERK1/2 and decreased total EGFR protein expression. Under basal conditions, downregulation of pEGFR and total EGFR was detected as early as 12 h following sulindac sulfide treatment and persisted through at least 48 h. Sulindac sulfone induced downregulation of pEGFR and total EGFR was detected as early as 1 h and 24 h, respectively, following drug treatment, and persisted through at least 72 h. EGFR downregulation by sulindac metabolites was observed in three different CRC cell lines, occurred prior to the observed downregulation of pERK1/2 and induction of apoptosis by these drugs, and was not dependent of caspase activation. Conclusion These results suggest that

  11. Maternally derived anti-fibroblast growth factor 23 antibody as new tool to reduce phosphorus requirement of chicks.

    Science.gov (United States)

    Ren, Zhouzheng; Bütz, Daniel E; Sand, Jordan M; Cook, Mark E

    2017-04-01

    Novel means to reduce phosphate input into poultry feeds and increase its retention would preserve world phosphate reserves and reduce environmental impact of poultry production. Here we show that a maternally derived antibody to a fibroblast growth factor-23 (FGF-23) peptide (GMNPPPYS) alleviated phosphorus deficiency in chicks fed low non-phytate phosphorus (nPP) diets. White Leghorn laying hens were vaccinated with either an adjuvant control or the synthetic FGF-23 peptide, and chicks with control or anti-FGF-23 maternal antibodies were fed a diet containing either 0.13 or 0.45% nPP (experiment 1), and 0.20 or 0.45% nPP (experiment 2) for 14 d. In both experiments, decreasing nPP from 0.45 to 0.13 or 0.20% decreased BW gain, G:F, excreta phosphorus, plasma phosphate, and plasma FGF-23 at all time periods examined (nPP main effect, P posture scores (d 7, 14) and bone lesion scores (d 14) decreased and plasma phosphate (d 14) increased in anti-FGF-23 chicks fed 0.13% nPP, compared to those with control antibody on the same diet (P < 0.05). In experiment 2, chicks with maternal anti-FGF-23 antibody had increased tibiotarsi ash (d 14), and plasma phosphate (d 14) and 1,25(OH)2D3 (d 14) levels, compared to chicks with control antibody (antibody main effect, P < 0.05). BW gain and G:F were increased in chicks with anti-FGF-23 antibody fed 0.20% nPP, compared to control antibody chicks on the same diet, at all time periods examined (P < 0.05). In conclusion, maternally-derived anti-FGF-23 antibody increased phosphorus retention in chicks fed diets containing either 0.13 or 0.20% nPP and thereby, reduced signs of phosphorus deficiency. © 2016 Poultry Science Association Inc.

  12. α7 Nicotinic acetylcholine receptor-specific antibody induces inflammation and amyloid β42 accumulation in the mouse brain to impair memory.

    Directory of Open Access Journals (Sweden)

    Olena Lykhmus

    Full Text Available Nicotinic acetylcholine receptors (nAChRs expressed in the brain are involved in regulating cognitive functions, as well as inflammatory reactions. Their density is decreased upon Alzheimer disease accompanied by accumulation of β-amyloid (Aβ42, memory deficit and neuroinflammation. Previously we found that α7 nAChR-specific antibody induced pro-inflammatory interleukin-6 production in U373 glioblastoma cells and that such antibodies were present in the blood of humans. We raised a hypothesis that α7 nAChR-specific antibody can cause neuroinflammation when penetrating the brain. To test this, C57Bl/6 mice were either immunized with extracellular domain of α7 nAChR subunit α7(1-208 or injected with bacterial lipopolysaccharide (LPS for 5 months. We studied their behavior and the presence of α3, α4, α7, β2 and β4 nAChR subunits, Aβ40 and Aβ42 and activated astrocytes in the brain by sandwich ELISA and confocal microscopy. It was found that either LPS injections or immunizations with α7(1-208 resulted in region-specific decrease of α7 and α4β2 and increase of α3β4 nAChRs, accumulation of Aβ42 and activated astrocytes in the brain of mice and worsening of their episodic memory. Intravenously transferred α7 nAChR-specific-antibodies penetrated the brain parenchyma of mice pre-injected with LPS. Our data demonstrate that (1 neuroinflammation is sufficient to provoke the decrease of α7 and α4β2 nAChRs, Aβ42 accumulation and memory impairment in mice and (2 α7(1-208 nAChR-specific antibodies can cause inflammation within the brain resulting in the symptoms typical for Alzheimer disease.

  13. Epidermal growth factor receptor VIII peptide vaccination is efficacious against established intracerebral tumors.

    Science.gov (United States)

    Heimberger, Amy B; Crotty, Laura E; Archer, Gary E; Hess, Kenneth R; Wikstrand, Carol J; Friedman, Allan H; Friedman, Henry S; Bigner, Darell D; Sampson, John H

    2003-09-15

    The epidermal growth factor receptor (EGFR) is often amplified and structurally rearranged in malignant gliomas and other tumors such as breast and lung, with the most common mutation being EGFRvIII. In the study described here, we tested in mouse models a vaccine consisting of a peptide encompassing the tumor-specific mutated segment of EGFRvIII (PEP-3) conjugated to keyhole limpet hemocyanin [KLH (PEP-3-KLH)]. C57BL/6J or C3H mice were vaccinated with PEP-3-KLH and subsequently challenged either s.c. or intracerebrally with a syngeneic melanoma cell line stably transfected with a murine homologue of EGFRvIII. Control mice were vaccinated with KLH. To test its effect on established tumors, C3H mice were also challenged intracerebrally and subsequently vaccinated with PEP-3-KLH. S.c. tumors developed in all of the C57BL/6J mice vaccinated with KLH in Freund's adjuvant, and there were no long-term survivors. Palpable tumors never developed in 70% of the PEP-3-KLH-vaccinated mice. In the C57BL/6J mice receiving the PEP-3-KLH vaccine, the tumors that did develop were significantly smaller than those in the control group (P PEP-3-KLH vaccination did not result in significant cytotoxic responses in standard cytotoxicity assays; however, antibody titers against PEP-3 were enhanced. The passive transfer of sera from the immunized mice to nonimmunized mice protected 31% of the mice from tumor development (P PEP-3-KLH-vaccinated mice. Peptide vaccination was also sufficiently potent to have marked efficacy against intracerebral tumors, resulting in a >173% increase in median survival time, with 80% of the C3H mice achieving long-term survival (P = 0.014). In addition, C3H mice with established intracerebral tumor that received a single treatment of PEP-3-KLH showed a 26% increase in median survival time, with 40% long-term survival (P = 0.007). Vaccination with an EGFRvIII-specific peptide is efficacious against both s.c. and established intracerebral tumors. The

  14. Regulation of insulin-like growth factor I receptors on vascular smooth muscle cells by growth factors and phorbol esters.

    Science.gov (United States)

    Ververis, J J; Ku, L; Delafontaine, P

    1993-06-01

    Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells. To characterize regulation of vascular IGF I receptors, we performed radioligand displacement experiments using rat aortic smooth muscle cells (RASMs). Serum deprivation for 48 hours caused a 40% decrease in IGF I receptor number. Exposure of quiescent RASMs to platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or angiotensin II (Ang II) caused a 1.5-2.0-fold increase in IGF I receptors per cell. After FGF exposure, there was a marked increase in the mitogenic response to IGF I. IGF I downregulated its receptors in the presence of platelet-poor plasma. Stimulation of protein kinase C (PKC) by exposure of quiescent RASMs to phorbol 12-myristate 13-acetate caused a biphasic response in IGF I binding; there was a 42% decrease in receptor number at 45 minutes and a 238% increase at 24 hours. To determine the role of PKC in growth factor-induced regulation of IGF I receptors, we downregulated PKC by exposing RASMs to phorbol 12,13-dibutyrate (PDBu) for 48 hours. PDGF- and FGF- but not Ang II-mediated upregulation of IGF I receptors was completely inhibited in PDBu-treated cells. Thus, acute PKC activation by phorbol esters inhibits IGF I binding, whereas chronic PKC activation increases IGF I binding. PDGF and FGF but not Ang II regulate vascular IGF I receptors through a PKC-dependent pathway. These data provide new insights into the regulation of vascular smooth muscle cell IGF I receptors in vitro and are of potential importance in characterizing vascular proliferative responses in vivo.

  15. Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01

    DEFF Research Database (Denmark)

    Geisler, C; Plesner, T; Pallesen, G

    1988-01-01

    demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20...

  16. Baseline HCV Antibody Prevalence and Risk Factors among Drug Users in China's National Methadone Maintenance Treatment Program.

    Directory of Open Access Journals (Sweden)

    Changhe Wang

    Full Text Available Hepatitis C virus (HCV is the most common viral infection among injecting drug users worldwide. We aimed to assess HCV antibody prevalence and associated risk factors among clients in the Chinese national methadone maintenance treatment (MMT program.Data from 296,209 clients who enrolled in the national MMT program between March 2004 and December 2012 were analyzed to assess HCV antibody prevalence, associated risk factors, and geographical distribution.Anti-HCV screening was positive for 54.6% of clients upon MMT entry between 2004 and 2012. HCV antibody prevalence at entry declined from 66.8% in 2005 to 45.9% in 2012. The most significant predictors of HCV seropositivity were injecting drug use (adjusted odds ratio [AOR]: 8.34, 95% confidence interval [CI]: 8.17-8.52, p<0.0001 and a history of drug use ≥9 years (AOR: 2.01, 95% CI: 1.96-2.06, p<0.0001. Being female, of Uyghur or Zhuang ethnicity, and unmarried were identified as demographic risk factors (all p-values<0.0001. Of the 28 provincial-level divisions included in the study, we found that 5 divisions had HCV antibody prevalence above 70% and 20 divisions above 50%. The HCV screening rate within 6 months after MMT entry greatly increased from 30.4% in 2004 to 93.1% in 2012.The current HCV antibody prevalence remains alarmingly high among MMT clients throughout most provincial-level divisions in China, particularly among injecting drug users and females. A comprehensive prevention strategy is needed to control the HCV epidemic among MMT clients in China.

  17. EphA2 is a functional receptor for the growth factor progranulin.

    Science.gov (United States)

    Neill, Thomas; Buraschi, Simone; Goyal, Atul; Sharpe, Catherine; Natkanski, Elizabeth; Schaefer, Liliana; Morrione, Andrea; Iozzo, Renato V

    2016-12-05

    Although the growth factor progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for progranulin. Recombinant progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for progranulin offers a new avenue for understanding the underlying mode of action of progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases. © 2016 Neill et al.

  18. A first‐in‐human pharmacodynamic and pharmacokinetic study of a fully human anti‐glucagon receptor monoclonal antibody in normal healthy volunteers

    Science.gov (United States)

    Kostic, Ana; King, Thomas Alexander; Yang, Feng; Chan, Kuo‐Chen; Yancopoulos, George D.; Gromada, Jesper

    2017-01-01

    Aims Glucagon receptor (GCGR) blockers are being investigated as potential therapeutics for type 1 and type 2 diabetes. Here we report the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of REGN1193, a fully human glucagon receptor blocking monoclonal antibody from a first‐in‐human healthy volunteer randomized double‐blinded trial. Methods Healthy men and women received single ascending doses of REGN1193 ranging from 0.05 to 0.6 mg/kg (n = 42) or placebo (n = 14) intravenously. Safety, tolerability and PK were assessed over 106 days. The glucose‐lowering effect of REGN1193 was assessed after induction of hyperglycaemia by serial glucagon challenges. Results REGN1193 was generally well tolerated. There were small (50 mg/dL, and did not require treatment or medical assistance. Concentration‐time profiles suggest a 2‐compartment disposition and marked nonlinearity, consistent with target‐mediated clearance. REGN1193 inhibited the glucagon‐stimulated glucose increase in a dose‐dependent manner. The 0.6 mg/kg dose inhibited the glucagon‐induced glucose area under the curve for 0 to 90 minutes (AUC0‐90 minutes) by 80% to 90% on days 3 and 15, while blunting the increase in C‐peptide. REGN1193 dose‐dependently increased total GLP‐1, GLP‐2 and glucagon, with plasma levels returning to baseline by day 29 in all dose groups. Conclusion REGN1193, a GCGR‐blocking monoclonal antibody, produced a safety, tolerability and PK/PD profile suitable for further clinical development. The occurrence of transient elevations in serum hepatic aminotransferases observed here and reported with several small molecule glucagon receptor antagonists suggests an on‐target effect of glucagon receptor blockade. The underlying mechanism is unknown. PMID:28755409

  19. Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies.

    Science.gov (United States)

    Bornholdt, Zachary A; Ndungo, Esther; Fusco, Marnie L; Bale, Shridhar; Flyak, Andrew I; Crowe, James E; Chandran, Kartik; Saphire, Erica Ollmann

    2016-02-23

    The filovirus surface glycoprotein (GP) mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics. Ebola virus uses its glycoprotein (GP) to enter new host cells. During entry, GP must be cleaved by human enzymes in order for receptor binding to occur. Here, we provide the crystal structure of the cleaved form of Ebola virus GP. We demonstrate that cleavage exposes a site at the top of GP and that this site binds the critical domain C of the receptor, termed Niemann-Pick C1 (NPC1). We perform mutagenesis to find parts of the site essential for binding NPC1 and map distinct roles for an upper, charged crest and lower, hydrophobic trough in cleaved GP. We find that this 3-dimensional site is conserved across the filovirus family and that antibody directed against this site is able to bind cleaved GP from every filovirus tested and neutralize viruses bearing those GPs. Copyright © 2016 Bornholdt et al.

  20. Signal transduction by growth factor receptors: signaling in an instant

    DEFF Research Database (Denmark)

    Dengjel, Joern; Akimov, Vyacheslav; Blagoev, Blagoy

    2007-01-01

    Phosphorylation-based signaling events happening within the first minute of receptor stimulation have so far only been analyzed by classical cell biological approaches like live-cell microscopy. The development of a quench flow system with a time resolution of one second coupled to a read...

  1. A Trematode Parasite Derived Growth Factor Binds and Exerts Influences on Host Immune Functions via Host Cytokine Receptor Complexes.

    Directory of Open Access Journals (Sweden)

    Azad A Sulaiman

    2016-11-01

    Full Text Available The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allowing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF superfamily, with a greater affinity for TGF-β RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-β RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory receptor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is dependent on TGF-β RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs in their evasion of antibody-dependent cell cytotoxicity (ADCC by reducing the NO response of macrophages-again dependent on TGF-β RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for dampened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow

  2. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    Directory of Open Access Journals (Sweden)

    Manetta J

    2014-08-01

    Full Text Available Joseph Manetta, Holly Bina, Paul Ryan, Niles Fox, Derrick R Witcher, Kristine Kikly Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: B-cell activating factor (BAFF is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. Keywords: autoimmunity, B-cell malignancies, B-cell survival factor, BAFF

  3. ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells

    NARCIS (Netherlands)

    Kol, Arjan; Terwisscha van Scheltinga, Anton; Pool, Martin; Gerdes, Christian; de Vries, Elisabeth; de Jong, Steven

    2017-01-01

    Imgatuzumab is a novel glycoengineered anti-epidermal growth factor receptor (EGFR) monoclonal antibody optimized to induce both antibody-dependent cellular cytotoxicity (ADCC) and EGFR signal transduction inhibition. We investigated antiEGFR monoclonal antibodies imgatuzumab and cetuximab-induced

  4. Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system.

    Science.gov (United States)

    Walsh, Matthew C; Lee, JangEun; Choi, Yongwon

    2015-07-01

    Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adapter protein that mediates a wide array of protein-protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of interleukin-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the Toll-like receptor (TLR) family, tumor growth factorreceptors (TGFβR), and T-cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system but also for maintaining immune tolerance, and more recent work has begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Tumor necrosis factor receptor associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system

    Science.gov (United States)

    Walsh, Matthew C.; Lee, JangEun; Choi, Yongwon

    2016-01-01

    Summary Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adaptor protein that mediates a wide array of protein-protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of IL-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the toll-like receptor (TLR) family, tumor growth factorreceptors (TGFβR), and T cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor (IRF) pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system, but also for maintaining immune tolerance, and more recent works have begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs. PMID:26085208

  6. 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)

  7. Monoclonal antibodies in the treatment of metastatic colorectal cancer: a review

    NARCIS (Netherlands)

    Tol, Jolien; Punt, Cornelis J. A.

    2010-01-01

    Two groups of agents targeting either the vascular endothelial growth factor (VEGF) receptor or the epidermal growth factor receptor (EGFR) have been added to the therapeutic arsenal against metastatic colorectal cancer (mCRC). Currently available agents in these groups are the anti-VEGF antibody

  8. Comparison of (/sup 125/I)beta-endorphin binding to rat brain and NG108-15 cells using a monoclonal antibody directed against the opioid receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bidlack, J.M.; O' Malley, W.E.; Schulz, R.

    1988-02-01

    The properties of (/sup 125/I)beta h-endorphin-binding sites from rat brain membranes and membranes from the NG108-15 cell line were compared using a monoclonal antibody directed against the opioid receptor and opioid peptides as probes. The binding of (/sup 125/I)beta h-endorphin to both rat brain and NG108-15 membranes yielded linear Scatchard plots with Kd values of 1.2 nM and 1.5 nM, respectively, and Bmax values of 865 fmol/mg rat brain membrane protein and 1077 fmol/mg NG108-15 membrane protein. A monoclonal antibody, OR-689.2.4, capable of inhibiting mu and delta binding but not kappa binding to rat brain membranes, noncompetitively inhibited the binding of 1 nM (/sup 125/I)beta h-endorphin to rat brain and NG108-15 membranes with an IC50 value of 405 nM for rat brain membranes and 543 nM for NG108-15 membranes. The monoclonal antibody also inhibited the binding of 3 nM (/sup 3/H) (D-penicillamine2, D-penicillamine5) enkephalin to NG108-15 membranes with an IC50 value of 370 nM. In addition to blocking the binding of (/sup 125/I)beta h-endorphin to brain membranes, the antibody also displaced (/sup 125/I)beta h-endorphin from membranes. Site-specific opioid peptides had large variations in their IC50 values depending on whether they were inhibiting (/sup 125/I)beta h-endorphin binding to rat brain or the NG108-15 membranes. When the peptides were tested with the monoclonal antibody for their combined ability to inhibit (/sup 125/I)beta h-endorphin binding to both membrane preparations, the peptides and antibody blocked binding as though they were acting at allosterically coupled sites, not two totally independent sites. These studies suggest that mu-, delta-, and beta-endorphin-binding sites share some sequence homology with the 35,000-dalton protein that the antibody is directed against.

  9. Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column.

    Science.gov (United States)

    Kiyoshi, Masato; Caaveiro, Jose M M; Tada, Minoru; Tamura, Hiroko; Tanaka, Toru; Terao, Yosuke; Morante, Koldo; Harazono, Akira; Hashii, Noritaka; Shibata, Hiroko; Kuroda, Daisuke; Nagatoishi, Satoru; Oe, Seigo; Ide, Teruhiko; Tsumoto, Kouhei; Ishii-Watabe, Akiko

    2018-03-02

    The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

  10. [ALAT and viral RNA as risk factors in 68 blood donors with anti-hepatitis C antibodies].

    Science.gov (United States)

    Tullen, E; De Saussure, P; Soulier-Lauper, M

    1993-01-23

    Determine the risk factors in blood donors with anti hepatitis C antibodies (anti-HCV ab) possible liver involvement and evaluation of their infectious potential by a search for viral RNA in blood. Between July 1990 and October 1991, 19,632 blood donors were screened for hepatitis C. Antibodies to HCV were detected in 74 donors (2nd generation ELISA, Abbott). We evaluated the risk factors, determined ALAT levels and looked for circulating RNA virus by amplification of the non-coding region of the viral genome (RTPCR) in 68 of these 74 donors screened. A control was chosen arbitrarily from 103 donors with high ALAT levels, but with no antibodies to HCV nor detectable circulating viral DNA. The prevalence of anti-HCV ab in blood donors in 0.37%. No risk factor was found in 29 donors (43%). Parenteral exposure (former i.v. drug addiction and history of transfusions) was found to be the mode of transmission of hepatitis C in 23 donors (34%). History of NANB jaundice (non-post transfusion) was reported in 1 donor (1%). The remaining 15 donors (22%) were found to have minor risk factors - either isolated or in combination (exposure, tatoos, multiple sexual partners). Former i.v. drug addiction (p = 0.0000006) as well as a history of transfusions (p = 0.0071) are significantly more frequent in the group of donors with antibodies to HCV. None of the 35 sexual partners of the tested donors proved to be positive. 21 donors (30%) had high ALAT (+2 SD). Viral RNA was detected in blood of 26 donors (38%). The proportion of cases with positive viral RNA was 61% if only those donors with high ALAT levels were taken into consideration (13 positive of 21). Risk factors were found in 39 donors (57%) with antibodies to HCV. History of parenteral exposure was found to be significantly more frequent than in the control group (p = 0.0000054). Sexual transmission within couples was not demonstrated in the population tested. A positive PCR test is a probable indicator of a continuous

  11. Growth inhibition of thyroid follicular cell-derived cancers by the opioid growth factor (OGF) - opioid growth factor receptor (OGFr) axis

    International Nuclear Information System (INIS)

    McLaughlin, Patricia J; Zagon