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Sample records for anti-human cd25 antibody

  1. Cloning and expression of canine CD25 for validation of an anti-human CD25 antibody to compare T regulatory lymphocytes in healthy dogs and dogs with osteosarcoma.

    Science.gov (United States)

    Rissetto, K C; Rindt, H; Selting, K A; Villamil, J A; Henry, C J; Reinero, C R

    2010-05-15

    T regulatory cells (Tregs) are a unique subset of T helper cells that serve to modify/inhibit effector cells of the immune system and thus are essential to prevent autoimmunity. Overzealous Treg activity may contribute to impaired immune responses to cancer. Tregs can be phenotypically identified by proteins expressed on the cell surface (CD4 and CD25) and inside the cell (forkhead box3 (FoxP3)), although in dogs, no anti-canine CD25 antibody exists. We hypothesized that a mouse anti-human CD25 antibody definitively recognizes the canine protein and can be used to identify Tregs in dogs. We describe cloning and transfection of the canine CD25 gene into human HeLa cells with subsequent expression of the canine protein on the cell surface detected using an anti-human CD25 antibody in a flow cytometric assay. Validation of this antibody was used to identify CD4+CD25+FoxP3+ Tregs in 39 healthy dogs and 16 dogs with osteosarcoma (OSA). Results were expressed in five different ways and showed significantly fewer %CD4+CD25+ T lymphocytes expressing FoxP3 in blood of older dogs (>/=7 years) compared with the other two age groups (dogs and those with OSA. While the CD25 antibody can be successfully used in a flow cytometric assay to identify Tregs, this study does not support clinical utility of phenotypic recognition of Tregs in dogs with OSA. Copyright 2010 Elsevier B.V. All rights reserved.

  2. In ovo injection of anti-chicken CD25 monoclonal antibodies depletes CD4+CD25+ T cells in chickens.

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    Shanmugasundaram, Revathi; Selvaraj, Ramesh K

    2013-01-01

    The CD4(+)CD25(+) cells have T regulatory cell properties in chickens. This study investigated the effect of in ovo injection of anti-chicken CD25 monoclonal antibodies (0.5 mg/egg) on CD4(+)CD25(+) cell depletion and on amounts of interleukin-2 mRNA and interferon-γ mRNA in CD4(+)CD25(-) cells posthatch. Anti-chicken CD25 or PBS (control) was injected into 16-d-old embryos. Chicks hatched from eggs injected with anti-chicken CD25 antibodies had a lower CD4(+)CD25(+) cell percentage in the blood until 25 d posthatch. The anti-chicken CD25 antibody injection nearly depleted CD4(+)CD25(+) cells in the blood until 16 d posthatch. At 30 d posthatch, the CD4(+)CD25(+) cell percentage in the anti-CD25-antibody-injected group was comparable with the percentage in the control group. At 16 d posthatch, the anti-chicken CD25 antibody injection decreased CD4(+)CD25(+) cell percentages in the thymus, spleen, and cecal tonsils. Chickens hatched from anti-CD25-antibody-injected eggs had approximately 25% of CD4(+)CD25(+) cells in the cecal tonsils and thymus compared with those in the cecal tonsils and thymus of the control group. The CD4(+)CD25(-) cells from the spleen and cecal tonsils of chicks hatched from anti-chicken-CD25-injected eggs had higher amounts of interferon-γ and interleukin-2 mRNA than CD4(+)CD25(-) cells from the control group. It could be concluded that injecting anti-chicken CD25 antibodies in ovo at 16 d of incubation nearly depleted the CD4(+)CD25(+) cells until 25 d posthatch.

  3. Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on regulatory T cell depletion and CD4+CD25- T cell properties in chickens.

    Science.gov (United States)

    Shanmugasundaram, Revathi; Selvaraj, Ramesh K

    2012-03-01

    Regulatory T cells (Tregs) are defined as CD4(+)CD25(+) cells in chickens. This study examined the effects of an anti-chicken CD25 monoclonal antibody injection (0.5 mg/bird) on in vivo depletion of Tregs and the properties of CD4(+)CD25(-) cells in Treg-depleted birds. The CD4(+)CD25(+) cell percentage in the blood was lower at 8 d post injection than at 0 d. Anti-CD25-mediated CD4(+)CD25(+) cell depletion in blood was maximum at 12 d post injection. The anti-CD25 antibody injection depleted CD4(+)CD25(+) cells in the spleen and cecal tonsils, but not in the thymus, at 12 d post antibody injection. CD4(+)CD25(-) cells from the spleen and cecal tonsils of birds injected with the anti-chicken CD25 antibody had higher proliferation and higher IL-2 and IFNγ mRNA amounts than the controls at 12 d post injection. At 20 d post injection, CD4(+)CD25(+) cell percentages in the blood, spleen and thymus were comparable to that of the 0 d post injection. It could be concluded that anti-chicken CD25 injection temporarily depleted Treg population and increased and IL-2 and IFNγ mRNA amounts in CD4(+)CD25(-) cells at 12d post injection. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells.

    Science.gov (United States)

    Law, Jacqueline P; Hirschkorn, Dale F; Owen, Rachel E; Biswas, Hope H; Norris, Philip J; Lanteri, Marion C

    2009-12-01

    Foxp3 is a key marker for CD4(+) regulatory T cells (T(regs)) and was used in developing a multiparameter flow cytometric panel to identify T(regs). Achieving reproducible staining and analysis first required optimization of Foxp3 staining. We present a comparative study of PCH101, 236A/E7, 3G3, 206D, 150D, and 259D/C7 clones of anti-human-Foxp3 antibodies used in combination with five different fixation/permeabilization buffers. Staining for CD25, CD152, and CD127 was also compared between fixation/permeabilization treatments. Promising antibody/buffer combinations were tested in a panel of peripheral blood mononuclear cells from 10 individuals, and then on fresh versus frozen cells from four individuals. Finally, different fluorochromes coupled to two representative antibodies were compared to optimize separation of Foxp3(+) from Foxp3(-) events. Foxp3 gates were set using two gating strategies based on CD127(+)CD25(-) "non-T(regs)" or based on isotype controls. For Foxp3 staining, the best conditions for fixation/permeabilization were obtained using the eBioscience Foxp3, Imgenex, BioLegend, and BD Foxp3 buffers. Comparing results from 10 subjects, 259D/C7, PCH101, 236A/E7, and 206D antibodies yielded statistically higher levels of Foxp3 cells than those by 150D and 3G3 antibodies (mean = 6.9, 5.1, 4.7, and 3.7% compared with 1.7, and 0.3% of CD25(+)Foxp3(+) events within CD4(+) cells, respectively). Importantly, the "nonspecificity" of some antibodies observed with a Foxp3 gate based on isotype controls could be eliminated by setting the Foxp3 gate on "non-T(regs)". Better separation of Foxp3(+) and Foxp3(-) populations was observed using the PCH101 clone coupled to Alexa647 compared with FITC or the 259D/C7 clone coupled to PE compared with Alexa488 fluorochrome. Foxp3 staining can be highly variable and depends on the choice of antibody/buffer pair and the fluorochrome used. Selecting the correct population for setting the Foxp3 gate is critical to avoid

  5. Biological Evaluation of 131I- and CF750-Labeled Dmab(scFv-Fc Antibodies for Xenograft Imaging of CD25-Positive Tumors

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    Qing Fan

    2014-01-01

    Full Text Available A Dmab(scFv-Fc antibody containing the single chain variable fragment of a humanized daclizumab antibody and the Fc fragment of a human IgG1 antibody was produced via recombinant expression in Pichia pastoris. The Dmab(scFv-Fc antibody forms a dimer in solution, and it specifically binds CD25-positive tumor cells and tumor tissues. For tumor imaging, the Dmab(scFv-Fc antibody was labeled with the 131I isotope and CF750 fluorescent dye, respectively. After intravenous injection of mice bearing CD25-positive tumor xenografts, tumor uptake of the 131I-Dmab(scFv-Fc antibody was visible at 1 h, and clear images were obtained at 5 h using SPECT/CT. After systemic administration of the CF750-Dmab(scFv-Fc antibody, tumor uptake was present as early as 1 h, and tumor xenografts could be kinetically imaged within 9 h after injection. These results indicate that the Dmab(scFv-Fc antibody rapidly and specifically targets CD25-positive tumor cells, suggesting the potential of this antibody as an imaging agent for the diagnosis of lymphomatous-type ATLL.

  6. [Preparation and preliminary application of rabbit anti-human PON2 antibodies(paraoxonase-2)].

    Science.gov (United States)

    Chen, Miao; Yang, Jin-Ju; Li, Shu-Zhen; Liu, Xiao-Lan; Liu, Ying; Zhang, Lin-Jie; Gao, Jian-En; Sun, Qi-Hong

    2008-07-01

    To preparation and characterize the rabbit polyclonal antibodies against human PON2 (paraoxonase-2). A fragment of human PON2 gene which was of low homology with rabbits but of higher hydrophilicity and immunogenicity was selected for recombinant expression in prokaryotic expression system. The rabbits were immunized with the purified GST fusion protein 3 times. The specificity and sensitivity of the anti-human PON2 polyclonal antibodies were detected by Western blot and indirect immunofluorescence. The GST-PON2 fusion protein was highly expressed in Ecoli with a molecular weight of 46 kDa. Western blot analysis proved the rabbit polyclonal antibodies could specifically recognize 39 kDa native PON2 protein expressed in several cells and tissues, such as HeLa cells, U937 cells, and human liver tissue. Indirect immunofluorescence analysis confirmed that PON2 protein was located in the cytoplasm of SY5Y cells. The rabbit polyclonal antibodies against human PON2 can specifically recognize natural protein expressed in human cells and tissues, Which can be used for further study and clinical detection of human PON2.

  7. Clinical significance of serum anti-human papillomavirus 16 and 18 antibodies in cervical neoplasia.

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    Chay, Doo Byung; Cho, Hanbyoul; Kim, Bo Wook; Kang, Eun Suk; Song, Eunseop; Kim, Jae-Hoon

    2013-02-01

    To estimate the clinical significance of serum anti-human papillomavirus (HPV) antibodies and high-risk cervical HPV DNA in cervical neoplasia. The study population comprised patients who were histopathologically diagnosed with cervical intraepithelial neoplasia (CIN) 1 (n=64), CIN 2 and 3 (n=241), cervical cancer (n=170), and normal control participants (n=975). Cervical HPV DNA tests were performed through nucleic acid hybridization assay tests, and serum anti-HPV 16 and 18 antibodies were measured by competitive immunoassay. The associations of HPV DNA and anti-HPV antibodies were evaluated with demographic characteristics and compared according to the levels of disease severity. Anti-HPV antibodies were also investigated with clinicopathologic parameters, including survival data. Among various demographic characteristics, factors involving sexual behavior had a higher tendency of HPV DNA positivity and HPV seropositivity. Human papillomavirus DNA mean titer and positivity were both increased in patients with cervical neoplasia compared with those with normal control participants, but there was no statistical difference among types of cervical neoplasia. Serum anti-HPV 16 antibodies were also able to differentiate cervical neoplasia from a normal control participant and furthermore distinguished CIN 1 from CIN 2 and 3 (odd ratio 2.87 [1.43-5.78], P=.002). In cervical cancer, HPV 16 seropositivity was associated with prolonged disease-free survival according to the univariable analysis (hazard ratio=0.12 [0.01-0.94], P=.044). Serum anti-HPV 16 antibodies can distinguish cervical neoplasia from a normal control and has the advantage of identifying high-grade CIN. Moreover, in cervical cancer, HPV 16 seropositivity may be associated with a more favorable prognosis. II.

  8. Immunohistochemical Analysis of Inflammatory Rheumatoid Synovial Tissues Using Anti-Human Podoplanin Monoclonal Antibody Panel.

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    Suzuki, Tomoto; Takakubo, Yuya; Oki, Hiroharu; Liu, Xing; Honma, Ryusuke; Naganuma, Yasushi; Goodman, Stuart B; Kaneko, Mika K; Kato, Yukinari; Takagi, Michiaki

    2018-02-01

    Podoplanin (PDPN) is a transmembrane sialoglycoprotein, which is expressed in several normal tissues and malignant tumors. Although PDPN expression in rheumatoid arthritis (RA) has been reported, the role of PDPN in RA and other arthritic conditions has not been fully elucidated. In this study, we examined PDPN expression in inflammatory synovial tissues using an anti-human PDPN (hPDPN) monoclonal antibody (mAb) panel to select the most useful one for evaluation of synovitis. Synovial tissue samples were obtained from 11 RA patients and 9 osteoarthritis (OA) patients undergoing joint surgery. PDPN-positive cells were immunostained by a panel of PDPN mAbs (NZ-1, LpMab-3, LpMab-7, LpMab-10, LpMab-12, LpMab-13, and LpMab-17), followed by cell grading of inflammation and cell counting of PDPN-positivity by a quantitative analyzer. Immunohistochemistry showed that PDPN was markedly expressed in both macrophage-like type A and fibroblast-like type B lining cells of the hyperplastic synovial lining cell layer, and macrophages and fibroblasts in the stroma of RA. Among anti-PDPN mAbs, LpMab-12 showed the highest score. In inflammatory OA synovium, PDPN expression was also detectable. Although LpMab-12 also showed the highest score in OA, the difference was not statistically significant. The inflammatory synovitis score of RA was significantly higher than that of OA. PDPN was expressed in inflammatory lining cells and sublining stroma of RA and OA synovium. In the seven anti-hPDPN antibodies examined, LpMab-12 was the most stainable antibody for PDPN in RA synovitis. Thus, LpMab-12 for PDPN has a possible and promising specific biomarker for evaluating synovitis in RA and inflammatory OA.

  9. Differential effects of IL-2 and IL-21 on expansion of the CD4+ CD25+ Foxp3+ T regulatory cells with redundant roles in natural killer cell mediated antibody dependent cellular cytotoxicity in chronic lymphocytic leukemia.

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    Gowda, Aruna; Ramanunni, Asha; Cheney, Carolyn; Rozewski, Darlene; Kindsvogel, Wayne; Lehman, Amy; Jarjoura, David; Caligiuri, Michael; Byrd, John C; Muthusamy, Natarajan

    2010-01-01

    CD4(+) CD25(+) regulatory T cells are expanded in solid and hematological malignancies including chronic lymphocytic leukemia (CLL). Several cytokines and co-stimulatory molecules are required for generation, survival and maintenance of their suppressive effect. We and others have shown direct cytotoxic effect of the novel common gamma chain cytokine interleukin (IL)-21 on primary B cells from CLL patients. Since members of this family of cytokines are known to exhibit their effects on diverse immune cells, we have examined the effects of IL-21 on CLL patient derived regulatory T cell (Treg) induction, expansion and the inhibitory effect on natural killer cells in vitro. We demonstrate here the expression of IL-21 receptor in CD4(+)CD25(High) regulatory cells from CLL patients. In contrast to IL-2, the IL-21 cytokine failed to mediate expansion of regulatory T cells or induced expression of Foxp3 in CD4(+)CD25(Intermediate) or CD4(+)CD25(Dim/-) T cells in whole blood derived from CLL patients. Interestingly, in contrast to their differential effects on expansion of the CD4(+)CD25(+)Foxp3(+)T cells, IL-2 and IL-21 exhibited a redundant role in Treg mediated suppression of NK cell mediated antibody dependent cytotoxicity function. Given the infusion related toxicities and pro-survival effect of IL-2 in CLL, these studies provide a rationale to explore IL-21 as an alternate gamma chain cytokine in CLL therapy.

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

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    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.

  11. 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.

  12. Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

    DEFF Research Database (Denmark)

    Aasted, Bent; Blixenkrone-Møller, Merete; Larsen, Else Bang

    1988-01-01

    Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four...... antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen......-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink....

  13. Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey) by Anti-Human Monoclonal Antibody

    Science.gov (United States)

    de Souza, Hugo Amorim dos Santos; Costa-Correa, Edmar Henrique; Bianco-Junior, Cesare; Andrade, Márcia Cristina Ribeiro; Lima-Junior, Josué da Costa; Pratt-Riccio, Lilian Rose; Daniel-Ribeiro, Cláudio Tadeu; Totino, Paulo Renato Rivas

    2017-01-01

    Non-human primates (NHP) are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors—such as the signal regulatory protein α (SIRPα)—in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC). As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model. PMID:29312325

  14. Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey by Anti-Human Monoclonal Antibody

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    Hugo Amorim dos Santos de Souza

    2017-12-01

    Full Text Available Non-human primates (NHP are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors—such as the signal regulatory protein α (SIRPα—in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC. As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model.

  15. Anti-human SIRPα antibody is a new tool for cancer immunotherapy.

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    Murata, Yoji; Tanaka, Daisuke; Hazama, Daisuke; Yanagita, Tadahiko; Saito, Yasuyuki; Kotani, Takenori; Oldenborg, Per-Arne; Matozaki, Takashi

    2018-02-23

    Interaction of signal regulatory protein α (SIRPα) expressed on the surface of macrophages with its ligand CD47 expressed on target cells negatively regulates phagocytosis of the latter cells by the former. We recently showed that blocking Abs to mouse SIRPα enhanced both the Ab-dependent cellular phagocytosis (ADCP) activity of mouse macrophages for Burkitt's lymphoma Raji cells opsonized with an Ab to CD20 (rituximab) in vitro as well as the inhibitory effect of rituximab on the growth of tumors formed by Raji cells in nonobese diabetic (NOD)/SCID mice. However, the effects of blocking Abs to human SIRPα in preclinical cancer models have remained unclear given that such Abs have failed to interact with endogenous SIRPα expressed on macrophages of immunodeficient mice. With the use of Rag2 -/- γ c -/- mice harboring a transgene for human SIRPα under the control of human regulatory elements (hSIRPα-DKO mice), we here show that a blocking Ab to human SIRPα significantly enhanced the ADCP activity of macrophages derived from these mice for human cancer cells. The anti-human SIRPα Ab also markedly enhanced the inhibitory effect of rituximab on the growth of tumors formed by Raji cells in hSIRPα-DKO mice. Our results thus suggest that the combination of Abs to human SIRPα with therapeutic Abs specific for tumor antigens warrants further investigation for potential application to cancer immunotherapy. In addition, humanized mice, such as hSIRPα-DKO mice, should prove useful for validation of the antitumor effects of checkpoint inhibitors before testing in clinical trials. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  16. 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

  17. Immunohistochemical evaluation of podoplanin in odontogenic tumours & cysts using anti-human podoplanin antibody.

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    Singhal, Namrata; Khanduri, Nitin; Kurup, Deepak; Gupta, Brijesh; Mitra, Pranjan; Chawla, Roshani

    2017-01-01

    Odontogenic Cysts & tumors originate through some aberration from the normal pattern of odontogenesis. Ameloblastoma is one of the most frequent intraosseous odontogenic tumors. However it is no longer appropriate to use the diagnosis of ameloblastoma without specifying the type. Varied-clinical entities of ameloblastoma differ in their biologic behaviour. Odontogenic cysts like dentigerous and radicular cysts are less aggressive in nature than odontogenic tumors. Recently, podoplanin commonly used as a lymphatic endothelial marker in cancers has recently been found to play a possible role in odontogenic tumorigenesis also. Therefore the purpose of this study was to immunohistochemically analyse the expression of podoplanin in ameloblastomas, KCOTs, dentigerous cysts, radicular cysts & dental follicles. Paraffin-embedded tissue specimens of 15 Ameloblastomas (7 follicular, 6 unicystic, 2 desmoplastic),10KCOTs, 5 dentigerous cysts, 5 radicular cysts & 5 dental follicles were immunohistochemically examined using antibody against podoplanin. All ameloblastomas displayed podoplanin expression in ameloblast-like cells of the epithelial islands while the stellate-reticulum like cells exhibited no or weak immunostaining. Expression of podoplanin in KCOTs was strongly positive in the cells of the basal and suprabasal layers & odontogenic epithelial nests. Positive immunoreaction for podoplanin was observed in the inflammatory radicular cysts and inflamed dentigerous cyst only and negative or weak expression in the lining epithelium of uninflamed dentigerous cysts and dental follicles. Our results suggest that podoplanin can be used as a potential proliferative marker to observe the aggressive behaviour of ameloblastomas and KCOTs.

  18. Serological analysis of human anti-human antibody responses in colon cancer patients treated with repeated doses of humanized monoclonal antibody A33.

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    Ritter, G; Cohen, L S; Williams, C; Richards, E C; Old, L J; Welt, S

    2001-09-15

    Mouse monoclonal antibody A33 (mAb A33) recognizes a M(r) 43,000 cell surface glycoprotein (designated A33) expressed in human colonic epithelium and colon cancer but absent from most other normal tissues. In patients, mAb A33 localizes with high specificity to colon cancer and is retained for up to 6 weeks in the cancer but cleared rapidly from normal colon (5-6 days). As a carrier of (125)I or (131)I, mAb A33 has shown antitumor activity. Induction of strong human anti-mouse antibody (immunoglobulin; HAMA) responses in patients, however, limits the use of the murine mAb A33 to very few injections. A humanized version of this antibody (huAb A33) has been prepared for Phase I and II clinical studies in patients with colon cancer. In those studies, immunogenicity of huAb A33 has been monitored using a novel, highly sensitive BIACORE method, which allows measurement of human anti-human antibodies (HAHAs) without the use of secondary reagents. We found that 63% (26 of 41) of the patients treated with repeated doses of huAb A33 developed HAHAs against a conformational antigenic determinant located in the V(L) and V(H) regions of huAb A33. Detailed serological analysis showed two distinct types of HAHAs. HAHA of type I (49% of patients) was characterized by an early onset with peak HAHA levels after 2 weeks of treatment, which declined with ongoing huAb A33 treatment. HAHA of type II (17% of patients) was characterized by a typically later onset of HAHA than in type I and by progressively increasing HAHA levels with each subsequent huAb A33 administration. Colon cancer patients with type I HAHAs did not develop infusion-related adverse events. In contrast, HAHA of type II was indicative of infusion-related adverse events. By using this new method, we were able to distinguish these two types of HAHAs in patients while on antibody treatment, allowing patients to be removed from study prior to the onset of severe infusion-related adverse events.

  19. 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

  20. Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the development aplastic crises.

    Science.gov (United States)

    Sant'Anna, Anadayr L M; Garcia, Rita de Cássia N Cubel; Marzoche, Mônica; da Rocha, Heloisa Helena A Gallo; Paula, Maria Tereza M; Lobo, Clarisse C; Nascimento, Jussara P

    2002-01-01

    The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE), Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140) have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05) was seen between IgG antibody prevalence in male (27.8%) and female (35.5%) patients. Anti-B19 IgG antibodies were more frequent in older (37.6%) than younger (28.2%) than 20 years old patients, although this difference had no statistical significance (p > 0.05). Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

  1. Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the developement aplastic crises

    Directory of Open Access Journals (Sweden)

    SANT'ANNA Anadayr L.M.

    2002-01-01

    Full Text Available The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE, Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140 have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05 was seen between IgG antibody prevalence in male (27.8% and female (35.5% patients. Anti-B19 IgG antibodies were more frequent in older (37.6% than younger (28.2% than 20 years old patients, although this difference had no statistical significance (p > 0.05. Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

  2. Application of four anti-human interferon-alpha monoclonal antibodies for immunoassay and comparative analysis of natural interferon-alpha mixtures

    International Nuclear Information System (INIS)

    Andersson, G.; Lundgren, E.; Ekre, H.P.

    1991-01-01

    Four different mouse monoclonal antibodies to human interferon-alpha (IFN-alpha) were evaluated for application in quantitative and comparative analysis of natural IFN-alpha mixtures. Binding to IFN-alpha subtypes in solution revealed individual reactivity patterns. These patterns changed if the IFN-alpha molecules were immobilized either passively to a surface or bound by another antibody. Also, substitution of a single amino acid in IFN-alpha 2 affected the binding, apparently by altering the conformation. Isoelectric focusing of three natural IFN-alpha preparations from different sources, followed by immunoblotting, resulted in individual patterns with each of the four mAbs and also demonstrated variation in the composition of the IFN-alpha preparations. None of the mAbs was subtype specific, but by combining the different mAbs, and also applying polyclonal anti-human IFN-alpha antibodies, it was possible to design sensitive sandwich ELISAs with broad or more limited IFN-alpha subtype specificity

  3. Influences of Pre-formed Donor-Specific Anti-Human Leukocyte Antigen Antibodies in Living-Donor Renal Transplantation: Results With Graft Immunocomplex Capture Fluorescence Analysis.

    Science.gov (United States)

    Nakamura, T; Ushigome, H; Watabe, K; Imanishi, Y; Masuda, K; Matsuyama, T; Harada, S; Koshino, K; Iida, T; Nobori, S; Yoshimura, N

    2017-06-01

    Advances in immunosuppressants enable organ transplantation for sensitized patients. However, influences of pre-formed donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) have not been fully understood in renal transplantation (RT). On the other hand, immunocomplex capture fluorescence analysis (ICFA) is a reliable method to detect donor-specific anti-HLA antibodies and HLA antigen complexes. Graft ICFA can detect DSA in an allograft (g-DSA). To elucidate the consequences of pre-formed DSA, 198 patients who underwent living-donor RT were enrolled for this study (observation period: 57.8 ± 34.9 months); 187 patients in the DSA- group (excluding ABO-incompatible cases) and 11 patients in the DSA+ group. Before RT, all DSA+ patients had undergone rituximab administration and plasmapheresis. For a graft ICFA, the biopsy specimen (1 × 10 5 cells) was dissolved, and HLA antigens were captured by anti-HLA beads. Finally, DSA-HLA complexes were detected by means of PE-conjugated anti-human IgG antibodies and analyzed by use of a Luminex system. A ratio (sample/blank beads, mean of fluorescence intensity) was calculated: ≥1.0 was determined as positive g-DSA. There were no significant differences in 5-year graft survival (87.9%/100% in the DSA-/DSA+ groups, respectively). In terms of antibody-mediated rejection (AMR), within 1 month after RT, pathologically determined AMR occurred 3.2% and 63.4% in the DSA- and DSA+ groups, respectively (P < .0001). However, interestingly, more than half of them (57.1%) indicated only subclinical AMR, that is, no fluctuation of S-Cr. As representative of 2 cases of subclinical AMR, g-DSA deposition could be confirmed (1.15 ± 0.04) at 1 hour after reperfusion by graft ICFA. Furthermore, g-DSA shifted to 2.20 ± 0.98 at 3 weeks after transplantation, along with a decline in s-DSA mean of fluorescence intensity (1718-506.5). Although pathologically determined AMR occurred more frequently in pre-formed DSA+ recipients, it

  4. Biological Validation of Plant-derived Anti-human Colorectal Cancer Monoclonal Antibody CO17-1A

    NARCIS (Netherlands)

    Jamal, Arshad; Ahn, Mi-Hyun; Song, Mira; Oh, Eun-Yi; Hong, Juyeon; Choo, Young-Kug; Ko, Kinarm; Han, Yeon Soo; Oh, Seung Han; Van Der Linden, Joke; Leusen, Jeanette H. W.; Ko, Kisung

    We validated expression and biological activities of plant-derived monoclonal antibody (MAb(P)) CO17-1A for its efficacy in cancer immunotherapy. PCR and immunoblot analyses demonstrated insertion and expression of heavy and light chains of MAb CO17-1A in transgenic plants, respectively. Confocal

  5. Transglutaminase-catalyzed covalent multimerization of Camelidae anti-human TNF single domain antibodies improves neutralizing activity.

    Science.gov (United States)

    Plagmann, Ingo; Chalaris, Athena; Kruglov, Andrei A; Nedospasov, Sergei; Rosenstiel, Philip; Rose-John, Stefan; Scheller, Jürgen

    2009-06-15

    Tumor necrosis factor (TNF) plays an important role in chronic inflammatory disorders, such as Rheumatoid Arthritis and Crohn's disease. Recently, monoclonal Camelidae variable heavy-chain domain-only antibodies (V(H)H) were developed to antagonize the action of human TNF (hTNF). Here, we show that hTNF-V(H)H does not interfere with hTNF trimerization, but competes with hTNF for hTNF-receptor binding. Moreover, we describe posttranslational dimerization and multimerization of hTNF-V(H)H molecules in vitro catalyzed by microbial transglutaminases (MTG). The ribonuclease S-tag-peptide was shown to act as a peptidyl substrate in covalent protein cross-linking reactions catalyzed by MTG from Streptomyces mobaraensis. The S-tag sequence was C-terminally fused to the hTNF-V(H)H and the fusion protein was expressed and purified from Escherichia coli culture supernatants. hTNF-V(H)H-S-tag fusion proteins were efficiently dimerized and multimerized by MTG whereas hTNF-V(H)H was not susceptible to protein cross-linking. Cell cytotoxicity assays, using hTNF as apoptosis inducing cytokine, revealed that dimerized and multimerized hTNF-V(H)H proteins were much more active than the monomeric hTNF-V(H)H. We hypothesize that improved inhibition by dimeric and multimeric single chain hTNF-V(H)H proteins is caused by avidity effects.

  6. Semi-high-throughput isolation and N-glycan analysis of human fibrinogen using monolithic supports bearing monoclonal anti-human fibrinogen antibodies.

    Science.gov (United States)

    Vidic, Urška; Trbojević-Akmačić, Irena; Černigoj, Urh; Albers, Malena; Gašperšič, Jernej; Pučić-Baković, Maja; Vidič, Jana; Štrancar, Aleš; Lauc, Gordan

    2017-11-01

    Fibrinogen (FIB) is a secretory glycoprotein synthesized by hepatocytes that has a key role in blood clotting. Its glycosylation has not been studied in detail and little is known about the biological variability of FIB N-glycosylation, mainly due to the lack of fast, simple, and robust approaches to purify FIB from blood plasma samples. In recent years, customised chromatographic monoliths have been used for a variety of biological applications due to their unique characteristics. Here we describe development and optimisation of monolithic supports bearing monoclonal anti-human fibrinogen antibodies in a single column as well as in multi-well plate formats with high FIB specificity and binding capacity for fast immunoaffinity purification of FIB from human blood samples. The developed semi-high-throughput workflow has been successfully applied for FIB immunoaffinity isolation and subsequent ultra performance liquid chromatography N-glycosylation analysis in ten healthy human individuals, demonstrating the potential of monolithic supports in glycomics studies. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Evaluation of an automated assay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline, and equine SAA.

    Science.gov (United States)

    Christensen, M; Jacobsen, S; Ichiyanagi, T; Kjelgaard-Hansen, M

    2012-12-01

    Major acute phase proteins (APPs) have proven diagnostically useful in dogs, cats and horses with routine use facilitated by commercially available automated heterologous assays. An automated assay applicable across all three species would highly facilitate further dissemination of routine use, and the aim of this study was to validate an automated latex agglutination turbidimetric immunoassay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline and equine SAA. Serum samples from 60 dogs, 40 cats and 40 horses were included. Intra- and inter-assay imprecision, linearity and detection limit (DL) were determined to assess analytical performance. To assess clinical performance, equine and feline SAA measurements were compared with parallel measurements using a previously validated automated SAA assay in a method comparison setting, and by assessing overlap performance of canine SAA in healthy dogs and diseased dogs with and without systemic inflammation. Intra- and inter-assay CVs ranged between 1.9-4.6% and between 3.0-14.5%, respectively. Acceptable linearity within a clinically relevant range of SAA concentrations was observed for all three species. The DL was 1.06 mg/L. Method comparison revealed acceptable agreement of the two assays measuring feline and equine SAA, and the overlap performance of canine SAA was acceptable. The tested assay measured SAA in canine, feline and equine serum with analytical and overlap performance acceptable for clinical purposes so improving practical aspects of clinical APP application. The monoclonal nature of the antibodies suggests strong, long-term inter-batch performance stability. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. CD25+ B-1a Cells Express Aicda

    Directory of Open Access Journals (Sweden)

    Hiroaki Kaku

    2017-06-01

    Full Text Available B-1a cells are innate-like B-lymphocytes producing natural antibodies. Activation-induced cytidine deaminase (AID, a product of the Aicda gene, plays a central role in class-switch recombination and somatic hypermutation in B cells. Although a role for Aicda in B-1a cells has been suggested on the basis of experiments with knock out (KO mice, whether B-1a cells express Aicda, and if so, which B-1a cell subpopulation expresses Aicda, remains unknown. Here, we demonstrate that B-1 cells express Aicda, but at a level below that expressed by germinal center (GC B cells. We previously reported that B-1a cells can be subdivided based on CD25 expression. We show here that B-1a cell Aicda expression is concentrated in the CD25+ B-1a cell subpopulation. These results suggest the possibility that previous studies of memory B cells identified on the basis of Aicda expression may have inadvertently included an unknown number of CD25+ B-1a cells. Although B-1a cells develop normally in the absence of Aicda, a competitive reconstitution assay reveals enhanced vigor for AID KO B-1a cell bone marrow (BM progenitors, as compared with wild-type BM B-1 cell progenitors. These results suggest that AID inhibits the development of B-1a cells from BM B-1 cell progenitors in a competitive environment.

  9. Subclinical antibody-mediated rejection due to anti-human-leukocyte-antigen-DR53 antibody accompanied by plasma cell-rich acute rejection in a patient with cadaveric kidney transplantation.

    Science.gov (United States)

    Katsuma, Ai; Yamamoto, Izumi; Komatsuzaki, Yo; Niikura, Takahito; Kawabe, Mayuko; Okabayashi, Yusuke; Yamakawa, Takafumi; Katsumata, Haruki; Nakada, Yasuyuki; Kobayashi, Akimitsu; Tanno, Yudo; Miki, Jun; Yamada, Hiroki; Ohkido, Ichiro; Tsuboi, Nobuo; Yamamoto, Hiroyasu; Yokoo, Takashi

    2016-07-01

    A 56-year-old man who had undergone cadaveric kidney transplantation 21 months earlier was admitted to our hospital for a protocol biopsy; he had a serum creatinine level of 1.2 mg/dL and no proteinuria. Histological features showed two distinct entities: (i) inflammatory cell infiltration, in the glomerular and peritubular capillaries and (ii) focal, aggressive tubulointerstitial inflammatory cell infiltration, predominantly plasma cells, with mild tubulitis (Banff 13 classification: i2, t1, g2, ptc2, v0, ci1, ct1, cg0, cv0). Immunohistological studies showed mildly positive C4d immunoreactivity in the peritubular capillaries. The patient had donor specific antibody to human-leucocyte-antigen-DR53. We diagnosed him with subclinical antibody-mediated rejection accompanied by plasma cell-rich acute rejection. Both antibody-mediated rejection due to anti- human-leucocyte-antigen -DR53 antibodies and plasma cell-rich acute rejection are known to be refractory and have a poor prognosis. Thus, we started plasma exchange with intravenous immunoglobulin and rituximab for the former and 3 days of consecutive steroid pulse therapy for the latter. Three months after treatment, a follow-up allograft biopsy showed excellent responses to treatment for both histological features. This case report considers the importance of an early diagnosis and appropriate intervention for subclinical antibody-mediated rejection due to donor specific antibody to human-leucocyte-antigen-DR53 and plasma cell-rich acute rejection. © 2016 Asian Pacific Society of Nephrology.

  10. HAHA--nothing to laugh about. Measuring the immunogenicity (human anti-human antibody response) induced by humanized monoclonal antibodies applying ELISA and SPR technology.

    Science.gov (United States)

    Nechansky, Andreas

    2010-01-05

    Immunogenicity induced by passively applied proteins is a serious issue because it is directly related to the patient's safety. The out-come of an immune reaction to a therapeutic protein can range from transient appearance of antibodies without any clinical significance to severe life threatening conditions. Within this article, enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) methodology to measure immunogenicity are compared and the pros and cons are discussed.

  11. Labelling of anti-human bladder tumor chimeric antibody with 99Tcm and radioimmunoimaging of bladder carcinoma xenograft in nude mice

    International Nuclear Information System (INIS)

    Zhang Chunli; Wang Rongfu; Fu Zhanli; Bai Yin; Ding Yi; Yu Lizhang

    2003-01-01

    Objective: To study the in vitro immunoreactivity and in vivo tissue distribution, tumor targeting property of anti-human bladder tumor human-murine chimeric antibody (ch-BDI) labeled with 99 Tc m and to investigate its possibility for being used in guiding diagnosis and guiding therapy of bladder cancer. Methods: The ch-BDI was labeled with 99 Tc m by improved Schwarz method and the labeled antibody was purified by Sephadex G-50. Labeling yield and radiochemical purity were measured by paper chromatography. The immunoreactive fraction and association constant (K a ) were measured by Lindmo method and Scatchard analysis, respectively. 11.1 MBq (30 μg) 99 Tc m -ch-BDI was intravenously injected into nude mice bearing human bladder cancer xenografts in the right thigh and radioimmunoimaging (RII) was performed 2, 6, 20 and 24 h postinjection. The images were processed by region of interest (ROI) method to acquire the counts of whole body and the tumor and the counts ratios of tumor to contralateral normal tissue or to tissues of other non-tumor bearing organs. The mice were killed after 24 h postinjection imaging and tissue distribution was measured. %ID/g and target to nontarget (T/NT) ratios were calculated. Results: The labeling yield and radiochemical purity of 99 Tc m -ch-BDI were (66.5±7.3)% and >90%, respectively. The immunoreactive fraction was 76% and K a was 3.56 x 10 9 L/mol. RII showed that the tumor was clearly visualized 6 h postinjection and becoming clearer along with time prolonging. The radioactivity of whole body decreased rapidly with time, whereas the radioactivity of the tumor decreased slowly. The T/NT ratios was increased with time. Biodistribution results showed that tumor uptake was 17.4%ID/g 24 h postinjection. T/NT ratios were very high except for the kidney. T/NT ratios for brain, muscle, intestinal wall, bone and heart wall were 136.0, 55.1, 39.3, 29.7 and 27.9, respectively. Conclusion: 99 Tc m -ch-BDI exhibits excellent

  12. Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

    Science.gov (United States)

    Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

    2017-02-01

    Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

  13. Immunity to experimental Salmonella typhimurium infections in rats. Transfer of immunity with primed CD4+CD25high and CD4+CD25low T lymphocytes

    DEFF Research Database (Denmark)

    Thygesen, P; Brandt, L; Jørgensen, T

    1994-01-01

    M and IgG antibodies. Cell sorting revealed that 2/3 of the primed CD4+ T lymphocytes expressed high levels of CD25. Cell transfer revealed that both CD25high and CD25low expression populations could induce immunity against a lethal dose of S. typhimurium, whilst antibody analysis revealed that antibody...... levels were not correlated with protection against S. typhimurium infections, although it showed that a higher and more persistent level of specific IgG antibodies was produced in animals receiving the CD4+CD25high fraction. It is concluded that 10(4) primed CD4+ T lymphocytes can induce immunity......The protective effect of primed CD4+ T lymphocytes against a lethal dose of 10(8) viable Salmonella typhimurium was studied in Lewis rats. Primed CD4+ T lymphocytes were obtained by inoculating Lewis rats with a non-lethal dose of 10(6) viable S. typhimurium. Four weeks after the infection, spleen...

  14. CD4+CD25Hi FoxP3+ regulatory T cells in long-term cardiac xenotransplantation.

    Science.gov (United States)

    Singh, Avneesh K; Chan, Joshua L; Seavey, Caleb N; Corcoran, Philip C; Hoyt, Robert F; Lewis, Billeta G T; Thomas, Marvin L; Ayares, David L; Horvath, Keith A; Mohiuddin, Muhammad M

    2018-03-01

    CD4+CD25 Hi FoxP3+ T (Treg) cells are a small subset of CD4+ T cells that have been shown to exhibit immunoregulatory function. Although the absolute number of Treg cells in peripheral blood lymphocytes (PBL) is very small, they play an important role in suppressing immune reactivity. Several studies have demonstrated that the number of Treg cells, rather than their intrinsic suppressive capacity, may contribute to determining the long-term fate of transplanted grafts. In this study, we analyzed Treg cells in PBL of long-term baboon recipients who have received genetically modified cardiac xenografts from pig donors. Heterotopic cardiac xenotransplantation was performed on baboons using hearts obtained from GTKO.hCD46 (n = 8) and GTKO.hCD46.TBM (n = 5) genetically modified pigs. Modified immunosuppression regimen included antithymocyte globulin (ATG), anti-CD20, mycophenolate mofetil (MMF), cobra venom factor (CVF), and costimulation blockade (anti-CD154/anti-CD40 monoclonal antibody). FACS analysis was performed on PBLs labeled with anti-human CD4, CD25, and FoxP3 monoclonal antibodies (mAb) to analyze the percentage of Treg cells in six baboons that survived longer than 2 months (range: 42-945 days) after receiving a pig cardiac xenograft. Total WBC count was low due to immunosuppression in baboons who received cardiac xenograft from GTKO.hCD46 and GTKO.hCD46.hTBM donor pigs. However, absolute numbers of CD4+CD25 Hi FoxP3 Treg cells in PBLs of long-term xenograft cardiac xenograft surviving baboon recipients were found to be increased (15.13 ± 1.50 vs 7.38 ± 2.92; P < .018) as compared to naïve or pre-transplant baboons. Xenograft rejection in these animals was correlated with decreased numbers of regulatory T cells. Our results suggest that regulatory T (Treg) cells may contribute to preventing or delaying xenograft rejection by controlling the activation and expansion of donor-reactive T cells, thereby masking the antidonor immune response

  15. Performance and reliability of five commercial enzyme-linked immunosorbent assay kits in screening for anti-human immunodeficiency virus antibody in high-risk subjects.

    OpenAIRE

    Ozanne, G; Fauvel, M

    1988-01-01

    Anti-human immunodeficiency virus enzyme-linked immunosorbent assay kits marketed by Electro-Nucleonics Inc. (ENI), Genetic Systems Corp. (GSC), Organon Teknika Inc. (OTI), Ortho Diagnostic Systems Inc. (ODSI), and Wellcome Diagnostics (WD) were evaluated by using 289 randomly selected serum samples from a high-risk population and 53 serum samples likely to produce false-positive results. The radioimmunoprecipitation assay was used as the reference test. Sensitivities ranged from 96.51% (ODSI...

  16. Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

    Directory of Open Access Journals (Sweden)

    Koushan Sineh sepehr

    2013-02-01

    Full Text Available Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells.

  17. CD4+ CD25+ cells in type 1 diabetic patients with other autoimmune manifestations

    Directory of Open Access Journals (Sweden)

    Dalia S. Abd Elaziz

    2014-11-01

    Full Text Available The existence of multiple autoimmune disorders in diabetics may indicate underlying primary defects of immune regulation. The study aims at estimation of defects of CD4+ CD25+high cells among diabetic children with multiple autoimmune manifestations, and identification of disease characteristics in those children. Twenty-two cases with type 1 diabetes associated with other autoimmune diseases were recruited from the Diabetic Endocrine and Metabolic Pediatric Unit (DEMPU, Cairo University along with twenty-one normal subjects matched for age and sex as a control group. Their anthropometric measurements, diabetic profiles and glycemic control were recorded. Laboratory investigations included complete blood picture, glycosylated hemoglobin, antithyroid antibodies, celiac antibody panel and inflammatory bowel disease markers when indicated. Flow cytometric analysis of T-cell subpopulation was performed using anti-CD3, anti-CD4, anti-CD8, anti-CD25 monoclonal antibodies. Three cases revealed a proportion of CD4+ CD25+high below 0.1% and one case had zero counts. However, this observation did not mount to a significant statistical difference between the case and control groups neither in percentage nor absolute numbers. Significant statistical differences were observed between the case and the control groups regarding their height, weight centiles, as well as hemoglobin percentage, white cell counts and the absolute lymphocytic counts. We concluded that, derangements of CD4+ CD25+high cells may exist among diabetic children with multiple autoimmune manifestations indicating defects of immune controllers.

  18. Anti-human platelet antigen (HPA)-1a antibodies may affect trophoblast functions crucial for placental development: a laboratory study using an in vitro model.

    Science.gov (United States)

    Eksteen, Mariana; Heide, Gøril; Tiller, Heidi; Zhou, Yan; Nedberg, Nora Hersoug; Martinez-Zubiaurre, Inigo; Husebekk, Anne; Skogen, Bjørn R; Stuge, Tor B; Kjær, Mette

    2017-04-21

    Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder caused by maternal antibodies against paternal human platelet antigens (HPAs) on fetal platelets. Antibodies against HPA-1a are accountable for the majority of FNAIT cases. We have previously shown that high levels of maternal anti-HPA-1a antibodies are associated with clinically significant reduced birth weight in newborn boys. Chronic inflammatory placental lesions are associated with increased risk of reduced birth weight and have previously been reported in connection with FNAIT pregnancies. The HPA-1a epitope is located on integrin β3 that is associated with integrin αIIb (the fibrinogen receptor) on platelets and megakaryocytes. Integrin β3 is also associated with integrin αV forming the αVβ3 integrin heterodimer, the vitronectin receptor, which is expressed on various cell types, including trophoblast cells. It is therefore thinkable that maternal anti-HPA-1a antibodies present during early pregnancy may affect placenta function through binding to the HPA-1a antigen epitope on invasive throphoblasts. The aim of the study was to examine whether interaction of a human anti-HPA-1a monoclonal antibody (mAb) with HPA-1a on trophoblast cells affect adhesion, migration and invasion of extravillous trophoblast cells. An in vitro model with human anti-HPA-1a mAb, clone 26.4, and the first trimester extravillous trophoblast cell line HTR8/SVneo was employed. The xCELLigence system was utilized to assess the possible effect of anti-HPA-1a mAb on adhesion and migration of HTR8/SVneo cells. Specially designed chambers precoated with Matrigel were used to assess the effect on the invasive capacity of cells. We found that human anti-HPA-1a mAb 26.4 partially inhibits adhesion and migratory capacity of HTR8/SVneo cells. Our findings suggest that anti-HPA-1a antibodies may affect trophoblast functions crucial for normal placental development. Future studies including primary throphoblast

  19. 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

  20. Use of CD25 as an immunohistochemical marker for acquired ocular toxoplasmosis

    Directory of Open Access Journals (Sweden)

    Cristina Miyamoto

    2010-10-01

    Full Text Available PURPOSE: Toxoplasmosis is the most common cause of posterior infectious uveitis worldwide. It is often impossible to determine its congenital or acquired nature. Interleukin-2 (IL-2 in peripheral blood has been described as a possible marker for acquired toxoplasmosis. The purpose of this study is to evaluate the histopathological characteristics of ocular toxoplasmosis cases using CD25 as a marker for the expression of interleukin-2. METHODS: Ten formalin-fixed, paraffin-embedded enucleated globes from ten immunocompetent patients with clinical diagnosis of toxoplasmosis were evaluated. Four patients had the acquired form of ocular toxoplasmosis (positive IgM while six were IgM negative and IgG positive for toxoplasmosis. Histopathological slides were reviewed for the extension of the retinal necrosis, number of toxo cysts, the granulomatous inflammatory reaction, the presence of T and B cells within the choroid and the IL-2 expression. Immunohistochemistry using monoclonal antibodies was performed to observe the expression of CD4, CD8, CD20, CD25, and CD68. RESULTS: The histopathological evaluation disclosed no differences between acquired and the other ocular toxoplasmosis cases regarding the characteristics studied. However, CD25 showed a higher expression of IL-2 on the 4 acquired cases of ocular toxoplasmosis compared to the remainders. CONCLUSIONS: To the best of our knowledge, this is the first report showing that the use of CD25 as a marker for interleukin-2 could differentiate acquired ocular toxoplasmosis.

  1. A single serum dilution enzyme-linked immunosorbent assay for determining anti-human papillomavirus (HPV) antibody titres in humans immunised with prophylactic HPV vaccines.

    Science.gov (United States)

    Jin, Yingji; Kim, Hyoung Jin; Yim, Ga Won; Kim, Young Tae; Chang, Don Yong; Kim, Hong-Jin

    2012-07-01

    Two types of prophylactic human papillomavirus (HPV) vaccines are currently available. However, there is no simple monitoring system for assessing acquired immunity that can cope simultaneously with large numbers of serum samples. Approximately 30% of women with normal cytology are known to be seropositive for HPV types 16 and 18 because of the high prevalence of these HPV types. Therefore, to be useful the monitoring system has to discriminate clearly between vaccine recipients and other serology groups. However, there has never been any focus on developing a method to satisfy this condition. In this study, we developed a high-throughput single-serum-dilution enzyme-linked immunoassay (ELISA) system for determining anti-HPV antibody titres following vaccination. We optimised the conditions for each ELISA step to increase its accuracy and precision and to avoid the high background of non-specific reactions that is a major problem for serology assays. The new ELISA system has superior linearity, accuracy and reproducibility. Moreover, it clearly discriminated between antibody levels in vaccine recipients and those in other serology groups such as individuals with normal cervical cytology and those with cervical cancer. Therefore, this single-serum-dilution ELISA should be very useful for assessing the acquired immunity of HPV vaccine recipients. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Factors associated with anti-human leukocyte antigen antibodies in patients supported with continuous-flow devices and effect on probability of transplant and post-transplant outcomes

    DEFF Research Database (Denmark)

    Alba, Ana C; Tinckam, Kathryn; Foroutan, Farid

    2015-01-01

    and outcomes. METHODS: We included 143 consecutive heart failure patients who received a CF-VAD as a bridge-to-transplant at 3 institutions. Factors associated with post-VAD peak panel reactive antibodies (PRA) among several measurements were identified using multivariable linear regression. A parametric...... survival model was used to assess transplant waiting time and probability, risk of rejection, and a composite outcome of rejection, graft failure, and death. RESULTS: Thirty-six patients (25%) were female; mean age was 47 ± 13 years. Eighty-one patients (57%) had a pre-VAD PRA of 0%, and 16 were highly...... sensitized (PRA > 80%). Age, female sex, and pre-VAD PRA were independently associated with post-VAD PRA. A 10-year increase in age was associated with a 5% decrease in post-VAD PRA (p = 0.03). Post-VAD PRA was 19% higher in women vs men (p PRA was associated with a 4...

  3. Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

    Science.gov (United States)

    Iwamoto, Ryo; Takagi, Mika; Akatsuka, Jun-Ichi; Ono, Ken-Ichiro; Kishi, Yoshiro; Mekada, Eisuke

    2016-04-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

  4. Donor-specific anti-human leukocyte antigen antibodies were associated with primary graft failure after unmanipulated haploidentical blood and marrow transplantation: a prospective study with randomly assigned training and validation sets.

    Science.gov (United States)

    Chang, Ying-Jun; Zhao, Xiang-Yu; Xu, Lan-Ping; Zhang, Xiao-Hui; Wang, Yu; Han, Wei; Chen, Huan; Wang, Feng-Rong; Mo, Xiao-Dong; Zhang, Yuan-Yuan; Huo, Ming-Rui; Zhao, Xiao-Su; Y, Kong; Liu, Kai-Yan; Huang, Xiao-Jun

    2015-07-10

    Small studies suggest an association of donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) with primary graft failure (GF) following haploidentical stem cell transplantation, but primary graft rejection (GR) was not discriminated from primary poor graft function (PGF). In this study, we aimed to determine the association of DSAs with primary GF, including GR and PGF, in patients who underwent unmanipulated haploidentical blood and marrow transplantation. A total of 345 subjects were prospectively recruited and randomly selected as training group (n = 173) and validation group (n = 172). Patient plasma/serum was screened. For HLA antibody positive samples with a median fluorescent intensity (MFI) >500, DSAs were further tested using a LABScreen Single Antigen Kit (One Lambda). A total of 342 patients (99.1%) achieved sustained myeloid engraftment. The median times to neutrophil engraftment and platelet engraftment were 13 days (range, 8-28 days) and 18 days (range, 6-330 days), respectively. The cumulative incidence of primary GF was 6.4 ± 1.3% and included GR (0.9 ± 0.5%) and PGF (5.5 ± .2%). Of the 345 cases tested, 39 (11.3%) were DSA positive. Multivariate models showed that DSAs (MFI ≥ 10,000) were correlated to primary GR (P < 0.001) and that DSAs (MFI ≥ 2000) were strongly associated with primary PGF (P = 0.005). All patients were classified into three groups for analysis. Group A included cases that were DSA negative and those with a DSA MFI <2000 (n = 316), group B included cases with a 2000 ≤ MFI < 10,000 (n = 19), and group C included cases with a MFI ≥ 10,000 (n = 10). The DSAs were associated with an increased incidence of the primary GF (3.2 vs. 31.6 vs. 60%, for groups A, B, and C, respectively, P < 0.001), transplant-related mortality (TRM) rate (17.2 vs. 14.7 vs. 33.3%, for groups A, B, and C, respectively, P = 0.022), and inferior overall survival (OS, 77.3 vs. 85.3 vs. 44.4%, for groups A, B, and C

  5. [THE INDUCTION OF CD25 EXPRESSION IN Jurkat T CELLS].

    Science.gov (United States)

    Shatrova, A N; Mityushova, E V; Aksenov, N V; Marakhova, L L

    2015-01-01

    The expression of an α-subunit of interleukin-2 receptor (IL-2Rα) was assessed by quantifying activation-induced upregulation of CD25 in IL-2-independent Jurkat leukemic cell line. It has been found that in growing Jurkat culture within 24 h, phytohemagglutinin (PHA, 5 μg/ml) or PHA in combination with 12,13-phorbol dibutirate (PDBu, 10(-8)M) increase the number of CD25+ cells to 32.3 ± 3.4% (n = 11) and 44.8 ± 8.6% (n = 6) respectively. Interleukin-2 (IL-2, 200 U/ml) alone or in combination with PDBu did not induce CD25 expression in Jurkat cells. All the tested stimulatory agencies affected neither the proliferation status no the growth of Jurkat cell cultures. In contrast to human blood T cells, WHI-P131, a selective pharmacological inhibitor of JAK/STAT signaling and CD25 expression, did not decrease the number of induced CD25+ cells in Jurkat culture. Flow cytometry analysis revealed a dose-dependent decrease in the proportion of cells in G1 phase and an increase in the proportion of cells in G2/M phase in WHI-P131-treated Jurkat cultures. It has been also found that WHI-P131 induces G2/M arrest in the absence of PHA or PDBu. We have concluded that (1) the IL-2-independent T cells of Jurkat line had not loss the mechanism for IL-2Rα expression in response to T cell receptor activation, (2) in the transformed T cells, WHI-P131 can arrest cell cycle at G2/M phase and has effects on targets other than IL-2 receptor-associated tyrosine kinase JAK3.

  6. Characterization of murine anti-human Fab antibodies for use in an immunoassay for generic quantification of human Fab fragments in non-human serum samples including cynomolgus monkey samples.

    Science.gov (United States)

    Stubenrauch, Kay; Wessels, Uwe; Essig, Ulrich; Kowalewsky, Frank; Vogel, Rudolf; Heinrich, Julia

    2013-01-01

    capturing. In conclusion, the two murine anti-human Fabs are versatile tools as capture and detection reagents for human antibodies in generic and specific PK ELISA formats for animal studies. Their use in specific ELISAs as detection reagents allows the usage of Fc-fusion proteins as capture reagents. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Identification and Functional Characterization of Human Cd4+Cd25+ T Cells with Regulatory Properties Isolated from Peripheral Blood

    Science.gov (United States)

    Jonuleit, Helmut; Schmitt, Edgar; Stassen, Michael; Tuettenberg, Andrea; Knop, Jurgen; Enk, Alexander H.

    2001-01-01

    A subpopulation of peripheral human CD4+CD25+ T cells that expresses CD45RO, histocompatibility leukocyte antigen DR, and intracellular cytotoxic T lymphocyte–associated antigen (CTLA) 4 does not expand after stimulation and markedly suppresses the expansion of conventional T cells in a contact-dependent manner. After activation, CD4+CD25+ T cells express CTLA-4 on the surface detectable for several weeks. These cells show a G1/G0 cell cycle arrest and no production of interleukin (IL)-2, IL-4, or interferon (IFN)-γ on either protein or mRNA levels. The anergic state of CD4+CD25+ T cells is not reversible by the addition of anti-CD28, anti–CTLA-4, anti–transforming growth factor β, or anti–IL-10 antibody. However, the refractory state of CD4+CD25+ T cells was partially reversible by the addition of IL-2 or IL-4. These data demonstrate that human blood contains a resident T cell population with potent regulatory properties. PMID:11390435

  8. Partial CD25 Antagonism Enables Dominance of Antigen-Inducible CD25high FOXP3+ Regulatory T Cells As a Basis for a Regulatory T Cell-Based Adoptive Immunotherapy.

    Science.gov (United States)

    Wilkinson, Daniel S; Ghosh, Debjani; Nickle, Rebecca A; Moorman, Cody D; Mannie, Mark D

    2017-01-01

    FOXP3 + regulatory T cells (Tregs) represent a promising platform for effective adoptive immunotherapy of chronic inflammatory disease, including autoimmune diseases such as multiple sclerosis. Successful Treg immunotherapy however requires new technologies to enable long-term expansion of stable, antigen-specific FOXP3 + Tregs in cell culture. Antigen-specific activation of naïve T cells in the presence of TGF-β elicits the initial differentiation of the FOXP3 + lineage, but these Treg lines lack phenotypic stability and rapidly transition to a conventional T cell (Tcon) phenotype during in vitro propagation. Because Tregs and Tcons differentially express CD25, we hypothesized that anti-CD25 monoclonal antibodies (mAbs) would only partially block IL-2 signaling in CD25 high FOXP3 + Tregs while completely blocking IL-2 responses of CD25 low-intermediate Tcons to enable preferential outgrowth of Tregs during in vitro propagation. Indeed, murine TGF-β-induced MOG-specific Treg lines from 2D2 transgenic mice that were maintained in IL-2 with the anti-CD25 PC61 mAb rapidly acquired and indefinitely maintained a FOXP3 high phenotype during long-term in vitro propagation (>90% FOXP3 + Tregs), whereas parallel cultures lacking PC61 rapidly lost FOXP3. These results pertained to TGF-β-inducible "iTregs" because Tregs from 2D2-FIG Rag1 - / - mice, which lack thymic or natural Tregs, were stabilized by continuous culture in IL-2 and PC61. MOG-specific and polyclonal Tregs upregulated the Treg-associated markers Neuropilin-1 (NRP1) and Helios (IKZF2). Just as PC61 stabilized FOXP3 + Tregs during expansion in IL-2, TGF-β fully stabilized FOXP3 + Tregs during cellular activation in the presence of dendritic cells and antigen/mitogen. Adoptive transfer of blastogenic CD25 high FOXP3 + Tregs from MOG35-55-specific 2D2 TCR transgenic mice suppressed experimental autoimmune encephalomyelitis in pretreatment and therapeutic protocols. In conclusion, low IL-2 concentrations

  9. CD25 shedding by human natural occurring CD4+CD25+ regulatory T cells does not inhibit the action of IL-2

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Lauritsen, Jens Peter Holst

    2009-01-01

    Tregs are known to inhibit CD4+ T cell in a contact-dependent manner, but at the same time, various suppressive factors are secreted. We, here, demonstrate that human naturally occurring CD4+CD25+ Tregs are able to shed large amounts of soluble CD25 upon activation. Secretion of sCD25 could add......Regulatory T (Treg) cells are important for the maintenance of peripheral tolerance and inhibition of pathogenic T-cell responses. Therefore, they are important for the limitation of chronic inflammation but can also be deleterious by e.g. limiting antitumour immune responses. Natural occurring...... to the inhibitory effect of Tregs as such secretion in other settings has been proposed to act as a sink for local IL-2. However, we here demonstrate that supernatant from human Tregs containing high concentration of sCD25 does not inhibit proliferation of CD4+CD25(-) T cells or inhibit the action of IL-2...

  10. Immunity to experimental Salmonella typhimurium infections in rats. Transfer of immunity with primed CD4+CD25high and CD4+CD25low T lymphocytes

    DEFF Research Database (Denmark)

    Thygesen, P; Brandt, L; Jørgensen, T

    1994-01-01

    levels were not correlated with protection against S. typhimurium infections, although it showed that a higher and more persistent level of specific IgG antibodies was produced in animals receiving the CD4+CD25high fraction. It is concluded that 10(4) primed CD4+ T lymphocytes can induce immunity......The protective effect of primed CD4+ T lymphocytes against a lethal dose of 10(8) viable Salmonella typhimurium was studied in Lewis rats. Primed CD4+ T lymphocytes were obtained by inoculating Lewis rats with a non-lethal dose of 10(6) viable S. typhimurium. Four weeks after the infection, spleen...... by a fluorescence-activated cell sorter. Untreated Lewis rats were injected with 10(4) different primed CD4+ T-cell populations 24 h prior to the lethal dose of 10(8) viable S. typhimurium. Blood samples were drawn from the orbital plexus 1, 2, 3, and 4 weeks after the infection, and analysed for specific Ig...

  11. CD4(+)CD25(+)CD127(-) and CD4(+)CD25(+)Foxp3(+) Regulatory T Cell Subsets in Mediating Autoimmune Reactivity in Systemic Lupus Erythematosus Patients.

    Science.gov (United States)

    Żabińska, Marcelina; Krajewska, Magdalena; Kościelska-Kasprzak, Katarzyna; Jakuszko, Katarzyna; Bartoszek, Dorota; Myszka, Marta; Klinger, Marian

    2016-10-01

    The available clinical as well as experimental studies implicate participation of T regulatory (Treg) subsets in the pathogenesis and course of systemic lupus erythematosus (SLE). Introduction of the CD4(+)CD25(+)CD127(-) and CD4(+)CD25(+)Foxp3(+) regulatory subpopulations analysis into immunological processes assessment and disease activation prognosis in patients with lupus nephritis (LN) may improve monitoring of disease activity and enable an early, and thus more effective, therapeutic treatment. The main goal of the study was to investigate whether the quantitative changes of Treg subpopulations are related to the clinical status of patients with LN. Fifty-four adult SLE patients divided into two groups according to their SLEDAI and renal SLEDAI scores were enrolled into the study. Subpopulations of CD4(+)CD25(+)CD127(-) and CD4(+)CD25(+)Foxp3(+) phenotypes were determined by flow cytometry. The control group had higher absolute number of CD4(+)CD25(+)Foxp3(+) cells compared with the study group (p < 0.001). Also, significant inverse correlation in the absolute number of CD4(+)CD25(+)Foxp3(+) cells and SLEDAI score was observed. There were significant differences in the percentage and absolute number of CD4(+)CD25(+)Foxp3(+) lymphocytes between active and non-active LN groups. The study group had statistically lower values of CD4(+)CD25(+)CD127(-) cells, both in the percentage (p < 0.001) as well as their absolute number (p = 0.014) compared to the control group. There were also statistically significant positive correlations between the absolute number of CD4(+)CD25(+)CD127(-) and CD4(+)CD25(+)Foxp3(+) Tregs. (1) reduction in the number of regulatory CD4(+)CD25(+)Foxp3(+) cells is a promising indicator of the activity of SLE, particularly of renal involvement; (2) determination of the number of regulatory cells using the CD4(+)CD25(+)CD127(-) phenotype is unreliable in patients with SLE.

  12. CD8+CD25+ T cells reduce atherosclerosis in apoE(−/−) mice

    International Nuclear Information System (INIS)

    Zhou, Jianchang; Dimayuga, Paul C.; Zhao, Xiaoning; Yano, Juliana; Lio, Wai Man; Trinidad, Portia; Honjo, Tomoyuki; Cercek, Bojan; Shah, Prediman K.; Chyu, Kuang-Yuh

    2014-01-01

    Highlights: •The role of a sub-population of CD8 + T cells with suppressor functions was investigated in atherosclerosis. •CD8 + CD25 + T cells from adult apoE(−/−) mice had phenotype characteristics of T suppressor cells. •These CD8 + CD25 + T cells reduced CD4 + T cell proliferation and CD8 + cytotoxic activity in vitro. •Adoptive transfer of CD8 + CD25 + T cells significantly reduced atherosclerosis. •CD8 + CD25 + T cells have a suppressive function in atherosclerosis. -- Abstract: Background: It is increasingly evident that CD8 + T cells are involved in atherosclerosis but the specific subtypes have yet to be defined. CD8 + CD25 + T cells exert suppressive effects on immune signaling and modulate experimental autoimmune disorders but their role in atherosclerosis remains to be determined. The phenotype and functional role of CD8 + CD25 + T cells in experimental atherosclerosis were investigated in this study. Methods and results: CD8 + CD25 + T cells were observed in atherosclerotic plaques of apoE(−/−) mice fed hypercholesterolemic diet. Characterization by flow cytometric analysis and functional evaluation using a CFSE-based proliferation assays revealed a suppressive phenotype and function of splenic CD8 + CD25 + T cells from apoE(−/−) mice. Depletion of CD8 + CD25 + from total CD8 + T cells rendered higher cytolytic activity of the remaining CD8 + CD25 − T cells. Adoptive transfer of CD8 + CD25 + T cells into apoE(−/−) mice suppressed the proliferation of splenic CD4 + T cells and significantly reduced atherosclerosis in recipient mice. Conclusions: Our study has identified an athero-protective role for CD8 + CD25 + T cells in experimental atherosclerosis

  13. Changes in Reactivity In Vitro of CD4+CD25+ and CD4+CD25− T Cell Subsets in Transplant Tolerance

    Directory of Open Access Journals (Sweden)

    Bruce M. Hall

    2017-08-01

    Full Text Available Transplant tolerance induced in adult animals is mediated by alloantigen-specific CD4+CD25+ T cells, yet in many models, proliferation of CD4+ T cells from hosts tolerant to specific-alloantigen in vitro is not impaired. To identify changes that may diagnose tolerance, changes in the patterns of proliferation of CD4+, CD4+CD25+, and CD4+CD25− T cells from DA rats tolerant to Piebald Virol Glaxo rat strain (PVG cardiac allografts and from naïve DA rats were examined. Proliferation of CD4+ T cells from both naïve and tolerant hosts was similar to both PVG and Lewis stimulator cells. In mixed lymphocyte culture to PVG, proliferation of naïve CD4+CD25− T cells was greater than naïve CD4+ T cells. In contrast, proliferation of CD4+CD25− T cells from tolerant hosts to specific-donor PVG was not greater than CD4+ T cells, whereas their response to Lewis and self-DA was greater than CD4+ T cells. Paradoxically, CD4+CD25+ T cells from tolerant hosts did not proliferate to PVG, but did to Lewis, whereas naïve CD4+CD25+ T cells proliferate to both PVG and Lewis but not to self-DA. CD4+CD25+ T cells from tolerant, but not naïve hosts, expressed receptors for interferon (IFN-γ and IL-5 and these cytokines promoted their proliferation to specific-alloantigen PVG but not to Lewis or self-DA. We identified several differences in the patterns of proliferation to specific-donor alloantigen between cells from tolerant and naïve hosts. Most relevant is that CD4+CD25+ T cells from tolerant hosts failed to proliferate or suppress to specific donor in the absence of either IFN-γ or IL-5. The proliferation to third-party and self of each cell population from tolerant and naïve hosts was similar and not affected by IFN-γ or IL-5. Our findings suggest CD4+CD25+ T cells that mediate transplant tolerance depend on IFN−γ or IL-5 from alloactivated Th1 and Th2 cells.

  14. Quantitative variations of CD4 + CD25 + cells in Peking duckwhite ...

    African Journals Online (AJOL)

    Purpose: To develop a chimera via microinjection of poultry xenogeneic bone marrow mesenchymal stem cells (BMMSCs), and to assess its immune tolerance based on variations in proportion of CD4+CD25+ cells in CD4+ cells (specific CD4+CD25+ cells). Methods: BMMSCs were flush out from femurs and tibias of ...

  15. The 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain and binding analysis to anti-human C2 domain antibodies and phospholipid surfaces.

    Directory of Open Access Journals (Sweden)

    Caileen M Brison

    Full Text Available The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the "non-classical" antibody epitopes. Furthermore, antibody-binding results illustrate that the "classical" 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.

  16. Passive immunotherapy in AIDS: a double-blind randomized study based on transfusions of plasma rich in anti-human immunodeficiency virus 1 antibodies vs. transfusions of seronegative plasma.

    Science.gov (United States)

    Vittecoq, D; Chevret, S; Morand-Joubert, L; Heshmati, F; Audat, F; Bary, M; Dusautoir, T; Bismuth, A; Viard, J P; Barré-Sinoussi, F

    1995-02-14

    A randomized double-blind controlled trial was conducted to determine the efficacy of passive immunotherapy in the treatment of symptomatic human immunodeficiency virus (HIV) infection. This trial included 86 symptomatic patients randomized to receive plasma rich in anti-HIV-1 antibody or standard seronegative plasma. Each patient in both groups received a 300-ml infusion every 14 days over a 1-year period, and every 28 days thereafter, in addition to zidovudine and other conventional prophylactic treatments. Plasma donors were selected among symptomless seropositive individuals with a CD4 lymphocyte count > or = 400 x 10(6) cells per liter, a negative p24 antigen assay, and a high concentration of anti-p24 antibody. The plasmas were heat-inactivated before infusion. During the study period (day 28-day 365) scheduled by the protocol, clinical benefit from passive immunotherapy was observed in delaying the appearance of the first AIDS-defining event (P group compared to the treatment group. Seven deaths occurred in the treatment group vs. 11 in the control group (P = 0.27). A total of 47 patients died or exhibited new AIDS-defining events, 18 in the treatment group and 29 in the control group (P = 0.009). No clinical benefit was observed after the 1-year period with infusions performed every 4 weeks. These results indicate a favorable effect of passive immunotherapy on the evolution of advanced AIDS.

  17. T cells and T-cell subsets in mycosis fungoides and parapsoriasis. A study of 18 cases with anti-human T-cell monoclonal antibodies and histochemical techniques.

    Science.gov (United States)

    Buechner, S A; Winkelmann, R K; Banks, P M

    1984-07-01

    Skin lesions from 15 patients with mycosis fungoides (MF) and from three with parapsoriasis were studied immunohistochemically with monoclonal antibodies against T cells (Leu 1) and against T-cell subsets (Leu 2a, Leu 3a). Lymphoid cell reactivity was diverse among these sampled cases. In two cases of parapsoriasis and nine of MF, there was a predominance of helper/inducer (Leu-3a-reactive) cells over suppressor/cytotoxic (Leu-2a-reactive) cells. In one case of parapsoriasis and one (advanced tumor stage) of MF, there was suppressor/cytotoxic cell predominance. One case of MF showed strong reactivity for both T-cell subset markers. Four cases of MF (two plaque-stage and two tumor-stage) featured a predominant cell type in the dermis which was nonreactive for all three antibodies. The intraepidermal lymphoid cellularity was Leu-1-reactive in ten cases of MF and two of parapsoriasis. Among these 12 cases, the intraepidermal cellularity was Leu-2a-reactive in four and Leu-3a-reactive in three. The use of such studies of T-cell subsets on in situ cutaneous lymphoid infiltrates may demonstrate a correlation with cytomorphology, clinical stage, and disease prognosis.

  18. Protective Effect of CXCR3+CD4+CD25+Foxp3+ Regulatory T Cells in Renal Ischemia-Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Cao Jun

    2015-01-01

    Full Text Available Regulatory T cells (Tregs suppress excessive immune responses and are potential therapeutic targets in autoimmune disease and organ transplantation rejection. However, their role in renal ischemia-reperfusion injury (IRI is unclear. Levels of Tregs and expression of CXCR3 in Tregs were analyzed to investigate their function in the early phase of renal IRI. Mice were randomly divided into Sham, IRI, and anti-CD25 (PC61 + IRI groups. The PC61 + IRI group was established by i.p. injection of PC61 monoclonal antibody (mAb to deplete Tregs before renal ischemia. CD4+CD25+Foxp3+ Tregs and CXCR3 on Tregs were analyzed by flow cytometry. Blood urea nitrogen (BUN, serum creatinine (Scr levels, and tubular necrosis scores, all measures of kidney injury, were greater in the IRI group than in the Sham group. Numbers of Tregs were increased at 72 h after reperfusion in kidney. PC61 mAb preconditioning decreased the numbers of Tregs and aggravated kidney injury. There was no expression of CXCR3 on Tregs in normal kidney, while it expanded at 72 h after reperfusion and inversely correlated with BUN, Scr, and kidney histology score. This indicated that recruitment of Tregs into the kidney was related to the recovery of renal function after IRI and CXCR3 might be involved in the migration of Tregs.

  19. Pancreatic stone protein and soluble CD25 for infection and sepsis in an emergency department.

    Science.gov (United States)

    García de Guadiana-Romualdo, Luis; Berger, Mario; Jiménez-Santos, Enrique; Rebollo-Acebes, Sergio; Jiménez-Sánchez, Roberto; Esteban-Torrella, Patricia; Hernando-Holgado, Ana; Ortín-Freire, Alejandro; Albaladejo-Otón, María Dolores

    2017-04-01

    Infection is a common problem in emergency departments (EDs) and is associated with high mortality, morbidity and costs. Identifying infection in ED patients can be challenging. Biomarkers can facilitate its diagnosis, enabling an early management and improving outcomes. In the critical care setting, two emerging biomarkers, pancreatic stone protein (PSP) and soluble CD25 (sCD25), have demonstrated to be useful for diagnosis of sepsis. We aimed to assess the diagnostic value of these biomarkers, in comparison with procalcitonin (PCT), for infection and sepsis in an ED population with suspected infection. Through a prospective, observational study, we investigated the utility of serum PCT, PSP and sCD25 levels, measured on admission, for diagnosis of infection and sepsis, defined according to the recently updated for sepsis (Sepsis-3), in patients presenting to the ED for suspected infection. Diagnostic accuracy was evaluated by using receiver operating characteristic curves (ROC) analysis. Of the 152 patients enrolled in this study, 129 had a final diagnosis of infection, including 82 with noncomplicated infection and 47 with sepsis. Median PCT, PSP and sCD25 levels were significantly higher in patients with infection and sepsis. The ROC curve analysis revealed a similar diagnostic accuracy for infection (ROC area under the curve (AUC) PCT: 0·904; sCD25: 0·869 and PSP: 0·839) and for sepsis (ROC AUC: PCT: 0·820; sCD25: 0·835 and PSP: 0·872). Pancreatic stone protein and sCD25 perform well as infection and sepsis biomarkers, with a similar performance than PCT, in ED patients with suspected infection. Further larger studies investigating use of PSP and sCD25 are needed. © 2017 Stichting European Society for Clinical Investigation Journal Foundation.

  20. 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

  1. Relationship between soluble CD25 and gene expression in healthy individuals and patients with multiple sclerosis

    DEFF Research Database (Denmark)

    Buhelt, Sophie; Ratzer, Rikke Lenhard; Christensen, Jeppe Romme

    2017-01-01

    Genome wide association studies and fine mapping has established a firm link between the IL2RA gene, encoding the interleukin-2 receptor α-chain CD25, and susceptibility to multiple sclerosis (MS). We hypothesized that gene expression in peripheral blood mononuclear cells (PBMCs) from healthy......-networks were focused around NFKB1, TNF, BCL6 and STAT1. Eighteen genes correlated with sCD25 with rho≥0.707 in relapsing remitting MS versus 33 in secondary progressive and 34 in primary progressive MS. None had a FDR

  2. Downregulation of IL-12 and a novel negative feedback system mediated by CD25+CD4+ T cells

    International Nuclear Information System (INIS)

    Sato, Kojiro; Tateishi, Shoko; Kubo, Kanae; Mimura, Toshihide; Yamamoto, Kazuhiko; Kanda, Hiroko

    2005-01-01

    CD25 + CD4 + regulatory T cells suppress immune responses and are believed to play roles in preventing autoimmune diseases. However, the mechanism(s) underlying the suppression and the regulation of their homeostasis remain to be elucidated. Here we show that these regulatory T cells downregulated CD25 - CD4 + T-cell-mediated production of IL-12 from antigen-presenting cells, which can act as a growth factor for CD25 - CD4 + T cells. We further found that CD25 + CD4 + T cells, despite their well-documented 'anergic' nature, proliferate significantly in vitro only when CD25 - CD4 + T cells are present. Notably, this proliferation was strongly dependent on IL-2 and relatively independent of IL-12. Thus, CD25 + CD4 + T cells suppress CD25 - CD4 + T-cell responses, at least in part, by inhibiting IL-12 production while they themselves can undergo proliferation with the mediation of CD25 - CD4 + T cells in vitro. These results offer a novel negative feedback system involving a tripartite interaction among CD25 + CD4 + and CD25 - CD4 + T cells, and APCs that may contribute to the termination of immune responses

  3. Evaluation of PLGA containing anti-CTLA4 inhibited endometriosis progression by regulating CD4+CD25+Treg cells in peritoneal fluid of mouse endometriosis model.

    Science.gov (United States)

    Liu, Qi; Ma, Pingchuan; Liu, Lanxia; Ma, Guilei; Ma, Jingjing; Liu, Xiaoxuan; Liu, Yijin; Lin, Wanjun; Zhu, Yingjun

    2017-01-01

    Our study investigated poly(lactic-co-glycolic acid) (PLGA) as protein delivery vehicles encapsulate CTLA-4-antibody (anti-CTLA-4) which is essential for CD4+CD25+Treg cells suppressive function exposing superior potential for inhibiting endometriosis progress in mouse model than single anti-CTLA-4. Anti-CTLA-4 loaded PLGA combined to ligands CTLA-4 in surface of CD4+CD25+Treg cells which distributed in peritoneal fluid of mouse endometriosis model. The particle size, zeta potential of the anti-CTLA-4 loaded nanoparticles was detected by dynamic light scattering. Morphology of nanoparticles was evaluated by transmission electron microscopy (TEM). Confocal laser scanning microscopy (CLSM) indicated distribution of anti-CTLA-4 with PLGA or without in peritoneal fluid. Cumulative anti-CTLA-4 release from nanoparticles was evaluated by Micro BCA assay. The percentage of CD4+CD25+Treg cells in peritoneal fluid was demonstrated by flow cytometer. In vitro experiment we co-culture ectopic endometrial cells (EEC) with isolated CD4+CD25+Treg cells in peritoneal fluid (PF), proliferation and invasion of ectopic endometrial cells (EEC) was measured by BrdU ELISA assay and Matrigel invasion assay. In comparison with anti-CTLA-4 without nanoparticles, the bioconjugates PLGA/anti-CTLA-4 were tolerated in peritoneal fluid with a controlled release of anti-CTLA-4 in 3, 7, 14days. Moreover, PLGA/anti-CTLA-4 had superior protective regulation ability to reduce level of CD4+CD25+Treg cells in peritoneal fluid. Most strikingly, in vitro experiment, PLGA/anti-CTLA-4 exhibited better ability in inhibiting proliferation and invasion of ectopic endometrial cells in co-culture system compared with anti-CTLA-4. Progressively, PLGA/anti-CTLA-4 had better suppressive activity to inhibited IL-10 and TGF-beta secreted by CD4+CD25+Treg cells which indicating that PLGA/anti-CTLA-4 suppressed cells proliferation and invasion through reduced IL-10 and TGF-beta production. Thus, PLGA/anti-CTLA-4 may

  4. Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

    International Nuclear Information System (INIS)

    Forward, Nicholas A.; Conrad, David M.; Power Coombs, Melanie R.; Doucette, Carolyn D.; Furlong, Suzanne J.; Lin, Tong-Jun; Hoskin, David W.

    2011-01-01

    Highlights: → Curcumin inhibits CD4 + T-lymphocyte proliferation. → Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4 + T-lymphocytes. → Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. → IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4 + T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca 2+ release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4 + CD25 + regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

  5. Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

    Energy Technology Data Exchange (ETDEWEB)

    Forward, Nicholas A.; Conrad, David M. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Power Coombs, Melanie R.; Doucette, Carolyn D. [Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Furlong, Suzanne J. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Lin, Tong-Jun [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia (Canada); Hoskin, David W., E-mail: d.w.hoskin@dal.ca [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Surgery, Dalhousie University, Halifax, Nova Scotia (Canada)

    2011-04-22

    Highlights: {yields} Curcumin inhibits CD4{sup +} T-lymphocyte proliferation. {yields} Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4{sup +} T-lymphocytes. {yields} Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. {yields} IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4{sup +} T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 ({alpha} chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca{sup 2+} release to inhibit I{kappa}B phosphorylation, which is required for nuclear translocation of the transcription factor NF{kappa}B. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4{sup +}CD25{sup +} regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

  6. [Expression of CD25 in Acute Myeloid Leukemia Is An Adverse Prognostic Factor Independent of the Chromosome Karyotype].

    Science.gov (United States)

    Liu, Yan-Fang; Dong, Li; Wang, Chong; Sun, Hui; Zhang, Qiu-Tang; Wang, Meng; Li, Tao; Xu, Yan; Ma, Jie; Xie, Xin-Sheng; Sun, Ling; Wan, Ding-Ming

    2016-04-01

    To investigate the CD25 expression in patients with acute myeloid leukemia (AML) and its significance. Clinical data of 168 newly diagnosed AML patients (except APL) were collected. The expression of CD25 in AML patients and its clinical characteristics were retrospectively analyzed. The leukemia cells of 29 out of 168 cases (17.26%) expressed CD25 antigen. Most of CD25 positive AML patients were occurred in patients with unfavourable or normal karyotype, higher WBC and Plt count at diagnosis and higher percentage of blasts in peripheral blood and bone marrow. Compared with CD25(-) AML patients, CD25(+) AML patients had lower CR rate (the CR rate of 1 course of treatment were 49.02% and 16.00%, respectively, P karyotype were not significantly different from that in patients with intermediate karyotype (P karyotype in terms of low complete remission rate and short survival time.

  7. CD4+CD25+FOXP3+ Regulatory T Cells In Allogeneic Hematopoietic Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Young-Ho Lee

    2011-06-01

    Full Text Available CD4+CD25+FOXP3+ regulatory T cells (Treg require activation through the T cell receptor for function. CD4+CD25+FOXP3+ regulatory T cells are believed to be key players of the immune tolerance network and control the induction and effector phase of the immune system. Although these cells require antigen-specific activation, they are generally able to suppress bystander T cell responses once activated. This raises the possibility that antigen-specific Treg may be useful therapeutically by localizing generalized suppressive activity to tissues expressing select target antigens. Treg can exert a potent suppressive effect on immune effector cells reactive to host antigens and prevent graft versus host disease (GVHD in allogeneic bone marrow transplantation (BMT. Here, we observed that co-transfer of CD4+CD25+FOXP3+ T cells derived from donor type along with the donor bone marrow cells could control GVHD-like reactions by suppressing effectors cells of host responding to the donor hematopoietic compartment, and resulted in prevention of autoimmunity and rejection. We further demonstrate that CD4+CD25+FOXP3+ regulatory T cells can control immune-based morbidity after allogeneic BMT by suppressing the development of granulocytes cells and increasing the level of B cell expression.

  8. Reactivity of naive CD4+CD25- T cells against gut microflora in healthy mice

    DEFF Research Database (Denmark)

    Gad, Monika; Lundsgaard, Dorthe; Kjellev, Stine

    2006-01-01

    We have previously shown that conventional as well as germ-free CD4+ T cells depleted of CD25+ cells from the gut-associated lymphoid tissue and the periphery proliferate specifically in response to enterobacterial antigen exposure whereas unfractionated CD4+ T cells are not reactive under...

  9. CD4+CD25+ regulatory T cells: I. Phenotype and physiology

    DEFF Research Database (Denmark)

    Holm, Thomas Lindebo; Nielsen, Janne; Claesson, Mogens H

    2004-01-01

    it has become increasingly clear that regulatory CD4+CD25+ T cells (Treg cells) play an important role in the maintenance of immunological self-tolerance, and that this cell subset exerts its function by suppressing the proliferation or function of autoreactive T cells. Based on human and murine...

  10. Tec protein tyrosine kinase inhibits CD25 expression in human T-lymphocyte.

    Science.gov (United States)

    Susaki, Kentaro; Kitanaka, Akira; Dobashi, Hiroaki; Kubota, Yoshitsugu; Kittaka, Katsuharu; Kameda, Tomohiro; Yamaoka, Genji; Mano, Hiroyuki; Mihara, Keichiro; Ishida, Toshihiko

    2010-01-04

    The Tec protein tyrosine kinase (PTK) belongs to a group of structurally related nonreceptor PTKs that also includes Btk, Itk, Rlk, and Bmx. Previous studies have suggested that these kinases play important roles in hematopoiesis and in the lymphocyte signaling pathway. Despite evidence suggesting the involvement of Tec in the T-lymphocyte activation pathway via T-cell receptor (TCR) and CD28, Tec's role in T-lymphocytes remains unclear because of the lack of apparent defects in T-lymphocyte function in Tec-deficient mice. In this study, we investigated the role of Tec in human T-lymphocyte using the Jurkat T-lymphoid cell line stably transfected with a cDNA encoding Tec. We found that the expression of wild-type Tec inhibited the expression of CD25 induced by TCR cross-linking. Second, we observed that LFM-A13, a selective inhibitor of Tec family PTK, rescued the suppression of TCR-induced CD25 expression observed in wild-type Tec-expressing Jurkat cells. In addition, expression of kinase-deleted Tec did not alter the expression level of CD25 after TCR ligation. We conclude that Tec PTK mediates signals that negatively regulate CD25 expression induced by TCR cross-linking. This, in turn, implies that this PTK plays a role in the attenuation of IL-2 activity in human T-lymphocytes.

  11. CD8{sup +}CD25{sup +} T cells reduce atherosclerosis in apoE(−/−) mice

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jianchang; Dimayuga, Paul C.; Zhao, Xiaoning; Yano, Juliana; Lio, Wai Man; Trinidad, Portia; Honjo, Tomoyuki; Cercek, Bojan; Shah, Prediman K.; Chyu, Kuang-Yuh, E-mail: Chyuk@cshs.org

    2014-01-17

    Highlights: •The role of a sub-population of CD8{sup +} T cells with suppressor functions was investigated in atherosclerosis. •CD8{sup +}CD25{sup +} T cells from adult apoE(−/−) mice had phenotype characteristics of T suppressor cells. •These CD8{sup +}CD25{sup +} T cells reduced CD4{sup +} T cell proliferation and CD8{sup +} cytotoxic activity in vitro. •Adoptive transfer of CD8{sup +}CD25{sup +} T cells significantly reduced atherosclerosis. •CD8{sup +}CD25{sup +} T cells have a suppressive function in atherosclerosis. -- Abstract: Background: It is increasingly evident that CD8{sup +} T cells are involved in atherosclerosis but the specific subtypes have yet to be defined. CD8{sup +}CD25{sup +} T cells exert suppressive effects on immune signaling and modulate experimental autoimmune disorders but their role in atherosclerosis remains to be determined. The phenotype and functional role of CD8{sup +}CD25{sup +} T cells in experimental atherosclerosis were investigated in this study. Methods and results: CD8{sup +}CD25{sup +} T cells were observed in atherosclerotic plaques of apoE(−/−) mice fed hypercholesterolemic diet. Characterization by flow cytometric analysis and functional evaluation using a CFSE-based proliferation assays revealed a suppressive phenotype and function of splenic CD8{sup +}CD25{sup +} T cells from apoE(−/−) mice. Depletion of CD8{sup +}CD25{sup +} from total CD8{sup +} T cells rendered higher cytolytic activity of the remaining CD8{sup +}CD25{sup −} T cells. Adoptive transfer of CD8{sup +}CD25{sup +} T cells into apoE(−/−) mice suppressed the proliferation of splenic CD4{sup +} T cells and significantly reduced atherosclerosis in recipient mice. Conclusions: Our study has identified an athero-protective role for CD8{sup +}CD25{sup +} T cells in experimental atherosclerosis.

  12. Demonstration of strong enterobacterial reactivity of CD4+CD25- T cells from conventional and germ-free mice which is counter-regulated by CD4+CD25+ T cells

    DEFF Research Database (Denmark)

    Gad, Monika; Pedersen, Anders Elm; Kristensen, Nanna N

    2004-01-01

    Unfractionated CD4+ T cells from the gut-associated lymphoid tissue (GALT) and peripheral lymph nodes are unresponsive when exposed to enterobacterial antigens in vitro. Under similar conditions, CD4+ T cells depleted in vivo or in vitro of CD4+CD25+ T cells proliferate extensively. The CD4+CD25- T...

  13. CD4+CD25+ regulatory T cells: II. Origin, disease models and clinical aspects

    DEFF Research Database (Denmark)

    Nielsen, Janne; Holm, Thomas Lindebo; Claesson, Mogens H

    2004-01-01

    Autoimmune diseases afflict approximately 5% of the population and reflect a failure in the immune system to discriminate between self and non-self resulting in the breakdown of self-tolerance. Regulatory CD4+CD25+ T cells (Treg cells) have been shown to play an important role in the maintenance ...... in disease models such as autoimmune gastritis and inflammatory bowel disease. Finally, we will consider some aspects of the therapeutic potential of Treg cells....

  14. Interleukin-7 optimizes FOXP3+CD4+ regulatory T cells reactivity to interleukin-2 by modulating CD25 expression.

    Directory of Open Access Journals (Sweden)

    Federico Simonetta

    Full Text Available The vast majority of Foxp3 regulatory T cells (Treg exhibits constitutive expression of CD25 (IL-2Rα, which allows the constitution of the high affinity IL-2Rαβγ receptor, ensuring efficient IL-2 binding by Treg. Maintenance of CD25 expression at Treg surface depends on both cell intrinsic factors and environmental stimuli such as IL-2 itself. Whether other factors can participate to maintenance of CD25 expression in vivo is at present unknown. In the present work we demonstrated that IL-7, a gamma-chain cytokine exerting a crucial role in T cell development and homeostasis, is able and necessary to sustain the expression of high levels of CD25 at Treg surface. We demonstrated that, during in vitro cultures performed in the absence of IL-2, IL-7 is able to sustain CD25 expression at Treg surface through a transcriptional mechanism. By studying mice in which IL-7 signaling is either genetically impaired or increased and by employing adoptive transfer murine models, we demonstrated that IL-7 is necessary for sustained expression of CD25 at Treg surface in vivo. To ascertain the biological impact of IL-7 mediated modulation of CD25 expression, we demonstrated that IL-7 modulation of CD25 expression at Treg surface affected their ability to efficiently bind IL-2 and transduce IL-2 signaling. Finally, we demonstrated that IL-7 dependent modulation of CD25 associated with potentiated IL-2 induced expansion of Treg in vivo. Collectively, our results identify IL-7 as a necessary factor contributing to sustained CD25 expression at Treg surface in vivo thereby affecting their ability to efficiently react to IL-2.

  15. Function and regulation of LAG3 on CD4+CD25- T cells in non-small cell lung cancer.

    Science.gov (United States)

    Ma, Qin-Yun; Huang, Da-Yu; Zhang, Hui-Jun; Wang, Shaohua; Chen, Xiao-Feng

    2017-11-15

    LAG3 is a surface molecule found on a subset of immune cells. The precise function of LAG3 appears to be context-dependent. In this study, we investigated the effect of LAG3 on CD4 + CD25 - T cells from non-small cell lung cancer (NSCLC) patients. We found that in the peripheral blood mononuclear cells of NSCLC patients, LAG3 was significantly increased in CD4 + T cells directly ex vivo and primarily in the CD4 + CD25 - fraction, which was regulated by prolonged TCR stimulation and the presence of IL-27. TCR stimulation also increased CD25 expression, but not Foxp3 expression, in LAG3-expressing CD4 + CD25 - cells Compared to LAG3-nonexpressing CD4 + CD25 - cells, LAG3-expressing CD4 + CD25 - cells presented significantly higher levels of PD1 and TIM3, two inhibitory receptors best described in exhausted CD8 + T effector cells. LAG3-expressing CD4 + CD25 - cells also presented impaired proliferation compared with LAG3-nonexpressing CD4 + CD25 - cells but could be partially rescued by inhibiting both PD1 and TIM3. Interestingly, CD8 + T cells co-incubated with LAG3-expressing CD4 + CD25 - cells at equal cell numbers demonstrated significantly lower proliferation than CD8 + T cells incubated alone. Co-culture with CD8 + T cell and LAG3-expressing CD4 + CD25 - T cell also upregulated soluble IL-10 level in the supernatant, of which the concentration was positively correlated with the number of LAG3-expressing CD4 + CD25 - T cells. In addition, we found that LAG3-expressing CD4 + CD25 - T cells infiltrated the resected tumors and were present at higher frequencies of in metastases than in primary tumors. Taken together, these data suggest that LAG3-expressing CD4 + CD25 - T cells represent another regulatory immune cell type with potential to interfere with anti-tumor immunity. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Phenotypic and functional analysis of CD4+ CD25- Foxp3+ T cells in patients with systemic lupus erythematosus.

    Science.gov (United States)

    Bonelli, Michael; Savitskaya, Anastasia; Steiner, Carl-Walter; Rath, Eva; Smolen, Josef S; Scheinecker, Clemens

    2009-02-01

    CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) that specialize in the suppression of immune responses might be critically involved in the pathogenesis of autoimmune diseases. Recent studies have described increased proportions of CD4(+)Foxp3(+) T cells that lacked expression of CD25 in systemic lupus erythematosus (SLE) patients but the suppressive capacity of these cells has not been analyzed so far. We therefore performed combined phenotypic and functional analyses of CD4(+)CD25(-)Foxp3(+) T cells in patients with autoimmune diseases and healthy controls (HC). Phenotypic analysis revealed increased proportions of CD4(+)CD25(-)Foxp3(+) T cells in SLE patients as compared with patients with systemic sclerosis, rheumatoid arthritis, (RA), or HC. In addition, increased proportions of CD4(+)CD25(-)Foxp3(+) T cells correlated with the clinical disease activity and the daily cortisone dose. According to phenotypic analysis, CD4(+)CD25(-)Foxp3(+) T cells resembled regulatory T cells rather than activated T cells. For functional analysis, a surrogate surface marker combination to substitute for intracellular Foxp3 was defined: CD4(+)CD25(-)CD127(-) T cells from SLE patients were isolated by FACS sorting and analyzed for their suppressive capacity in vitro. CD4(+)CD25(-)CD127(-) T cells, that contained up to 53% Foxp3(+) T cells, were found to suppress T cell proliferation but not IFN-gamma production in vitro. In summary, CD4(+)CD25(-)Foxp3(+) T cells phenotypically and to a certain extent also functionally resemble conventional Treg. Despite increased proportions, however, their selective functional defects might contribute to the failure of Treg to control autoimmune dysregulation in SLE patients.

  17. CD4+CD25+ regulatory T cells: II. Origin, disease models and clinical aspects

    DEFF Research Database (Denmark)

    Nielsen, Janne; Holm, Thomas Lindebo; Claesson, Mogens H

    2004-01-01

    of immune homeostasis and self-tolerance by counteracting the development and effector functions of potentially autoreactive T cells. We have in the previous APMIS review described the phenotype and physiology of Treg cells. The present overview deals with the thymic origin of Treg cells and their role......Autoimmune diseases afflict approximately 5% of the population and reflect a failure in the immune system to discriminate between self and non-self resulting in the breakdown of self-tolerance. Regulatory CD4+CD25+ T cells (Treg cells) have been shown to play an important role in the maintenance...

  18. Chemokines involved in protection from colitis by CD4+CD25+ regulatory T cells

    DEFF Research Database (Denmark)

    Kristensen, Nanna Ny; Brudzewsky, Dan; Gad, Monika

    2006-01-01

    Chemokines are small proteins involved in the direction of migration of immune cells both during normal homeostasis and inflammation. Chemokines have been implicated in the pathology of many different inflammatory disorders and are therefore appealing therapeutic targets. Using a chemokine....../chemokine receptor-specific gene expression profiling system of 67 genes, the authors have determined the expression profile of chemokine and chemokine receptor genes in the rectum of colitic mice and in mice that have been protected fromcolitis by CD4CD25 regulatory T cells. In mice protected from colitis...

  19. CD 4 + CD 25 + T cells maintain homeostasis by promoting TER - 119 cell development and inhibiting T cell activation

    Directory of Open Access Journals (Sweden)

    Muhaimin Rifa’i

    2014-05-01

    Full Text Available CD4+ CD25+ regulatory T cells involved in the regulation of self- tolerance and normality of homeostasis. CD122 deficient mice are model animals that have an abnormal immune system characteristically have a high number of activated T cells and TER-119 cell decreased. Here we showed evidence that the transfer of CD4+ CD25+ regulatory T cells derived from normal mice to CD122- defficient neonates prevent the development of activated memory T cells and elicit TER-119 differentiation. Bone marrow reconstitution derived from CD122-/- mice to normal mice resulting tolerance to individual that genetically different. Importantly, CD4+ CD25+ regulatory T cells derived from normal mice can replace CD4+ CD25+ cells derived from CD122-/- mice. The results of this experiment suggest that regulatory T cells from normal mice exert a critical role in maintaining peripheral tolerance and controlling hematopoietic disorder.

  20. Growth Arrest-Specific 6 Enhances the Suppressive Function of CD4+CD25+ Regulatory T Cells Mainly through Axl Receptor

    Directory of Open Access Journals (Sweden)

    Guang-ju Zhao

    2017-01-01

    Full Text Available Background. Growth arrest-specific (Gas 6 is one of the endogenous ligands of TAM receptors (Tyro3, Axl, and Mertk, and its role as an immune modulator has been recently emphasized. Naturally occurring CD4+CD25+ regulatory T cells (Tregs are essential for the active suppression of autoimmunity. The present study was designed to investigate whether Tregs express TAM receptors and the potential role of Gas6-TAM signal in regulating the suppressive function of Tregs. Methods. The protein and mRNA levels of TAM receptors were determined by using Western blot, immunofluorescence, flow cytometry, and RT-PCR. Then, TAM receptors were silenced using targeted siRNA or blocked with specific antibody. The suppressive function of Tregs was assessed by using a CFSE-based T cell proliferation assay. Flow cytometry was used to determine the expression of Foxp3 and CTLA4 whereas cytokines secretion levels were measured by ELISA assay. Results. Tregs express both Axl and Mertk receptors. Gas6 increases the suppressive function of Tregs in vitro and in mice. Both Foxp3 and CTLA-4 expression on Tregs are enhanced after Gas6 stimulation. Gas6 enhances the suppressive activity of Tregs mainly through Axl receptor. Conclusion. Gas6 has a direct effect on the functions of CD4+CD25+Tregs mainly through its interaction with Axl receptor.

  1. CD4+CD25+ T regulatory cells from FIV+ cats induce a unique anergic profile in CD8+ lymphocyte targets

    Directory of Open Access Journals (Sweden)

    Tompkins Mary B

    2010-11-01

    Full Text Available Abstract Background Using the FIV model, we reported previously that CD4+CD25+ T regulatory (Treg cells from FIV+ cats are constitutively activated and suppress CD4+CD25- and CD8+ T cell immune responses. In an effort to further explore Treg-mediated suppression, we asked whether Treg cells induce anergy through the alteration of production of cyclins, cyclin-dependent kinases and their inhibitors. Results Lymphocytes were obtained from control or FIV+ cats and sorted by FACS into CD4+CD25+ and CD8+ populations. Following co-culture with CD4+CD25+ cells, CD8+ targets were examined by Western blot for changes in cyclins D3, E and A, retinoblastoma (Rb protein, as well as the cyclin dependent kinase inhibitor p21cip1. Following co-culture with CD4+CD25+cells, we observed up-regulation of p21cip1 and cyclin E, with down-regulation of cyclin D3, in CD8+ cells from FIV+ cats. As expected, CD8+ targets from control cats were quiescent with little up-regulation of p21cip1 and cyclin E. There was also a lack of Rb phosphorylation in CD8+ targets consistent with late G1 cell cycle arrest. Further, IL-2 mRNA was down regulated in CD8+ cells after co-culture with CD4+CD25+ Treg cells. Following CD4+CD25+ co-culture, CD8+ targets from FIV+ cats also had increased Foxp3 mRNA expression; however, these CD8+Foxp3+ cells did not exhibit suppressor function. Conclusions Collectively, these data suggest that CD4+CD25+ Treg cells from FIV+ cats induce CD8+ anergy by disruption of normal G1 to S cell cycle progression.

  2. CD25+ T-lymphocytes induce CD11b on eosinophils in allergic nasal mucosa

    Directory of Open Access Journals (Sweden)

    S. Horiguchi

    1995-01-01

    Full Text Available In the allergic mucosa, there is a significant increase in numbers of CD25+ cells and activated eosinophils. To determine whether a link exists between the activated T-lymphocytes and tissue eosinophils in nasal allergy, we studied CD25+ cells in the nasal mucosa and compared the levels of soluble IL-2 receptor (sIL-2R both in the serum and the nasal secretions, and further investigated expression of CD11b on eosinophils in the nasal lavage fluids and peripheral blood of patients with nasal allergy. We also examined the effects of the culture supernatant of Con A- and IL-2-activated T-lymphocytes on CD11b expression on eosinophils in the present study. The concentration of sIL-2R in the nasal secretions from patients with Japanese cedar pollinosis (JCP was significantly higher than that from normal subjects (p < 0.01. The sIL-2R level was significantly higher in the nasal secretions than in the sera in patients (p < 0.01, and CD11b expression on eosinophils from nasal hvage fluid was significandy higher than that of eosinophils from peripheral blood of the same individuals (p < 0.01. The activated T-lymphocytes promoted eosinophil activation with upregulation of CD11b in vitro, and eosinophils in the nasal secretions from patients significantly expressed more CD11b in vivo. These results indicate that activation of T-lymphocytes is linked to eosinophil activation in nasal allergy.

  3. CD25+ FoxP3+ Memory CD4 T Cells Are Frequent Targets of HIV Infection In Vivo.

    Science.gov (United States)

    Chachage, Mkunde; Pollakis, Georgios; Kuffour, Edmund Osei; Haase, Kerstin; Bauer, Asli; Nadai, Yuka; Podola, Lilli; Clowes, Petra; Schiemann, Matthias; Henkel, Lynette; Hoffmann, Dieter; Joseph, Sarah; Bhuju, Sabin; Maboko, Leonard; Sarfo, Fred Stephen; Eberhardt, Kirsten; Hoelscher, Michael; Feldt, Torsten; Saathoff, Elmar; Geldmacher, Christof

    2016-10-15

    Interleukin 2 (IL-2) signaling through the IL-2 receptor alpha chain (CD25) facilitates HIV replication in vitro and facilitates homeostatic proliferation of CD25(+) FoxP3(+) CD4(+) T cells. CD25(+) FoxP3(+) CD4(+) T cells may therefore constitute a suitable subset for HIV infection and plasma virion production. CD25(+) FoxP3(+) CD4(+) T cell frequencies, absolute numbers, and the expression of CCR5 and cell cycle marker Ki67 were studied in peripheral blood from HIV(+) and HIV(-) study volunteers. Different memory CD4(+) T cell subsets were then sorted for quantification of cell-associated HIV DNA and phylogenetic analyses of the highly variable EnvV1V3 region in comparison to plasma-derived virus sequences. In HIV(+) subjects, 51% (median) of CD25(+) FoxP3(+) CD4(+) T cells expressed the HIV coreceptor CCR5. Very high frequencies of Ki67(+) cells were detected in CD25(+) FoxP3(+) memory CD4(+) T cells (median, 27.6%) in comparison to CD25(-) FoxP3(-) memory CD4(+) T cells (median, 4.1%; P HIV DNA content was 15-fold higher in CD25(+) FoxP3(+) memory CD4(+) T cells than in CD25(-) FoxP3(-) T cells (P = 0.003). EnvV1V3 sequences derived from CD25(+) FoxP3(+) memory CD4(+) T cells did not preferentially cluster with plasma-derived sequences. Quasi-identical cell-plasma sequence pairs were rare, and their proportion decreased with the estimated HIV infection duration. These data suggest that specific cellular characteristics of CD25(+) FoxP3(+) memory CD4(+) T cells might facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. The contribution of this cell population to plasma virion production remains unclear. Despite recent advances in the understanding of AIDS virus pathogenesis, which cell subsets support HIV infection and replication in vivo is incompletely understood. In vitro, the IL-2 signaling pathway and IL-2-dependent cell cycle induction are essential for HIV infection of stimulated T

  4. An upregulation of CD8+CD25+Foxp3+T cells with suppressive function through interleukin 2 pathway in pulmonary arterial hypertension.

    Science.gov (United States)

    Zhu, Rong; Chen, Liang; Xiong, Yaqiong; Wang, Nana; Xie, Xiaochen; Hong, Yongqing; Meng, Zili

    2017-09-15

    Accumulating evidence suggests that abnormal inflammation plays a critical role in the pathogenesis of pulmonary arterial hypertension (PAH). CD8 + CD25 + Foxp3 + T cell is a novel cell subtype, and its role in PAH is not yet investigated. Here, we observed that PAH patients presented a significant upregulation of CD8 + CD25 + Foxp3 + T cells and a downregulation of CD4 + CD25 + Foxp3 + T cells compared to healthy controls. Regardless, the total number of CD25 + Foxp3 + T cells in PAH patients was still smaller than that in healthy controls. Compared to CD8 + CD25 - T cells, CD8 + CD25 + T cells presented higher Foxp3 expression, lower interferon (IFN)-γ expression and higher transforming growth factor (TGF)-β expression, in both healthy and PAH individuals. The CD8 + CD25 + T cells in PAH patients also demonstrated regulatory function by suppressing the proliferation of CD4 + CD25 - and CD8 + CD25 - effector T cells, albeit at lower efficiency than CD4 + CD25 + T cells from PAH patients and healthy volunteers. CD8 + CD25 + T cells from PAH responded to interleukin (IL)-2 supplement by expansion and upregulating Foxp3 expression. In PAH patients, IL-2-treated CD8 + CD25 + T cells were more potent at inhibiting CD4 + CD25 - effector T cell proliferation than IL-2-untreated CD8 + CD25 + T cells. Together, we found an upregulation of CD8 + CD25 + Foxp3 + T cells in PAH patients, and this T cell population presented suppressive activity that could be enhanced by IL-2 treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Dexamethasone Regulates Macrophage and Cd4+Cd25+ Cell Numbers in the Chicken Spleen

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    AS Calefi

    2016-03-01

    Full Text Available Abstract Dexamethasone (DEX is a corticoid hormone that is experimentally used to mimic the effects of increased levels of endogenous corticosterone observed during the stress response. Currently, stress is considered one of the major predisposing factors for diseases in the poultry industry. The aim of this study was to analyze the effects of DEX and/or of a 20-fold coccidial vaccine dose on leukocyte phenotypes in the spleen and cecal tonsils of chickens. Twenty specific-pathogen-free (SPF Leghorn chickens were divided into four groups: a non-treated group (NT, a DEX-treated group (Dex, a vaccinated group (V and a DEX-treated+vaccinated group (Dex+V. On experimental day (ED 42, each bird in the vaccinated groups received a anti-coccidial vaccine. DEX was injected in the birds of the Dex and Dex+V groups (0.9 mg/kg onED42 and ED45. The immunophenotyping was performed by flow cytometry analysis of splenocytes and cecal tonsils cells onED48. DEX treatment per se was unable to change CD4+CD8+, CD4+CD8+ and CD4-CD8+ populations with TCRgd or CD28 in the spleen, or macrophages and T lymphocytes in the cecal tonsils. V group birds presented higher numbers of splenic macrophages compared with those measured in the Dex+V group. The number of CD4+CD25+ cells in the spleen of birds of the V group was higher than those measured in the other experimental groups. Our data suggest that CD4+CD25+ cells and macrophages might be influenced by DEX treatment in spleen, but not in the cecal tonsils of chickens inoculated with Eimeria.

  6. Identification and Functional Characterization of Human Cd4+Cd25+ T Cells with Regulatory Properties Isolated from Peripheral Blood

    OpenAIRE

    Jonuleit, Helmut; Schmitt, Edgar; Stassen, Michael; Tuettenberg, Andrea; Knop, Jurgen; Enk, Alexander H.

    2001-01-01

    A subpopulation of peripheral human CD4+CD25+ T cells that expresses CD45RO, histocompatibility leukocyte antigen DR, and intracellular cytotoxic T lymphocyte–associated antigen (CTLA) 4 does not expand after stimulation and markedly suppresses the expansion of conventional T cells in a contact-dependent manner. After activation, CD4+CD25+ T cells express CTLA-4 on the surface detectable for several weeks. These cells show a G1/G0 cell cycle arrest and no production of interleukin (IL)-2, IL-...

  7. Immunophenotype and increased presence of CD4+CD25+ regulatory T cells in patients with acute lymphoblastic leukemia

    OpenAIRE

    WU, CUI-PING; QING, XI; WU, CUI-YUN; ZHU, HONG; ZHOU, HAI-YAN

    2011-01-01

    Acute lymphoblastic leukemia (ALL), cancer of the white blood cells, is a heterogeneous disease that mainly occurs due to the malignant cloning of original and naive lymphocytes. The aim of this study was to explore the immunophenotype, the percentage of CD4+CD25+ regulatory T cells (Tregs) and the expression of cytokines interleukin (IL)-2, IL-10 and TGF-β in patients with ALL. The immunophenotype and levels of CD4+CD25+ Tregs were detected using flow cytometry in the peripheral blood of 35 ...

  8. CD4+CD25+ and CD4+CD25high regulatory T cells in disseminated and localized forms of allergic contact dermatitis: relation to specific cytokines

    Directory of Open Access Journals (Sweden)

    Halina Laudańska

    2011-07-01

    Full Text Available The aim of this study was to evaluate regulatory T lymphocytes (Tregs in the course of allergic contact dermatitis (ACD and to elucidate the role of IL-10 and TGF-b in Tregs activity. Peripheral blood CD4+CD25+ and CD4+CD25high cells were determined by flow cytometry in patients with acute disseminated ACD (‘ad’, n = 36, acute localized ACD (‘al’, n = 26, and disseminated ACD during remission (‘rd’, n = 27 as well as in controls (n = 22. Serum levels of cytokines were measured using ELISA. The mean percentage of CD4+CD25+ and CD4+CD25high cells in patients with ad ACD was significantly higher than in controls (p < 0.01 and the remaining patients (p < 0.05. Both cell populations were significantly elevated in persons with widespread skin lesions (p < 0.05. In ad patients the CD4+CD25+ increased during three weeks of disease, although the significant increase of CD4+CD25high was noted only in the third week. Patients with ad ACD showed a significantly decreased serum level of TGF-b1 as compared with controls and the remaining ACD patients. IL-10 level did not differ between all groups. The elevated population of CD4+CD25high cells in ad ACD patients, and its dependence on the extension of skin lesions, suggest a role of Tregs in regulating the course of ACD. The growing Tregs percentages may indicate their peripheral generation during ACD. The development of lesions despite an increased population of Tregs suggests their functional defect. The role of TGF-b1 in the suppressive activity of Tregs cannot be excluded. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 2, pp. 255–262

  9. Regulatory CD4+CD25+ T cells restrict memory CD8+ T cell responses.

    Science.gov (United States)

    Kursar, Mischo; Bonhagen, Kerstin; Fensterle, Joachim; Köhler, Anne; Hurwitz, Robert; Kamradt, Thomas; Kaufmann, Stefan H E; Mittrücker, Hans-Willi

    2002-12-16

    CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.

  10. Effects of up-regulation of lymphocyte HO-1 expression on CD4+CD25+ Treg differentiation in vitro

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    Rui CHEN

    2013-09-01

    Full Text Available Objective To explore the effect of heme oxygenase-1 (HO-1 on differentiation of CD4+CD25+Treg derived from spleen T lymphocytes in mice in vitro and its underlying mechanism. Methods Lymphocytes were prepared from spleen of mice and pretreated with PBS, Copp (cobalt protoporphyrin, HO-1 inducer, 50μmol/L and Znpp (zinc protoporphyrin, HO-1 inhibitor, 50μmol/L respectively, for 12h. The expressions of HO-1 mRNA and protein in each group were determined by real-time qPCR and Western blotting. The ratio of CD4+CD25+Tregs was detected by flow cytometry. The level of Th1 and Th2 cytokines in the supernatant was determined by ELISA. Results Compared with PBS group, the expressions of HO-1 mRNA and protein, and the rates of CD4+CD25+Tregs increased in Copp group. At the same time, the cytokine of Th1, IL-2 and IFN-γ, significantly decreased, and the Th2 cytokines, TGF-β and IL-10, increased significantly (P<0.05. The results of Znpp group showed an opposite trend. Conclusion HO-1 could promote differentiation of T lymphocytes to CD4+CD25+Treg, and it may be related to an adjustment of the balance of Th1/Th2 cytokines.

  11. Ascaris suum infection modulates inflammation: Implication of CD4+CD25highFoxp3+T cells and IL-10.

    Science.gov (United States)

    Titz, T de O; de Araújo, C A A; Enobe, C S; Rigato, P O; Oshiro, T M; de Macedo-Soares, M F

    2017-09-01

    Helminth infections have the ability to modulate host's immune response through mechanisms that allow the chronic persistence of the worms in the host. Here, we investigated the mechanisms involved on the suppressive effect of Ascaris suum infection using a murine experimental model of LPS-induced inflammation. We found that infection with A. suum markedly inhibited leucocyte influx induced by LPS into air pouches, suppressed secretion of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6) and induced high levels of IL-10 and TGF-β. Augmented frequency of CD4 + CD25 high Foxp3 + T cells was observed in the mesenteric lymph nodes of infected mice. Adoptive transfer of purified CD4 + CD25 + T cells to recipient uninfected mice demonstrated that these cells were able to induce a suppressive effect in the LPS-induced inflammation in air pouch model. In addition, adoptive transfer of CD4 + CD25 + T cells derived from IL-10 knockout mice suggests that this suppressive effect of A. suum infection involves IL-10 cytokine. In conclusion, our results demonstrated that A. suum experimental infection was capable of suppressing LPS-induced inflammation by mechanisms, which seem to be dependent on responses of CD4 + CD25 + T cells and secretion of IL-10 cytokine. © 2017 John Wiley & Sons Ltd.

  12. Expression of FOXP3 mRNA is not confined to CD4+CD25+ T regulatory cells in humans.

    NARCIS (Netherlands)

    Morgan, M.E.; Bilsen, J.H. van; Bakker, A.M.; Heemskerk, B.; Schilham, M.W.; Hartgers, F.C.; Elferink, B.G.; Zanden, L.F.M. van der; Vries, R.R.P. de; Huizinga, T.W.J.; Ottenhoff, T.H.; Toes, R.E.

    2005-01-01

    Expression of the transcription factor Foxp3 (forkhead box P3) has been implicated as a key element for CD25(+) T regulatory cell function in mice. However, literature over similar involvement of FOXP3 expression in human T regulatory cells is limited. We found that, unlike murine cells, FOXP3 mRNA

  13. An increased CD25-positive intestinal regulatory T lymphocyte population is dependent upon Cox-2 activity in the Apcmin/+ model.

    Science.gov (United States)

    Faluyi, O O; Fitch, P; Howie, S E M

    2018-01-01

    Only mismatch repair (MMR)-deficient colorectal cancer (CRC) appears to respond well to programmed death (PD)-1 inhibition at the present time. Emerging evidence suggests a role for micro-environmental factors such as CD25 + cells modulating response to PD-1 inhibition. In the Apc Min/+ model of familial adenomatous polyposis (MMR-proficient CRC), increased Cyclooxygenase-2 (Cox-2) expression by cells which include alternatively activated mononuclear phagocytes promotes intestinal tumorigenesis by mechanisms which may include immune suppression. To gain insight into this, we compared regulatory T cell (T reg ) populations between Apc Min/+ and wild-type mice prior to and after the phase of increased intestinal Cox-2-dependent prostaglandin E 2 (PGE 2 ) production. There was no difference in systemic T reg function or numbers between Apc Min/+ and wild-type mice. However, increased numbers of small intestinal CD25 + T regs were observed with increased Cox-2 activity in the absence of any difference in the expression of Tgf-β or Tslp between Apc Min/+ and wild-type mice. Cox-2 inhibitor therapy (Celecoxib) reversed the increase in Apc Min/+ intestinal CD25 + T reg numbers, without decreasing numbers of CD25 + systemic T regs . Forkhead box protein 3 (FoxP3 + ) and Cox-2 + cells were co-localized to the interstitium of adenomas of Apc min/+ mice. These results suggest selective dependence of an 'activated T reg ' phenotype on paracrine Cox-2 activity in Apc Min/+ small intestine. For therapeutic potential, further studies are required to evaluate the relevance of these findings to human cancer as well as the functional significance of CD25 + intestinal T regs in cancer. © 2017 British Society for Immunology.

  14. CD4+CD25+ T cells expressing FoxP3 in Icelandic horses affected with insect bite hypersensitivity.

    Science.gov (United States)

    Hamza, Eman; Steinbach, Falko; Marti, Eliane

    2012-07-15

    Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-β1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Culicoides extract or a control antigen (Tetanus Toxoid). There were no differences between healthy and IBH horses either in the proportion of FoxP3(+)CD4(+)CD25(+) cells in freshly isolated PBMC or in the following stimulation with Tetanus Toxoid. However, upon stimulation of PBMC with the allergen, expression of FoxP3 by CD4(+)CD25(+high) and CD4(+)CD25(+dim) cells was significantly higher in healthy than in IBH horses. Addition of recombinant IL-4 to PBMC from healthy horses stimulated with the allergen significantly decreased the proportion of FoxP3 expressing cells within CD4(+)CD25(+high). These results suggest that IBH is associated with a decreased number of allergen-induced Tregs. This could be a consequence of the increased IL-4 production by PBMC of IBH-affected horses. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. CD4+CD25+CD127low FoxP3+ regulatory T cells in Crohn's disease.

    Science.gov (United States)

    Khalili, Ali; Ebrahimpour, Soheil; Maleki, Iradj; Abediankenari, Saeid; Mohammadnia Afrouzi, Mousa

    2018-02-16

    Regulatory T (Treg) cell play a key role in autoimmune diseases. We evaluated the regulatory function and frequency of Treg cells and secreted IL-10, IL-35 concentration in Crohn's disease (CD). Twenty-three patients with CD and 25 healthy controls (HC) were included in this study. We analysed the alteration of Tregs frequency using flow cytometry for CD4, CD25, CD127 and FoxP3 markers. Surface expression of CD4, CD25 and CD127 markers were used for isolation of a relatively pure Treg cells. Suppressive activity of Tregs was determined by measuring their ability to inhibit the proliferation of T responder (Tres) cells. In addition, the amounts of IL-10 and IL-35 cytokines in co-culture supernatants were measured by ELISA assay after stimulation with anti-CD2/CD3/CD28. CD patients had significantly lower frequency of CD4+ CD25+ CD127low FoxP3+ Treg cells in comparison with controls (‎2.17 ± 1.04 vs.2.83 ± 1.07, p=0.0352)‎. Additionally, Treg cells mediated suppression was not significantly different in CD patients compared to controls. There was a significant difference in IL-10 secretion in response to anti-CD2/CD3/CD28 stimulation compared with HC (p= 0.0074). The frequency of CD4+ CD25+ CD127 low FoxP3+ Tregs decreased in active stage of CD but there was no impaired suppressive function of CD4+ CD25+ ‎CD127low FoxP3+Treg cells. We suggest that an alteration in the ‎balance of Tregs and T effectors may contribute to pathogenesis of CD.‎.

  16. Urinary and serum soluble CD25 complements urinary soluble CD163 to detect active renal anti-neutrophil cytoplasmic autoantibody-associated vasculitis: a cohort study.

    Science.gov (United States)

    Dekkema, Gerjan J; Abdulahad, Wayel H; Bijma, Theo; Moran, Sarah M; Ryan, Louise; Little, Mark A; Stegeman, Coen A; Heeringa, Peter; Sanders, Jan-Stephan F

    2018-03-01

    Early detection of renal involvement in anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is of major clinical importance to allow prompt initiation of treatment and limit renal damage. Urinary soluble cluster of differentiation 163 (usCD163) has recently been identified as a potential biomarker for active renal vasculitis. However, a significant number of patients with active renal vasculitis test negative using usCD163. We therefore studied whether soluble CD25 (sCD25), a T cell activation marker, could improve the detection of renal flares in AAV. sCD25 and sCD163 levels in serum and urine were measured by enzyme-linked immunosorbent assay in 72 patients with active renal AAV, 20 with active extrarenal disease, 62 patients in remission and 18 healthy controls. Urinary and blood CD4+ T and CD4+ T effector memory (TEM) cell counts were measured in 22 patients with active renal vasculitis. Receiver operating characteristics (ROC) curves were generated and recursive partitioning was used to calculate whether usCD25 and serum soluble CD25 (ssCD25) add utility to usCD163. usCD25, ssCD25 and usCD163 levels were significantly higher during active renal disease and significantly decreased after induction of remission. A combination of usCD25, usCD163 and ssCD25 outperformed all individual markers (sensitivity 84.7%, specificity 95.1%). Patients positive for sCD25 but negative for usCD163 (n = 10) had significantly higher C-reactive protein levels and significantly lower serum creatinine and proteinuria levels compared with the usCD163-positive patients. usCD25 correlated positively with urinary CD4+ T and CD4+ TEM cell numbers, whereas ssCD25 correlated negatively with circulating CD4+ T and CD4+ TEM cells. Measurement of usCD25 and ssCD25 complements usCD163 in the detection of active renal vasculitis.

  17. The alternation of autophagy/apoptosis in CD4+CD25+Foxp3+ Tregs on the developmental stages of atherosclerosis.

    Science.gov (United States)

    Tian, Yuling; Liang, Xiao; Wu, Yue

    2018-01-01

    Naturally regulatory T cells (Tregs) play a critical role in the regulation of T cell-mediated immune responses in atherosclerosis. However, the regulatory mechanism underlying Tregs upon long-term development of atherosclerosis remains unknown. Therefore, in this study, atherosclerotic model was induced in ApoE-/- mice by feeding fat-diet for 10 weeks. Quantification of atherosclerotic lesions was done by calculating the lesion size in the aortic sinus every 2 weeks. The lipid levels and inflammatory mediators were detected in serum sample. The populations of CD4+CD25+Foxp3+ Tregs were compared between ApoE-/- mice (ApoE-/-) and wild type C57BL/6 littermates (WT). The expression levels of autophagy and apoptosis signaling related regulators were determined by flow cytomery, RT-qPCR, and western blot assays in the CD4+CD25+Foxp3+ Tregs isolated from ApoE-/- and WT. We found that the sizes of plaque lesions in atherosclerotic ApoE-/- mice were larger than those in WT group during 10 weeks' detection (all P<0.05); Whereas, flow cytometry assay showed that the populations of CD4+CD25+Foxp3+ Tregs were significantly reduced in atherosclerotic ApoE-/- mice compared with those in corresponding WT group from the 4th weeks' detection (all P<0.05). The lipid accumulation and increased pro-inflammatory mediators were correlated with the developmental progression of atherosclerosis. Furthermore, compared to WT group, the functional properties of CD4+CD25+Foxp3+ Tregs from ApoE-/- mice showed a gradually decreased autophagic activity with aberrant expressions of LC3, Beclin1, ATG5, ATG7, p62 (all P<0.05), and a gradually increased apoptotic activity with abnormal expressions of cleaved caspase 3, Bim, Bcl-2 (all P<0.05) during the 10 weeks' detection period. Taken together, our data demonstrated that the population of CD4+CD25+Foxp3+ Tregs was reversely correlated with plaque forming in atherosclerotic ApoE-/- mice during atherosclerosis development. And the autophagy

  18. Dynamic Detection of Anti-Human Leukocyte Antigen (HLA) Antibodies but not HLA-DP Loci Mismatches Can Predict Acute Graft-versus-Host Disease and Overall Survival in HLA 12/12-Matched Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation for Hematological Malignancies.

    Science.gov (United States)

    Pan, Zhijuan; Yuan, Xiaoni; Li, Yang; Wu, Xiaojin; Zhu, Wenjuan; Bao, Xiaojin; Zhao, Qinqin; He, Jun

    2016-01-01

    The National Marrow Donor Program and Center for International Blood and Marrow Transplant Research provided guidelines for the use of anti-HLA antibodies and HLA-DP-mismatched loci in unrelated donor hematopoietic stem cell transplantation (HSCT). However, a deeper understanding of other potentially useful biomarkers for predicting clinical outcomes in HLA-A, -B, -C, -DRB1, -DQB1, and -DQA1 (12/12)-matched unrelated donor HSCT is needed to further improve clinical outcomes. We tested HLA genotyping for 123 pairs of patients and donors. Anti-HLA antibodies using the Luminex method was applied to 123, 117, and 106 serum samples collected before and 1 month and 3 months after transplantation. The presences of anti-HLA antibodies at the 3 time points were 37.4% (46 of 123), 40.2% (47 of 117), and 22.6% (24 of 106). Mismatch of HLA-DPB1 and/or DPA1 allele between patient-donor pairs was 83.6% (92 of 110). Patients with anti-HLA antibodies had delayed platelet recovery. The presence of anti-HLA antibodies and their dynamic changes after transplantation were associated with increased occurrence of grades II to IV acute and chronic graft-versus-host disease (GVHD), higher treatment-related mortality, and reduced overall survival (OS) and disease-free survival, especially in acute myeloid leukemia and myelodysplastic syndrome patients. Multivariate analysis showed that presence of anti-HLA antibodies before transplantation was a risk factor for GVHD and OS. Furthermore, HLA-DP loci-matched subgroup showed a trend towards a lower rate of acute GVHD and a higher OS in the anti-HLA Abs-negative group. Our results suggest that dynamic changes of anti-HLA antibodies independently predict for a negative outcome of HSCT, independent of HLA-DP loci mismatches. Routine monitoring for anti-HLA antibody dynamics should be conducted before and after HSCT. Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  19. The calcineurin inhibitor tacrolimus allows the induction of functional CD4+CD25+ regulatory T cells by rabbit anti-thymocyte globulins

    NARCIS (Netherlands)

    V.D.K.D. Sewgobind (Varsha); L.J.W. van der Laan (Luc); M.M.L. Kho (Marcia); R. Kraaijeveld (Rens); S.S. Korevaar (Sander); W.M. Mol (Wendy)

    2010-01-01

    textabstractRabbit anti-thymocyte globulins (rATG) induce CD4+CD25 +forkhead box P3 (FoxP3+) regulatory T cells that control alloreactivity. In the present study, we investigated whether rATG convert T cells into functional CD4+CD25+FoxP3+CD127 -/low regulatory T cells in the presence of drugs that

  20. CD4+CD25+ regulatory T cells control CD8+ T-cell effector differentiation by modulating IL-2 homeostasis

    Science.gov (United States)

    McNally, Alice; Hill, Geoffrey R.; Sparwasser, Tim; Thomas, Ranjeny; Steptoe, Raymond J.

    2011-01-01

    CD4+CD25+ regulatory T cells (Treg) play a crucial role in the regulation of immune responses. Although many mechanisms of Treg suppression in vitro have been described, the mechanisms by which Treg modulate CD8+ T cell differentiation and effector function in vivo are more poorly defined. It has been proposed, in many instances, that modulation of cytokine homeostasis could be an important mechanism by which Treg regulate adaptive immunity; however, direct experimental evidence is sparse. Here we demonstrate that CD4+CD25+ Treg, by critically regulating IL-2 homeostasis, modulate CD8+ T-cell effector differentiation. Expansion and effector differentiation of CD8+ T cells is promoted by autocrine IL-2 but, by competing for IL-2, Treg limit CD8+ effector differentiation. Furthermore, a regulatory loop exists between Treg and CD8+ effector T cells, where IL-2 produced during CD8+ T-cell effector differentiation promotes Treg expansion. PMID:21502514

  1. Studie van CD4+CD25+ regulatoire T-cellen bij patiënten met multiple sclerose

    OpenAIRE

    DOUMEN, Kathleen

    2005-01-01

    Multiple sclerose (MS) wordt gekenmerkt door een immuungemedieerde afbraak van de myelinelaag rondom de zenuwen in het centrale zenuwstelsel (demyelinisatie). Sinds enkele jaren is er vernieuwde aandacht gekomen voor de rol van regulatoire T-cellen in autoimmune ziekten, waarbij een belangrijke subpopulatie van CD4+ T-cellen met constitutieve CD25 expressie (Tregs) een kritische functie vervult in de perifere immuuntolerantie. Echter zijn de ontwikkeling en functionele eigensch...

  2. Induction of Immunosuppressive CD8+CD25+FOXP3+ Regulatory T Cells by Suboptimal Stimulation with Staphylococcal Enterotoxin C1.

    Science.gov (United States)

    Lee, Juyeun; Park, Nogi; Park, Joo Youn; Kaplan, Barbara L F; Pruett, Stephen B; Park, Juw Won; Park, Yong Ho; Seo, Keun Seok

    2018-01-15

    Superantigens (SAgs) produced by Staphylococcus aureus at high concentrations induce proliferation of T cells bearing specific TCR Vβ sequences and massive cytokinemia that cause toxic shock syndrome. However, the biological relevance of SAgs produced at very low concentrations during asymptomatic colonization or chronic infections is not understood. In this study, we demonstrate that suboptimal stimulation of human PBMCs with a low concentration (1 ng/ml) of staphylococcal enterotoxin C1, at which half-maximal T cell proliferation was observed, induced CD8 + CD25 + T cells expressing markers related to regulatory T cells (Tregs), such as IFN-γ, IL-10, TGF-β, FOXP3, CD28, CTLA4, TNFR2, CD45RO, and HLA-DR. Importantly, these CD8 + CD25 + T cells suppressed responder cell proliferation mediated in contact-dependent and soluble factor-dependent manners, involving galectin-1 and granzymes, respectively. In contrast, optimal stimulation of human PBMCs with a high concentration (1 μg/ml) of staphylococcal enterotoxin C1, at which maximal T cell proliferation was observed, also induced similar expression of markers related to Tregs, including FOXP3 in CD8 + CD25 + cells, but these T cells were not functionally immunosuppressive. We further demonstrated that SAg-induced TCR Vβ-restricted and MHC class II-restricted expansion of immunosuppressive CD8 + CD25 + T cells is independent of CD4 + T cells. Our results suggest that the concentration of SAg strongly affects the functional characteristics of activated T cells, and low concentrations of SAg produced during asymptomatic colonization or chronic S. aureus infection induce immunosuppressive CD8 + Tregs, potentially promoting colonization, propagation, and invasion of S. aureus in the host. Copyright © 2018 by The American Association of Immunologists, Inc.

  3. Immunophenotype and increased presence of CD4(+)CD25(+) regulatory T cells in patients with acute lymphoblastic leukemia.

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    Wu, Cui-Ping; Qing, Xi; Wu, Cui-Yun; Zhu, Hong; Zhou, Hai-Yan

    2012-02-01

    Acute lymphoblastic leukemia (ALL), cancer of the white blood cells, is a heterogeneous disease that mainly occurs due to the malignant cloning of original and naive lymphocytes. The aim of this study was to explore the immunophenotype, the percentage of CD4(+)CD25(+) regulatory T cells (Tregs) and the expression of cytokines interleukin (IL)-2, IL-10 and TGF-β in patients with ALL. The immunophenotype and levels of CD4(+)CD25(+) Tregs were detected using flow cytometry in the peripheral blood of 35 ALL patients, with 18 healthy individuals being selected as controls. The results suggested that 22 patients had B cell ALL (B-ALL) and 13 had T cell ALL (T-ALL) among the 35 ALL patients. In B-ALL patients, the surface antigen CD19 was most commonly expressed; in T-ALL patients, CD7 was most common. Furthermore, the percentage of CD4(+)CD25(+) Treg cells in the peripheral blood of B-ALL and T-ALL patients was higher compared to that of healthy individuals (Pcell culture supernatants from B-ALL and T-ALL patients were higher compared to those in the controls (Pcells, IL-2, IL-10 or TGF-β in B-ALL versus T-ALL patients. The authors concluded that CD19 and CD7 may serve as diagnostic markers of B-ALL and T-ALL, respectively. The increased presence of CD4(+)CD25(+) Treg cells and the altered levels of secreted cytokines are indicative of an immunosuppressive mechanism in the pathogenesis of ALL.

  4. CD4+CD25+ regulatory T cells have divergent effects on intestinal inflammation in IL-10 gene-deficient mice.

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    Sydora, Beate C; MacFarlane, Sarah M; Tavernini, Michele M; Doyle, Jason S G; Fedorak, Richard N

    2008-06-01

    The regulatory effect of murine CD4+CD25+ T-cells in vivo appears to be dependent on the secretion of IL-10. The lack of IL-10 in the IL-10 gene-deficient mouse has a profoundly negative effect on the mouse's regulation of the response to intestinal bacteria, resulting in severe enterocolitis. We investigated the effect of neonatal injection with wild-type CD4+CD25+ T-cells on the intestinal immune response in IL-10 gene-deficient mice. At the time of analysis, 8-15 weeks later, all mice demonstrated an increased, antigen-stimulated systemic response. However, the intestinal response was divergent with about half of the mice developing an intestinal inflammation with a high injury score, the other half demonstrating a remarkable reduction in injury score with a marked decrease in intestinal IFNgamma release. Our data demonstrate that CD4+CD25+ T-cells can be activated in IL-10 gene-deficient mice and that this stimulation under stringent conditions has the potential to reduce intestinal inflammation.

  5. [Effect of Coixenolide on Foxp3+ CD4+ CD25+ Regulatory T Cells in Collagen-induced Arthritis Mice].

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    Zheng, Hong-xia; Zhang, Wei-ming; Zhou, Hong-juan; Zhang, Wen; Yu, Jian-ning; Wang, Wei

    2016-03-01

    To study the effect of coixenolide on Foxp3+ CD4+ CD25+ regulatory T cells (Treg) in collagen induced arthritis (CIA) mice, and to explore its possible mechanism for treating rheumatiol arthritis. Five mice were recruited as a normal control group from 25 mice, and the rest 20 were used in CIA modeling. After successful modeling they were randomly divided in the model control group and the coixenolide group, 10 in each group. Coixenolide injection at 25 mL/kg was intraperitoneally injected to mice in the coixenolide group, while normal saline at 25 mL/kg was intraperitoneally injected to mice in the normal control group and the model control group. The injection lasted for 21 days. Scoring for CIA was performed after injection and arthritis index was calculated. The peripheral blood Foxp3+ CD4+ CD25+ Treg ratio was determined by flow cytometry (FCM). Compared with the normal control group, the arthritis index obviously increased in the model control group (P coixenolide group than in the model control group (P coixenolide control group than in the model control group (P Coixenolide could up-regulate Foxp3+ CD4+ CD25+ Treg ratios in CIA mice, which might play certain immunoregulation roles in the incidence of CIA.

  6. Ex vivo generation of human alloantigen-specific regulatory T cells from CD4(posCD25(high T cells for immunotherapy.

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    Jorieke H Peters

    Full Text Available BACKGROUND: Regulatory T cell (Treg based immunotherapy is a potential treatment for several immune disorders. By now, this approach proved successful in preclinical animal transplantation and auto-immunity models. In these models the success of Treg based immunotherapy crucially depends on the antigen-specificity of the infused Treg population. For the human setting, information is lacking on how to generate Treg with direct antigen-specificity ex vivo to be used for immunotherapy. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that in as little as two stimulation cycles with HLA mismatched allogeneic stimulator cells and T cell growth factors a very high degree of alloantigen-specificity was reached in magnetic bead isolated human CD4(posCD25(high Treg. Efficient increases in cell numbers were obtained. Primary allogeneic stimulation appeared a prerequisite in the generation of alloantigen-specific Treg, while secondary allogeneic or polyclonal stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies enriched alloantigen-specificity and cell yield to a similar extent. CONCLUSIONS/SIGNIFICANCE: The ex vivo expansion protocol that we describe will very likely increase the success of clinical Treg-based immunotherapy, and will help to induce tolerance to selected antigens, while minimizing general immune suppression. This approach is of particular interest for recipients of HLA mismatched transplants.

  7. H9N2-specific IgG and CD4+CD25+ T cells in broilers fed a diet supplemented with organic acids.

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    Lee, In Kyu; Bae, Suhan; Gu, Min Jeong; You, Sun Jong; Kim, Girak; Park, Sung-Moo; Jeung, Woon-Hee; Ko, Kwang Hyun; Cho, Kyung Jin; Kang, Jung Sun; Yun, Cheol-Heui

    2017-05-01

    Organic acids have long been known for their beneficial effects on growth performance in domestic animals. However, their impact on immune responses against viral antigens in chickens is unclear. The present study aimed to investigate immunological parameters in broilers immunized with a H9N2 vaccine and/or fed a diet containing organic acids (citric, formic, and lactic acids). We allotted 1-day-old broilers into 4 groups: control (C), fed a diet supplemented with organic acids (O), administered a H9N2 vaccine (V), and fed a diet supplemented with organic acids and administered a H9N2 vaccine (OV). Blood and spleen samples were taken at 2, 7 and 14 d post vaccination (DPV). At 14 DPV, total and H9N2-specific IgG levels were significantly lower in the OV group than in the V group. However, it was intriguing to observe that at 2 DPV, the percentage of CD4+CD25+ T cells was significantly higher in the OV group than in the other groups, indicating the potential induction of regulatory T cells by organic acids. In contrast, at 2 DPV, the percentage of CD4+CD28+ T cells were significantly lower in the OV group than in the other groups, suggesting that CD28 molecules are down-regulated by the treatment. The expression of CD28 on CD4+ T cells, up-regulated by the stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Iono), was inhibited upon organic acid treatment in OV group. In addition, the proliferation of lymphocytes, stimulated with formalin-inactivated H9N2, was significantly higher in the V group than in the OV group. Alpha 1-acid glycoprotein (AGP) production was significantly lower in the OV group than in the V group, suggesting that the organic acids inhibited the inflammation caused by the vaccination. Overall, induction of regulatory CD4+CD25+ T cells, coinciding with the decrease of H9N2-specific antibodies, was observed in broilers fed organic acids. © 2016 Poultry Science Association Inc.

  8. The Nature of Increased Circulating CD4+CD25-Foxp3+ T Cells in Patients with Systemic Lupus Erythematosus: A Novel Hypothesis

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    Yan, Bing; Liu, Yi

    2009-01-01

    The forkhead family transcriptional factor (Foxp3) is an important lineage marker for regulatory T (Treg) cells. Foxp3 expression is primarily restricted to CD4+CD25+ cell population. Recently, an intriguing phenomenon is highlighted that there is a considerable amount of CD4+CD25-Foxp3+ T cells present in the peripheral blood of patients with systemic lupus erythematosus (SLE). Up to now, it is still an open question as to the nature of this cell subset. Following an analyses of the available phenotypic characteristics of CD4+CD25-Foxp3+ T cell subset along with some new findings in research of Treg in human SLE, we propose the hypothesis: the increased circulating CD4+CD25-Foxp3+ T cells in patients with SLE may constitute a peripheral reservoir of CD4+CD25+ Foxp3+ Treg cells. Under the condition of autoimmune response reactivated, CD4+CD25-Foxp3+ T cells could be recruited to expand the Treg pool upon CD25 regaining, for the effort to try to reverse a homeostatic imbalance shift to more aggressive expansion of autoreactive T cells and B cells. This hypothesis, if confirmed, would provide a new strategy for the treatment of SLE via the generation of therapeutic regulatory T cells. PMID:19590592

  9. Modulation of phenotype and function of human CD4+CD25+ T regulatory lymphocytes mediated by cAMP elevating agents

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    Antonella Riccomi

    2016-09-01

    Full Text Available We have shown that Cholera Toxin (CT and other cyclic AMP (cAMP elevating agents induce up-regulation of the inhibitory molecule CTLA-4 in human resting CD4+ T lymphocytes, which following the treatment acquired suppressive functions. In this study, we evaluated the effect of cAMP elevating agents on human CD4+CD25+ T cells, which include the T regulatory (Treg cells that play a pivotal role in the maintenance of immunological tolerance. We found that cAMP elevating agents induce up-regulation of CTLA-4 in CD4+CD25- and further enhance its expression in CD4+CD25+ T cells. We observed an increase of two isoforms of mRNA coding for the membrane and the soluble CTLA-4 molecules, suggesting that the regulation of CTLA-4 expression by cAMP is at the transcriptional level. In addition, we found that the increase of cAMP in CD4+CD25+ T cells converts the CD4+CD25+Foxp3- T cells in CD4+CD25+Foxp3+ T cells, whereas the increase of cAMP in CD4+CD25- T cells did not up-regulate Foxp3 in the absence of activation stimuli. To investigate the function of these cells, we performed an in vitro suppression assay by culturing CD4+CD25+ T cells untreated or pre-treated with CT with anti-CD3 mAbs-stimulated autologous PBMC. We found that CT enhances the inhibitory function of CD4+CD25+ T cells, CD4+ and CD8+ T cell proliferation and IFNγ production are strongly inhibited by CD4+CD25+ T cells pre-treated with cAMP elevating agents. Furthermore, we found that CD4+CD25+ T lymphocytes pre-treated with cAMP elevating agents induce the up-regulation of CD80 and CD86 co-stimulatory molecules on immature dendritic cells (DCs in the absence of antigenic stimulation, however without leading to full DC maturation. These data show that the increase of intracellular cAMP modulates the phenotype and function of human CD4+CD25+ T cells.

  10. Mast cells down-regulate CD4+CD25+ T regulatory cell suppressor function via histamine H1 receptor interaction.

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    Forward, Nicholas A; Furlong, Suzanne J; Yang, Yongjun; Lin, Tong-Jun; Hoskin, David W

    2009-09-01

    Mast cells promote both innate and acquired immune responses, but little is known about the effect of mast cells on T regulatory (T(reg)) cell function. In this study, we show for the first time that the capacity of murine CD4(+)CD25(+) T(reg) cells to suppress in vitro proliferation by CD4(+)CD25(-) T responder (T(resp)) cells in response to anti-CD3/anti-CD28 mAb-coated beads was reduced in the presence of syngeneic bone marrow-derived mast cells (BMMC) activated by FcepsilonR cross-linking. Activated BMMC culture supernatants or exogenous histamine also inhibited T(reg) cell suppressor function while the histamine H1 receptor-specific antagonist loratadine, but not the H2 receptor-specific antagonist famotidine, restored T(reg) cell suppressor function in the presence of activated BMMC or activated BMMC culture supernatants. Moreover, treatment of T(reg) cells with loratadine, but not famotidine, rescued T(reg) cell suppressor function in the presence of exogenous histamine. In addition, the H1 receptor-specific agonist 2-pyridylethylamine dihydrochloride inhibited T(reg) cell suppressor function to an extent that was comparable to histamine, whereas the H2 receptor-specific agonist amthamine dihydrobromide was without effect. Both T(reg) cells and T(resp) cells expressed H1 receptors. Exposure to histamine caused T(reg) cells to express lower levels of CD25 and the T(reg) cell-specific transcription factor Foxp3. Taken together, these data indicate that BMMC-elaborated histamine inhibited T(reg) cell suppressor function by signaling through the H1 receptor. We suggest that histamine released as a result of mast cell activation by microbial products might cause a transient decrease in T(reg) cell suppressor function, thereby enhancing the development of protective immunity.

  11. Central muscarinic cholinergic activation alters interaction between splenic dendritic cell and CD4+CD25- T cells in experimental colitis.

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    Peris Munyaka

    Full Text Available The cholinergic anti-inflammatory pathway (CAP is based on vagus nerve (VN activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR signaling. Inflammatory bowel disease (IBD patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs and sequential CD4+/CD25-T cell activation in the context of experimental colitis.The dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4+CD25-T cell co-culture were determined.McN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4+CD25-T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy.Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD.

  12. Evaluation of CD4+ CD25+ FoxP3+ regulatory T cells during treatment of patients with brucellosis.

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    Hasanjani Roushan, M R; Bayani, M; Soleimani Amiri, S; Mohammadnia-Afrouzi, M; Nouri, H R; Ebrahimpour, S

    2016-01-01

    Cell-mediated immunity (CMI) plays a critical role in the control of brucellosis. Regulatory T cells (Tregs) have a functional character in modulating the balance between host immune response and tolerance, which can eventually lead to chronic infection or relapse. The aim of this study was to assess the alteration of Tregs in cases of brucellosis before and after treatment. Thirty cases of acute brucellosis with the mean age of 41.03±15.15 years (case group) and 30 healthy persons with the mean age of 40.63±13.95 years (control group) were selected and assessed. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of all individuals. We analyzed the alteration of Treg cell count using flow cytometry for CD4, CD25, and FoxP3 markers. The level of CD4+ CD25+ FoxP3+ Treg cells was increased in active patients compared with controls (2.5±0.99% vs 1.6±0.84%, p= 0.0004), but it had declined in the treated cases (1.83±0.73%, p=0.02). The level of Tregs was elevated in three relapsed cases. The frequency of Tregs and Treg/Teff (effector T cell) ratio was correlated with inverse serum agglutination test (SAT) and, 2-mercaptoethanol (2-ME) titers as markers of treatment in brucellosis. Based on our findings, we suggest that regulatory cells, such as CD4+ CD25+ FoxP3+ Treg cells, may contribute to the development of infection processes involving immune responses in brucellosis, and evaluation of regulatory T-cell levels may be a potential diagnostic strategy for the treatment outcome in chronic and relapsed cases of brucellosis.

  13. In vitro induction of functional allergen-specific CD4+ CD25high Treg cells in horses affected with insect bite hypersensitivity.

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    Hamza, E; Akdis, C A; Wagner, B; Steinbach, F; Marti, E

    2013-08-01

    Insect bite hypersensitivity (IBH) is a recurrent allergic dermatitis of horses with similarities to human atopic eczema, caused by bites of insects of the genus Culicoides. Previous studies suggested a dysregulated T cell tolerance to Culicoides allergen in IBH-affected horses. We have investigated whether the suppressive function of CD4(+) CD25(high) cells is impaired in IBH-affected horses and possible ways to restore it. CD4(+) CD25(-) cells sorted from peripheral blood mononuclear cells (PBMC) were stimulated with irradiated autologous PBMC pulsed with Culicoides or tetanus toxoid as control antigen, in the presence of CD4(+) CD25(high) cells. Furthermore, Culicoides-specific CD4(+) CD25(high) regulatory cells were expanded or induced from CD4(+) CD25(-) cells in vitro in the presence of a combination of rIL-2 and rTGF-β1 (rIL-2/rTGF-β1) or of retinoic acid and rapamycin (RetA/Rapa). Proliferation was determined by [(3) H] thymidine incorporation and cytokine production measured by flow cytometry. The ability of Culicoides- but not tetanus-stimulated CD4(+) CD25(high) cells to suppress proliferation of CD4(+) CD25(-) cells was significantly lower in IBH-affected horses (28%) than in healthy controls (86%). The decreased suppression in IBH-affected horses was associated with a significantly higher proportion of IL-4(+) cells and a lower percentage of FoxP3(+) IL-10(+) compared to controls. Addition of rIL-2/rTGF-β1 or of RetA/Rapa to Culicoides-stimulated CD4(+) CD25(high) cells from IBH-affected horses significantly increased the proportion of FoxP3(+) IL-10(+) cells. We also found that RetA/Rapa induced a more significant decrease in the frequency of IL-4(+) cells than rIL-2/rTGF-β1. Moreover, the suppressive activity of Culicoides-stimulated CD4(+) CD25(high) cells was significantly restored by both rIL-2/rTGF-β1and RetA/Rapa, albeit in an antigen-unspecific manner. In contrast, in vitro induced Culicoides-specific CD4(+) CD25(high) cells suppressed

  14. CD4⁺CD25⁻Foxp3⁺ T cells: a marker for lupus nephritis?

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    Bonelli, Michael; Göschl, Lisa; Blüml, Stephan; Karonitsch, Thomas; Steiner, Carl-Walter; Steiner, Günter; Smolen, Josef S; Scheinecker, Clemens

    2014-04-28

    Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease, which can affect different organs. Increased proportions of CD4⁺CD25-Foxp3⁺ T cells have been described in SLE patients. The exact role of this cell population in SLE patients still remains unclear. We therefore analyzed this T cell subset in a large cohort of SLE patients with different organ manifestations. Phenotypic analyses, proportions and absolute cell numbers of CD4⁺CD25-Foxp3⁺ T cells were determined by flow cytometry (FACS) in healthy controls (HC) (n = 36) and SLE patients (n = 61) with different organ manifestations. CD4⁺CD25⁻Foxp3⁺ T cells were correlated with clinical data, the immunosuppressive therapy and different disease activity indices. In patients with active glomerulonephritis, CD4⁺CD25⁻Foxp3⁺ T cells were analyzed in urine sediment samples. Time course analyses of CD4⁺CD25⁻Foxp3⁺ T cells were performed in patients with active disease activity before and after treatment with cyclophosphamide and prednisone. CD4⁺CD25⁻Foxp3⁺ T cells were significantly increased in active SLE patients and the majority expressed Helios. Detailed analysis of this patient cohort revealed increased proportions of CD4⁺CD25⁻Foxp3⁺ T cells in SLE patients with renal involvement. CD4⁺CD25⁻Foxp3⁺ T cells were also detected in urine sediment samples of patients with active glomerulonephritis and correlated with the extent of proteinuria. CD4⁺CD25⁻Foxp3⁺ T cells resemble regulatory rather than activated T cells. Comparative analysis of CD4⁺CD25⁻Foxp3⁺ T cells in SLE patients revealed a significant association of this newly described cell population with active nephritis. Therefore CD4⁺CD25⁻Foxp3⁺ T cells might serve as an important tool to recognize and monitor SLE patients with renal involvement.

  15. CD4+ CD25− FoxP3+ regulatory cells are the predominant responding regulatory T cells after human rotavirus infection or vaccination in gnotobiotic pigs

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    Wen, Ke; Li, Guohua; Yang, Xingdong; Bui, Tammy; Bai, Muqun; Liu, Fangning; Kocher, Jacob; Yuan, Lijuan

    2012-01-01

    The distribution and dynamic changes of CD4+ CD25+ FoxP3+ and CD4+ CD25− FoxP3+ regulatory T (Treg) cells induced by human rotavirus (HRV) infection and vaccination were examined in neonatal gnotobiotic pigs infected with virulent HRV (VirHRV) or vaccinated with attenuated HRV (AttHRV). Subsets of gnotobiotic pigs in the AttHRV and control groups were challenged with VirHRV at post-inoculation day (PID) 28. We demonstrated that VirHRV infection or AttHRV vaccination reduced frequencies and numbers of tissue-residing Treg cells, and decreased the frequencies of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) producing CD4+ CD25− Treg cells in ileum, spleen and blood at PID 28. The frequencies of IL-10 and TGF-β producing CD4+ CD25− Treg cells in all sites at PID 28 were significantly inversely correlated with the protection rate against VirHRV-caused diarrhoea (r = −1, P protective immunity against rotavirus. Our results highlighted the importance of CD4+ CD25− Treg cells over CD4+ CD25+ Treg cells in rotavirus infection and immunity. AttHRV vaccination (induction of immune effector responses) reduced the expansion of CD4+ CD25− Treg cells in ileum seen in the challenged naive pigs during the acute phase of VirHRV infection and preserved normal levels of intestinal TGF-β producing Treg cells post-challenge. The reduced suppressive effect of Treg cells in AttHRV-vaccinated pigs would unleash effector/memory T-cell activation upon challenge. Preserving TGF-β producing CD4+ CD25− Treg cells is important in maintaining homeostasis. Based on our findings, a model is proposed to depict the dynamic equilibrium course of Treg and effector T-cell responses after primary rotavirus infection/vaccination and challenge. PMID:22716916

  16. The cellular prion protein is preferentially expressed by CD4+ CD25+ Foxp3+ regulatory T cells

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    Isaacs, Jeremy D; Garden, Oliver A; Kaur, Gurman; Collinge, John; Jackson, Graham S; Altmann, Daniel M

    2008-01-01

    Post-translational modification of the cellular prion protein (PrPC) is intimately associated with the pathogenesis of prion disease, yet the normal function of the protein remains unclear. PrPC is expressed in lymphoid cells and is known to be a T-cell activation antigen. Further, transcription profiling studies of regulatory T cells have shown preferential overexpression of PrPC, suggesting a possible role in regulatory function. We report that both the expression of PrP message and cell surface PrPC levels are increased in murine CD4+ CD25+ regulatory T cells compared with CD4+ CD25− cells. However, PrP0/0 mice do not show altered regulatory T-cell numbers or forkhead box P3 (Foxp3) expression levels, or impaired regulatory T-cell function in vitro. Nevertheless, the preferential expression of surface PrPC by regulatory T cells raises the possibility that therapeutic ligation of PrPC might alter immune regulation. PMID:18462346

  17. Evaluation of the Association between CD4, CD8 and CD25 Cell Counts and SLE in Active Disease and in Remission.

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    Sonawale, Archana; Bohara, Vinay; Bichile, L S

    2017-04-01

    To evaluate the correlation between the levels of CD4, CD8 and CD25 cells and SLE disease in active phase and in remission. A total of 25 SLE patients, aged between 18-60 years, and fulfilling the ACR criteria with preferential Renal and CNS involvement were included in this study. Baseline CD4/CD8 and CD25 counts, lab parameters etc were conducted. Approximately at the end of 6 months with the settlement of the disease activity blood sample was drawn for the CD4, CD8 and CD25 counts and other lab parameters. ESR showed a statistical significant decrease while the SLEDAI score and proteinuria showed a decreasing trend as the patients underwent remission. The C3 showed an increasing trend, while the C4 showed more or less a stable pattern. Rise in %CD4 and %CD25 count was statistically significant. There was negative correlation between % CD4 count and SLEDAI score, while positive correlation between % CD25 count and SLEDAI score. %CD4 count is a sensitive, specific, reliable and valid marker of active disease in SLE and can be used to follow disease activity. %CD25 count can also be used as a marker to follow disease activity.

  18. An experimental study on inhibiting graft rejection following high-risk penetrating keratoplasty by CD25 siRNA nanocarrier in rats

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    Yun-jie SHI

    2015-06-01

    Full Text Available Objective To investigate the effects of CD25 siRNA nanoparticles against immune rejection and prolongation of corneal graft survival time after high-risk corneal grafting in rats. Methods Orthotopic corneal transplantation was performed in SD rats with alkali burned corneas to mimic high-risk rat models. Donor cornea (Wistar rats was grafted into the right cornea of SD recipients on day 14 after alkali burn. The grafted rats were randomly divided into control group (Group A, EntransterTM-control CD25siRNA instillation treatment (Group B, EntransterTM-CD25siRNA instillation treatment (Group C and EntransterTM-CD25siRNA twice instillation treatment (Group D, first administration at 2-hour post-surgery and second on day 7 post-surgery. The recipient eyes were examined using a slit lamp microscope. Then, the mean survival time and rejection index (RI were calculated. The morphologies of grafts were microscopically examined with HE staining, and TEM. CD25 expression after operation was determined by quantitative RT-PCR and immunohistochemistry. Results The survival curves of transplanted cornea showed that the mean survival time in rats of groups C and D was significantly longer than that in groups A and B (P<0.05. No significant difference was found in survival time between group A and group B, and the same between group C and group D. The grafts in groups A and B showed obvious edema and thickening, with irregular arrangement of collagen fibers and infiltration of a large amount of inflammatory cells. Immunohistochemical results showed that expression of CD25 was found in the corneal epithelium, stroma and endothelium in all rats, and higher CD25 expression was observed in groups A and B. Transmission electron microscopy revealed that the degree of stromal fibroblast apoptosis and necrosis in corneal graft was obviously lower in groups C and D than that of groups A and B, with a significant statistical difference. The expression of CD25 m

  19. Changes in Th1 cells and CD4+CD25+Treg cells in non-obese diabetic mice at early stage of diabetes

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    Hong-jun WANG

    2013-11-01

    Full Text Available Objective To investigate the changes in Th1 cells and CD4+CD25+Treg cells in non-obese diabetic (NOD mice at early stage of diabetes, and to evaluate the significance of these changes. Methods Four week- (group A, 8 week- (group B and 16 week-old (group C female NOD mice (8 each were used in present study. The spleen, thymus and pancreas were harvested. Th1 and CD4+CD25+Treg cells in spleen were determined by flow cytometer, and the ratios of Th1/CD4+T, CD4+CD25+Treg/CD4+T and Th1/CD4+CD25+Treg were calculated. Subsequently, CD4–CD8–T, CD4+CD8+T, CD4–CD8+T and CD4+CD8–T cells in thymus were determined by flow cytometer, and the ratio of CD25+Treg/CD4+CD8–T was calculated. The histopathological changes in pancreas were also evaluated by HE staining and immunohistochemistry staining. Results The proportion of Th1 cells in spleen and the ratios of Th1/CD4+T and Th1/CD4+CD25+Treg were higher significantly in group C than in group A and B. However, no significant differences were found in the proportion of spleen CD4+CD25+Treg cells and the ratio of CD4+CD25+Treg/CD4+T among the three groups. Compared with group A, no obvious changes were found in thymus CD4–CD8–T, CD4+CD8+T, CD4–CD8+T and CD4+CD8–T cells in group B and C, but the ratio of thymus CD25+Treg/CD4+CD8–T increased significantly in group B and C. Lymphocytic infiltration was observed in pancreatic islets of group B and C as shown with HE staining, but Foxp3+T cells were not seen in pancreatic islets by immunohistochemistry. Conclusion Th1 cells are gradually increased at early stage of diabetes in NOD mice, but CD4+CD25+Treg cells are relatively default. These changes may play an important role in the progress of diabetes. DOI: 10.11855/j.issn.0577-7402.2013.11.004

  20. A new approach to the role of IL-7 and TGF-ß in the in vitro generation of thymus-derived CD4+CD25+Foxp3+ regulatory T cells.

    Science.gov (United States)

    Bieńkowska, Anna; Kiernozek, Ewelina; Kozlowska, Ewa; Bugajski, Łukasz; Drela, Nadzieja

    2018-02-01

    Thymus-derived regulatory T cells of CD4+CD25+Foxp3+ phenotype develop as a functional, mature population playing an essential role in self-tolerance and immune homeostasis, and exhibiting therapeutic potential to inhibit adverse immune response. Despite intensive research on thymus-derived Tregs, the knowledge about agents involved in their generation, survival, proliferation, and biological functions is still insufficient. In this research we have focused on the role of selected cytokines in previously developed in vitro model based on the application of anti-CD3 monoclonal antibodies. We have demonstrated an essential role of IL-7 and TGF-β in the generation of thymus-derived Tregs in the co-culture of thymocytes and JAWS II cells. In addition, in vitro generated Tregs exhibited their suppressive function similarly to Tregs sorted from freshly isolated thymus. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Study on immobilizations of ovine anti-human IgG and MCAb against EHF on radiation-modified silicone films

    International Nuclear Information System (INIS)

    Guo Jinhui; Ha Hongfei; Zhang Yuhua

    1990-01-01

    Films of silicone (silastic) were grafted by monomer acrylamide vis γ-radiation technology and then the ovine anti-human IgG, Epidemic hemorrhagic fever (EHF)-MCAb were immobilized on the silastic-AAM films with different grafting yields passthrough associate reactions. Measruements of relationships between grafting yields. Contents of immobilized antibodies and immunoactivities for immobilized silastic-AAM films were performed by using 125 I method ELISA method was used to measure the immunoactivities for the immobilized monoantibody. The results showed that the antibodies used can be immobilized on radiation-grafted silicone films and this immobilization method has its potential significance in clinical practice

  2. CD4+Foxp3+CD25+/- Tregs characterize liver tissue specimens of patients suffering from drug-induced autoimmune hepatitis: A clinical-pathological study.

    Science.gov (United States)

    Qu, Li-Mei; Wang, Shu-Hua; Yang, Kun; Brigstock, David R; Sun, Li; Gao, Run-Ping

    2018-02-19

    The diagnosis of drug-induced autoimmune hepatitis (DIAIH) and its differentiation from idiopathic autoimmune hepatitis (AIH) is challenging. This study aimed to differentiate DIAIH from AIH by comparing the biochemical changes, histological features, and frequencies of CD4 + Foxp3 + CD25 +/- regulatory T cells (Tregs) in liver tissues or peripheral blood lymphocytes. A total of 15 DIAIH patients and 24 AIH patients who underwent liver biopsies at initial presentation were enrolled in this study. The liver histological changes were assessed by HE staining. The phenotypic recognition and distribution of CD4 + Foxp3 + CD25 +/- Tregs in liver tissues were evaluated by single/double immunostains in serial sections. The CD4 + Foxp3 + CD25 +/- Tregs in peripheral blood were analyzed by flow cytometry. The median values of ALT and AST were 404.50 U/L and 454.10 U/L in DIAIH patients and 309.50 U/L and 315.00 U/L in AIH patients, respectively. More importantly, for the first time we found that patients with DIAIH had higher levels of serum ALT and AST, more severe degree of lobular inflammation, higher frequencies of zone 3 necrosis and higher number of lobular CD4 + Foxp3 + CD25 - Tregs compared with AIH (P < 0.05). Furthermore, there were positive correlations in DIAIH between the degree of lobular inflammation and either the AST/ALT level or the number of lobular CD4 + Foxp3 + CD25 - Tregs (P < 0.05). However, the frequency of peripheral blood CD4 + Foxp3 + CD25 +/- Tregs were not significantly different between DIAIH and AIH. The differences of ALT, AST and the number of lobular CD4 + Foxp3 + CD25 - Tregs between patients with DIAIH and those with AIH are clinically helpful in differentiating these two diseases in their early stage. Copyright © 2018. Published by Elsevier B.V.

  3. Non-traditional CD4+CD25-CD69+ regulatory T cells are correlated to leukemia relapse after allogeneic hematopoietic stem cell transplantation.

    Science.gov (United States)

    Zhao, Xiao-su; Wang, Xu-hua; Zhao, Xiang-yu; Chang, Ying-jun; Xu, Lan-ping; Zhang, Xiao-hui; Huang, Xiao-jun

    2014-07-01

    Non-traditional CD4+CD25-CD69+ T cells were found to be involved in disease progression in tumor-bearing mouse models and cancer patients recently. We attempted to define whether this subset of T cells were related to leukemia relapse after allogeneic hematopoietic cell transplantation (allo-HSCT). The frequency of CD4+CD25-CD69+ T cells among the CD4+ T cell population from the bone marrow of relapsed patients, patients with positive minimal residual disease (MRD+) and healthy donors was examined by flow cytometry. The CD4+CD25-CD69+ T cells were also stained with the intracellular markers to determine the cytokine (TGF-β, IL-2 and IL-10) secretion. The results showed that the frequency of CD4+CD25-CD69 + T cells was markedly increased in patients in the relapsed group and the MRD + group compared to the healthy donor group. The percentage of this subset of T cells was significantly decreased after effective intervention treatment. We also analyzed the reconstitution of CD4+CD25-CD69+ T cells at various time points after allo-HSCT, and the results showed that this subset of T cells reconstituted rapidly and reached a relatively higher level at +60 d in patients compared to controls. The incidence of either MRD+ or relapse in patients with a high frequency of CD4+CD25-CD69+ T cells (>7%) was significantly higher than that of patients with a low frequency of CD4+CD25-CD69+ T cells at +60 d, +90 d and +270 d after transplant. However, our preliminary data indicated that CD4+CD25-CD69+ T cells may not exert immunoregulatory function via cytokine secretion. This study provides the first clinical evidence of a correlation between non-traditional CD4+CD25-CD69+ Tregs and leukemia relapse after allo-HSCT and suggests that exploration of new methods of adoptive immunotherapy may be beneficial. Further research related to regulatory mechanism behind this phenomenon would be necessary.

  4. Increased natural CD4+CD25+ regulatory T cells and their suppressor activity do not contribute to mortality in murine polymicrobial sepsis.

    Science.gov (United States)

    Scumpia, Philip O; Delano, Matthew J; Kelly, Kindra M; O'Malley, Kerri A; Efron, Philip A; McAuliffe, Priscilla F; Brusko, Todd; Ungaro, Ricardo; Barker, Tolga; Wynn, James L; Atkinson, Mark A; Reeves, Westley H; Salzler, Michael J Clare; Moldawer, Lyle L

    2006-12-01

    Regulatory T cells (Tregs), including natural CD4+CD25+ Tregs and inducible IL-10 producing T regulatory type 1 (T(R)1) cells, maintain tolerance and inhibit autoimmunity. Recently, increased percentages of Tregs have been observed in the blood of septic patients, and ex vivo-activated Tregs were shown to prevent polymicrobial sepsis mortality. Whether endogenous Tregs contribute to sepsis outcome remains unclear. Polymicrobial sepsis, induced by cecal ligation and puncture, caused an increased number of splenic Tregs compared with sham-treated mice. Splenic CD4+CD25+ T cells from septic mice expressed higher levels of Foxp3 mRNA and were more efficient suppressors of CD4+CD25- T effector cell proliferation. Isolated CD4+ T cells from septic mice displayed increased intracellular IL-10 staining following stimulation, indicating that T(R)1 cells may also be elevated in sepsis. Surprisingly, Ab depletion of total CD4+ or CD4+CD25+ populations did not affect mortality. Furthermore, no difference in survival outcome was found between CD25 or IL-10 null mice and wild-type littermates, indicating that Treg or T(R)1-generated IL-10 are not required for survival. These results demonstrate that, although sepsis causes a relative increase in Treg number and increases their suppressive function, their presence does not contribute significantly to overall survival in this model.

  5. Deficiency of Mouse CD4+CD25+Foxp3+ Regulatory T Cells in Xenogeneic Pig Thymus-Grafted Nude Mice Suffering from Autoimmune Diseases

    Science.gov (United States)

    Zhang, Baojun; Sun, Chenming; Qu, Yanyan; Zhang, Aijun; Liu, Jun; Zhang, Lianjun; Niu, Zeqing; Zhao, Yong

    2008-01-01

    Xenogeneic thymus transplantation can efficiently induce specific immune tolerance to donor antigens in athymic recipients. However, many nude mice suffer from autoimmune diseases (AID) for over 10 weeks after xenogeneic thymus transplantation. CD4+CD25+Foxp3+ regulatory T (Treg) cells were recently determined to play a pivotal role in keeping immune tolerance in humans and mice. Thus, we investigated this subpopulation of Treg cells in the periphery of pig thymus-grafted nude mice suffering from AID. Our results showed that the expression of Foxp3, CTLA-4 and GITR on mouse CD4+CD25+ T cells and the ratio of CD4+CD25+Foxp3+ Treg cells to CD4+ T cells were significantly decreased in the periphery of pig thymus-grafted nude mice suffering from AID, compared with healthy pig or mouse thymus-grafted nude mice. Furthermore, mouse CD4+CD25+ T cells in pig thymus-grafted nude mice suffering from AID showed more severe deficiency in immunosuppressive function compared with the counterpart in xenogeneic pig or syngeneic thymus-grafted nude mice without AID. Thus, the decreased frequency, altered phenotype and functional deficiency of mouse CD4+CD25+ Treg cells in pig thymus-grafted nude mice may contribute to the development of AID in this model. PMID:18954555

  6. 21 CFR 660.50 - Anti-Human Globulin.

    Science.gov (United States)

    2010-04-01

    ... which shall consist of one or more antiglobulin antibodies identified in § 660.55(d). (b) Source. The... globulins or protein-rich fluids derived from stable immunoglobulin-secreting cell lines maintained either...

  7. Increased sensitivity of CD4+ T-effector cells to CD4+CD25+ Treg suppression compensates for reduced Treg number in asymptomatic HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Georgina Thorborn

    Full Text Available BACKGROUND: In HIV infection, uncontrolled immune activation and disease progression is attributed to declining CD4+CD25+FoxP3+ regulatory T-cell (Treg numbers. However, qualitative aspects of Treg function in HIV infection, specifically the balance between Treg cell suppressive potency versus suppressibility of effector cells, remain poorly understood. This report addresses this issue. METHODOLOGY/PRINCIPAL FINDINGS: A classic suppression assay to measure CD4+CD45RO+CD25hi Treg cells to suppress the proliferation of CD4+CD45RO+CD25- effectors cells (E following CD3/CD28 polyclonal stimulation was employed to compare the suppressive ability of healthy volunteers (N = 27 and chronic, asymptomatic, treatment naïve, HIV-infected subjects (N = 14. HIV-infected subjects displayed significantly elevated Treg-mediated suppression compared to healthy volunteers (p = 0.0047. Cross-over studies comparing Treg cell potency from HIV-infected versus control subjects to suppress the proliferation of a given population of allogeneic effector cells demonstrated increased sensitivity of CD4+CD25- effector cells from HIV-infected subjects to be suppressed, associated with reduced production of the Treg counter-regulatory cytokine, IL-17, rather than an increase in the suppressive potential of their CD4+CD25+ Treg cells. However, compared to controls, HIV+ subjects had significantly fewer absolute numbers of circulating CD4+CD25+FoxP3+ Treg cells. In vitro studies highlighted that one mechanism for this loss could be the preferential infection of Treg cells by HIV. CONCLUSIONS/SIGNIFICANCE: Together, novel data is provided to support the contention that elevated Treg-mediated suppression may be a natural host response to HIV infection.

  8. FoxP3+CD4+CD25+ T cells with regulatory properties can be cultured from colonic mucosa of patients with Crohn's disease

    Science.gov (United States)

    Kelsen, J; Agnholt, J; Hoffmann, H J; Rømer, J L; Hvas, C L; Dahlerup, J F

    2005-01-01

    CD4+CD25+ regulatory T cells (Tregs) are involved in the maintenance of peripheral tolerance and ensure a balanced immune response competent of fighting pathogens and at the same time recognizing commensals as harmless. This feature is lost in Crohn's disease (CD). The forkhead/winged helix transcription factor FoxP3 is a master gene for Treg function and defects in the FoxP3 gene lead to a clinical picture similar to inflammatory bowel disease (IBD). Murine colitis can be cured by adoptive transfer of Tregs and ex vivo-generated gut-specific Tregs represent an attractive option for therapy in CD. Thus, defective Tregs could contribute to the development of CD. We cultured biopsies of colonic mucosa in the presence of high concentrations of interleukin (IL)-2 and IL-4 to overcome the anergic nature of naturally occurring CD4+CD25+ Tregs in the mucosa. We investigated the expression of FoxP3 and regulatory potential of gut-derived CD4+CD25+ T cells cultured from patients with CD and healthy individuals. The FoxP3 expression was analysed by reverse transcriptase polymerase chain reaction (RT-PCR), and the suppressive effect of FoxP3+CD4+CD25+ T cells on proliferation and cytokine production of autologous CD4+ T cells was assessed by flow cytometry. Cultured gut-derived T cells with CD4+CD25+ phenotype expressed FoxP3 and were able as the freshly isolated Tregs from peripheral blood to suppress proliferation and cytokine production of autologous CD4+ T cells. Thus, we demonstrate that FoxP3+CD4+CD25+ T cells with regulatory properties can be propagated in vitro from inflamed mucosa of CD patients, which may be of interest in adoptive immunotherapy. PMID:16045746

  9. [Change of CD4(+) CD25(+) regulatory T cells and NK Cells in peripheral blood of children with acute leukemia and its possible significance in tumor immunity].

    Science.gov (United States)

    Wu, Ze-Lin; Hu, Guan-Yu; Chen, Fu-Xiong; Lu, Hui-Min; Wu, Zi-Liang; Li, Hua-Mei; Wei, Feng-Gui; Guan, Jing-Ming; Wu, Li-Ping

    2010-06-01

    This study was purposed to investigate the changes of CD4(+) CD25(+) regulatory T cells and NK cells in peripheral blood of acute leukemia children at different stages, the function of immune system and the possible roles of the CD4(+) CD25(+) regulatory T cells as well as NK cells in leukemia immunity. The number and proportion of CD4(+) CD25(+) regulatory T cells and NK cells were detected by flow cytometry in the peripheral blood of 53 acute leukemia children, including 25 patients in new diagnosis and 28 patients in continuous complete remission (CCR), and were compared with that of 20 normal children. The results indicated that the mean proportion of CD4(+) CD25(+) CD127(+) in CD4(+) T cells of peripheral blood in newly diagnosed patients, patients with CCR and normal children were (9.55 +/- 2.41)%, (8.54 +/- 2.51)% and (6.25 +/- 0.85)% respectively, the mean proportions of CD4(+)CD25(+)CD127(+) in newly diagnosed patients and patients with CCR were higher than that in normal children, the mean proportion of CD4(+)CD25(+)CD127(+) in newly diagnosed patients were higher than that in patients with CCR (p cell count in patients with acute leukaemia decreased as compared with normal control, while after achieving CCR, the NK cell count in patients were also less than that in normal control (4.11 +/- 3.87% and 10.41 +/- 7.20% vs 14.06 +/- 5.95%, p regulatory T cells is a simple, reproductive and accurate method, and the CD4(+) CD25(+) CD127(+) T cells can better reflect the proportion of CD4(+)CD25(+) regulatory T cells. The increase of regulatory T cells and decrease of NK cells in pediatric patients with acute leukemia indicate that the function of NK cells may be depressed. Treg T cells play a role in occurrence and development of leukemia, and are involved in down-regulating NK cell function.

  10. Reconstitution of Scid mice with CD4+CD25- T cells leads to rapid colitis: an improved model for pharmacologic testing

    DEFF Research Database (Denmark)

    Kjellev, Stine; Lundsgaard, Dorthe; Poulsen, Steen Seier

    2006-01-01

    was paralleled by increased fecal soluble tumor necrosis factor receptor II content. Cytokines in colonic tissue homogenates exhibited a Th1-like profile. We conclude that adoptive transfer of CD4+CD25- T cells results in colitis resembling IBD with a rapid onset and limited variability between individuals....... Purification of CD4+CD25- T cells is a simple procedure, and does not require flow-cytometric sorting. Fecal consistency score and colonic weight:length ratio are readily measurable and consistent disease parameters. This model is thus highly suitable for pharmacological testing of intervention strategies....

  11. Expansion of CD25-Negative Forkhead Box P3-Positive T Cells during HIV and Mycobacterium tuberculosis Infection

    Directory of Open Access Journals (Sweden)

    Matías T. Angerami

    2017-05-01

    Full Text Available Tuberculosis (TB and HIV alter the immune system, and coinfected (HIV-TB individuals usually present deregulations of T-lymphocytic immune response. We previously observed an increased frequency of “unconventional” CD4+CD25−FoxP3+ Treg (uTreg population during HIV-TB disease. Therefore, we aimed to explore the phenotype and function of uTreg and conventional CD4+CD25+FoxP3+ Treg subsets (cTreg in this context. We evaluated the expression of CD39, programmed cell death protein 1 (PD1, glucocorticoid-induced tumor necrosis factor receptor (GITR, and the effector/memory distribution by flow cytometry in cTreg and uTreg. Also, IL-10, TGF-β, IFN-γ production, and the suppressor capacity of uTregs were analyzed in cocultures with effector lymphocytes and compared with the effect of regulatory T cells (Tregs. We found diminished expression of CD39 and higher levels of PD1 on uTreg compared to cTreg in both HIV-TB and healthy donors (HD. In addition, uTreg and cTreg showed differences in maturation status in both HIV-TB and HD groups, due to the expansion of effector memory uTregs. Interestingly, both HIV-TB and HD showed a pronounced production of IFN-γ in uTreg population, though no significant differences were observed for IL-10 and TGF-β production between uTreg and cTreg. Moreover, IFN-γ+ cells were restricted to the CD39− uTreg population. Finally, when the suppressor capacity was evaluated, both uTreg and cTreg inhibited polyclonal T cell-proliferation and IFN-γ production in a similar extent. These findings suggest that uTregs, which are expanded during HIV-TB coinfection, exert regulatory functions in a similar way to cTregs despite an altered surface expression of Treg characteristic markers and differences in cytokine production.

  12. TOGOG & SEMAR: DEHUMANIZATION, ANTI-HUMAN, POST-HUMAN

    Directory of Open Access Journals (Sweden)

    Imam Setyobudi

    2018-01-01

    Full Text Available The core of Wayang stories (Mahabharata, Bharatayuda, Ramayana is the reflection of human that is always continuously in crisis and emergency situations and conditions. Tragic. Wayang stories raise humanity problems so that the existences of human conception experience the perpetuity of dehumanization, existential instability and ambiguous. The killing of the essence of the human existence purpose on earth. It is an issue fundamentally of anti-human. Shadow play perspective does not rest on human understanding or Knight  (Pandawa, Kurawa, Rama, Giant, the heaven God (Bathara Guru, and/or Prabu Khresna, incarnation of Wisnhu God. Wayang stories evoke Togog and Semar angle. Liyan. Grassroots. The general public. Amorphous: No Man is not a knight instead of a brahmana is not god instead of giant. Arises a question that is closely related to the presence of Togog and Semar. Why do shadow play stories present the figure of the them, while the authentic story of the Mahabharata and Ramayana India version does not exist at all? Are Togog and Semar truly actualization of post-human aesthetic and post-human anthropology ideas? The substance of this writing ask us to discuss regarding the idea of Togog and Semar in shadow play into the realm of post-human discourse as a result of dehumanization signs in the frame of shadow play stories. The main focus of the discussion focused on the position of Togog and Semar as the figure as linuwih or great beyond human (knight and god. Study restriction on the context of Purwa Java Gagrak Jogja and Solo wayang; similarly in East Java, Sunda and Bali, there are also those two figures.

  13. Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

    Science.gov (United States)

    Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

    2016-06-10

    Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

  14. Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

    Directory of Open Access Journals (Sweden)

    Begoña Barriuso

    2016-06-01

    Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

  15. GITR overexpression on CD4+CD25+ HTLV-1 transformed cells: detection by massively parallel signature sequencing.

    Science.gov (United States)

    Bal, Harshawardhan P; Cheng, Jihua; Murakami, Akikazu; Tallarico, Aimee St Claire; Wang, Wei; Zhou, Daixing; Vasicek, Thomas J; Marasco, Wayne A

    2005-07-01

    HTLV-I is the etiologic agent of adult T-cell leukemia (ATL), a fatal T-cell malignancy that is associated with profound immunosuppression. In this study, comprehensive gene expression profiling was performed using massively parallel signature sequencing (MPSS) to investigate virus-host interactions in acutely HTLV-1 transformed cells. The analysis revealed the modulation of numerous genes across different functional classes, many of which have not been previously implicated in HTLV-1 transformation or ATL. Differences in the transcriptomes of transformed cell lines were observed that have provided clues on how different clonal populations of cells respond to virus transformation. Quantitation of HTLV-1 transcription was possible, thus making MPSS a useful tool to study emerging pathogens and unknown microbial causes of human diseases. Importantly, overexpression of GITR, an activation marker that has not been previously reported to be upregulated by HTLV-1-infection or in transformed/leukemic cells and that is associated with the suppressor phenotype of CD4+CD25+ regulatory T-cells (Tregs), was also observed. The deep and quantitative gene expression profile generated by MPSS should provide additional leads for discovery research that can be applied to better understand the pathobiology of HTLV-1 transformation and ATL as well as to developing new therapies.

  16. Increased bronchial density of CD25+Foxp3+ regulatory T cells in occupational asthma: relationship to current smoking.

    Science.gov (United States)

    Sjåheim, T B; Bjørtuft, Ø; Drabløs, P A; Kongerud, J; Halstensen, T S

    2013-05-01

    To identify activated T cell subset in the asthmatic bronchia, we developed a triple-colour immunohistofluorescence labelling technique on cryo-section to discriminate activated CD4+CD25+ T cells, (effector T cells) from Foxp3+ regulatory T cells (Treg). Additional coexpression of activation and proliferation markers was also examined in situ. Bronchial biopsies were taken from 20 aluminium potroom workers (12 smokers) with asthma (>12% reversibility), 15 non-asthmatic potroom workers (7 smokers) and 10 non-smoking, non-exposed controls. Non-smoking asthmatics had significantly higher subepithelial density of both Tregs, effector T cells, activated (HLA-DR+) CD8+ and activated CD4+ T cells. Moreover, both Tregs, effector T cells and CD8+ T cells proliferated in the non-smoking asthmatics, only. Although smoking asthmatics had no asthma-associated increase in bronchial T cell, both had a significantly increase in effector T cell to Treg ratios. The significantly increased bronchial density of Tregs, effector T cells, proliferative T cells and activated CD8+ T cells in non-smoking asthmatics clearly showed that both the effector T cells and the inhibitory Treg system were activated in asthma. © 2013 The Authors. Scandinavian Journal of Immunology © 2013 Blackwell Publishing Ltd.

  17. CD25, CD28 and CD38 expression in peripheral blood lymphocytes as a tool to predict acute rejection after liver transplantation.

    Science.gov (United States)

    Boleslawski, Emmanuel; BenOthman, Samia; Grabar, Sophie; Correia, Leonor; Podevin, Philippe; Chouzenoux, Sandrine; Soubrane, Olivier; Calmus, Yvon; Conti, Filomena

    2008-01-01

    The aim of this study was to determine whether the expression of CD25, CD28 and CD38 (which reflects the degree of T-cell activation) by peripheral blood mononuclear cells constitutes a useful means of measuring the immune status of liver transplant recipients. Fifty-two patients enrolled in a prospective randomized study comparing cyclosporine and tacrolimus as the principal immunosuppressive drugs were monitored prospectively. The expression of CD25, CD28 and CD38 was analyzed on CD3-, CD4- and CD8-positive cells from whole blood using flow cytometry. The prognostic value of baseline and day 14 measurements regarding acute rejection was examined using Kaplan-Meier estimates for univariate analyses and the Cox model for multivariate analyses. The mean frequencies of CD28 and CD38-expressing T cells were significantly higher in patients with acute rejection (p = 0.01 and p = 0.001, respectively), whereas the frequency CD25-expressing T cells did not differ significantly. Under univariate analysis, baseline CD25 levels, the type of calcineurin inhibitor, as well as the CD28 and CD38 frequencies obtained at day 14 were associated with the subsequent development of acute rejection. Under multivariate analysis, only CD28 and CD38 frequencies obtained at day 14 were independently associated with acute rejection. The evaluation of CD28 and CD38 expression in peripheral blood lymphocytes is a simple marker that could be used routinely in clinical practice to assess the level of immunosuppression.

  18. FoxP3(+)CD4(+)CD25(+) T cells with regulatory properties can be cultured from colonic mucosa of patients with Crohn's disease

    DEFF Research Database (Denmark)

    Rømer, Johanne Lade

    2005-01-01

    Summary CD4(+)CD25(+) regulatory T cells (T(regs)) are involved in the maintenance of peripheral tolerance and ensure a balanced immune response competent of fighting pathogens and at the same time recognizing commensals as harmless. This feature is lost in Crohn's disease (CD). The forkhead...... option for therapy in CD. Thus, defective T(regs) could contribute to the development of CD. We cultured biopsies of colonic mucosa in the presence of high concentrations of interleukin (IL)-2 and IL-4 to overcome the anergic nature of naturally occurring CD4(+)CD25(+) T(regs) in the mucosa. We...... investigated the expression of FoxP3 and regulatory potential of gut-derived CD4(+)CD25(+) T cells cultured from patients with CD and healthy individuals. The FoxP3 expression was analysed by reverse transcriptase polymerase chain reaction (RT-PCR), and the suppressive effect of FoxP3(+)CD4(+)CD25(+) T cells...

  19. FOXP3, CBLB and ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4(+) CD25(high) T cells in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, F; Krakauer, M; Khademi, M

    2012-01-01

    the phenotype of CD4(+) CD25(high) T cells in MS by flow cytometry and its relationship with expression of the FOXP3, ITCH and CBLB genes. We found that untreated MS patients had lower cell surface expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) on CD4(+) CD25(high) T cells and higher intracellular CTLA......Expression of the forkhead box protein 3 (FoxP3) transcription factor is regulated by the E3 ubiquitin ligases Itch and Cbl-b and induces regulatory activity CD4(+) CD25(high) T cells. Treatment with interferon (IFN)-β enhances regulatory T cell activity in multiple sclerosis (MS). We studied......-4 expression than healthy controls. Cell surface expression of CTLA-4 on CD4(+) CD25(high) T cells correlated with expression of FOXP3 mRNA in untreated patients and increased significantly with time from most recent injection in patients treated with IFN-β. FOXP3 mRNA expression correlated...

  20. In vitro inhibition of enterobacteria-reactive CD4+CD25- T cells and suppression of immunoinflammatory colitis in mice by the novel immunomodulatory agent VGX-1027

    DEFF Research Database (Denmark)

    Mangano, Katia; Sardesai, Niranjan; D'Alcamo, Maria

    2008-01-01

    of innate immunity in inflammatory bowel diseases prompted us to consider the use of VGX-1027 in these diseases leading us to in vitro and in vivo test the drug in related experimental conditions. These consist, respectively, of the proliferation assay of CD4+CD25- T cells to enterobacteria, and the acute...

  1. The relative values of CD8+CD25+Foxp3brigh Treg cells correlate with selected lung function parameters in asthma.

    Science.gov (United States)

    Eusebio, M; Kuna, P; Kraszula, L; Kupczyk, M; Pietruczuk, M

    2015-06-01

    The study aimed to detect CD8(+)CD25(+)FoxP3(brigh) Tregs and investigate their possible association with selected lung function values. CD8(+)CD25(+)FoxP3(brigh) Tregs were detected by flow cytometry in the peripheral blood of 25 patients with severe asthma (SA), 25 patients with mild-to-moderate asthma (MA), and 25 age-matched healthy donors (NC). The percentages of CD8(+)CD25(+)FoxP3(brigh) Tregs of the patients with severe (3.4 ± 4.55), and mild-to-moderate asthma (7.5 ± 8.15), were markedly lower than those of controls (12.1 ± 13.2). The mean forced expiratory volume in 1 s (FEV1) % predicted value in severe asthma subpopulation was significantly lower (67.05 ± 15.98%) when compared with that of mild-to-moderate asthma subgroup (87.71 ± 16.12%). Interestingly, the percentages of CD8(+)CD25(+)FoxP3(brigh) Tregs correlate with mean peak expiratory flow (PEF)% predicted values in severe (r = 0.7, P <0.01) and mild-to-moderate (r = 0.73, P <0.01) asthma. In contrast, this parameter was positively correlated with FEV1% predicted values in the severe asthmatics only (r = 0.71, P <0.01). In summary, this study establishes a link between the percentage of CD8(+)CD25(+)FoxP3(brigh) Tregs and selected lung function parameters, suggesting that this parameter has potential as a marker for inflammation and airflow obstruction. © The Author(s) 2015.

  2. Ozone and allergen exposure during postnatal development alters the frequency and airway distribution of CD25+ cells in infant rhesus monkeys

    International Nuclear Information System (INIS)

    Miller, Lisa A.; Gerriets, Joan E.; Tyler, Nancy K.; Abel, Kristina; Schelegle, Edward S.; Plopper, Charles G.; Hyde, Dallas M.

    2009-01-01

    The epidemiologic link between air pollutant exposure and asthma has been supported by experimental findings, but the mechanisms are not understood. In this study, we evaluated the impact of combined ozone and house dust mite (HDM) exposure on the immunophenotype of peripheral blood and airway lymphocytes from rhesus macaque monkeys during the postnatal period of development. Starting at 30 days of age, monkeys were exposed to 11 cycles of filtered air, ozone, HDM aerosol, or ozone + HDM aerosol. Each cycle consisted of ozone delivered at 0.5 ppm for 5 days (8 h/day), followed by 9 days of filtered air; animals received HDM aerosol during the last 3 days of each ozone exposure period. Between 2-3 months of age, animals co-exposed to ozone + HDM exhibited a decline in total circulating leukocyte numbers and increased total circulating lymphocyte frequency. At 3 months of age, blood CD4+/CD25+ lymphocytes were increased with ozone + HDM. At 6 months of age, CD4+/CD25+ and CD8+/CD25+ lymphocyte populations increased in both blood and lavage of ozone + HDM animals. Overall volume of CD25+ cells within airway mucosa increased with HDM exposure. Ozone did not have an additive effect on volume of mucosal CD25+ cells in HDM-exposed animals, but did alter the anatomical distribution of this cell type throughout the proximal and distal airways. We conclude that a window of postnatal development is sensitive to air pollutant and allergen exposure, resulting in immunomodulation of peripheral blood and airway lymphocyte frequency and trafficking

  3. High soluble CD30, CD25 and IL-6 may identify patients with worse survival in CD30+ cutaneous lymphomas and early mycosis fungoides

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    Kadin, Marshall E.; Pavlov, Igor; Delgado, Julio C.; Vonderheid, Eric C.

    2011-01-01

    Histopathology alone cannot predict outcome of patients with CD30+ primary cutaneous lymphoproliferative disorders (CD30CLPD) and early mycosis fungoides (MF). To test the hypothesis that serum cytokines/cytokine receptors provide prognostic information in these disorders, we measured soluble CD30 (sCD30), sCD25, and selected cytokines in cell cultures and sera of 116 patients with CD30CLPD and 96 patients with early MF followed up to 20 years. Significant positive correlation was found between sCD30 levels and sCD25, CD40L, IL-6, and IL-8, suggesting CD30+ neoplastic cells secrete these cytokines, but not Th2 cytokines. In vitro studies confirmed sCD30, sCD25, IL-6 and IL-8 are secreted by CD30CLPD-derived cell lines. CD30CLPD patients with above normal sCD30 and sCD25 had worse overall and disease-related survivals, but only sCD30 retained significance in Cox models that included advanced age. High sCD30 also identified patients with worse survival in early MF. Increased IL-6 and IL-8 correlated with poor disease-related survival in CD30CLPD patients, We conclude that: (1) neoplastic cells of some CD30CLPD patients do not resemble Th2 cells, (2) high serum sCD30, sCD25, IL-6, and perhaps IL-8 levels may provide prognostic information useful for patient management. PMID:22071475

  4. Unbalanced expression of aryl hydrocarbon receptor in peripheral blood CCR6+CD4+ and CD4+CD25+T cells of rheumatoid arthritis

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    Lin Cheng

    Full Text Available ABSTRACT Objective: The goal of this study was to analyze the role of aryl hydrocarbon receptor in peripheral blood CCR6+CD4+ and CD4+CD25+T cells of patients with rheumatoid arthritis. Methods: Flow cytometry was applied to determine the proportion of AhR positive cells in CCR6+CD4+T, CD4+CD25+T and peripheral blood peripheral mononuclear cells from each subject. AhR mRNA and CYP1A1 mRNA relative expression levels were tested by real-time PCR. Results: The percentage of AhR positive cells in peripheral blood mononuclear cells was higher in RA group than that in healthy cases [(35.23 ± 10.71% vs. (18.83 ± 7.32%, p < 0.01]. The expression levels of AhR and CYP1A1 were both increased in patients with RA while compared to controls [(3.71 ± 1.63 vs. (2.00 ± 1.27, p = 0.002; (2.62 ± 2.08 vs. (0.62 ± 0.29, p < 0.01, respectively]. In RA patients, the percentage of AhR positive cells in CD4+CD25+T cells was significantly lower than that from controls [17.90 (6.10 ± 80.10% vs. (52.49 ± 19.18%, p < 0.01]; In healthy controls, the percentage of AhR positive cells in CD4+CD25+T cells was significantly higher than that in CCR6+CD4+T cells, and was also significantly higher than that in PBMCs [(52.49 ± 19.18% vs. (23.18 ± 5.62% vs. (18.06 ± 7.80%, X 2 = 24.03, p < 0.01]; in RA patients, the percentage of AhR positive cells in CCR6+CD4+T cells was significantly increased than that in CD4+CD25+T cells and PBMCs [(46.02 ± 14.68% vs. 17.90 (6.10 ± 80.10% vs. (34.22 ± 10.33%, X 2 = 38.29, p < 0.01]; Nevertheless, no statistically significant relationship was found between clinical data and AhR positive cells in CCR6+CD4+T and CD4+CD25+T cells. Conclusion: AhR may participate in the pathological progress of RA by controlling the differentiation of Th17 and Treg cells in peripheral blood.

  5. Unbalanced expression of aryl hydrocarbon receptor in peripheral blood CCR6+CD4+and CD4+CD25+T cells of rheumatoid arthritis.

    Science.gov (United States)

    Cheng, Lin; Qian, Long; Tan, Yue; Wang, Guo-Sheng; Li, Xiao-Mei; Li, Xiang-Pei; Luo, Chao-Yin

    The goal of this study was to analyze the role of aryl hydrocarbon receptor in peripheral blood CCR6 + CD4 + and CD4 + CD25 + T cells of patients with rheumatoid arthritis. Flow cytometry was applied to determine the proportion of AhR positive cells in CCR6 + CD4 + T, CD4 + CD25 + T and peripheral blood peripheral mononuclear cells from each subject. AhR mRNA and CYP1A1 mRNA relative expression levels were tested by real-time PCR. The percentage of AhR positive cells in peripheral blood mononuclear cells was higher in RA group than that in healthy cases [(35.23±10.71)% vs. (18.83±7.32)%, p<0.01]. The expression levels of AhR and CYP1A1 were both increased in patients with RA while compared to controls [(3.71±1.63) vs. (2.00±1.27), p=0.002; (2.62±2.08) vs. (0.62±0.29), p<0.01, respectively]. In RA patients, the percentage of AhR positive cells in CD4 + CD25 + T cells was significantly lower than that from controls [17.90 (6.10±80.10)% vs. (52.49±19.18)%, p<0.01]; In healthy controls, the percentage of AhR positive cells in CD4 + CD25 + T cells was significantly higher than that in CCR6 + CD4 + T cells, and was also significantly higher than that in PBMCs [(52.49±19.18)% vs. (23.18±5.62)% vs. (18.06±7.80)%, X 2 =24.03, p<0.01]; in RA patients, the percentage of AhR positive cells in CCR6 + CD4 + T cells was significantly increased than that in CD4 + CD25 + T cells and PBMCs [(46.02±14.68)% vs. 17.90 (6.10±80.10)% vs. (34.22±10.33)%, X 2 =38.29, p<0.01]; Nevertheless, no statistically significant relationship was found between clinical data and AhR positive cells in CCR6 + CD4 + T and CD4 + CD25 + T cells. AhR may participate in the pathological progress of RA by controlling the differentiation of Th17 and Treg cells in peripheral blood. Copyright © 2016 Elsevier Editora Ltda. All rights reserved.

  6. Regulatory T cells in early life: comparative study of CD4+CD25high T cells from foals and adult horses.

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    Eman Hamza

    Full Text Available The immune system of mammals is subject to continuous development during the postnatal phase of life. Studies following the longitudinal development of the immune system in healthy children are limited both by ethical considerations and sample volumes. Horses represent a particular valuable large animal model for T regulatory (Treg cells and allergy research. We have recently characterised Treg cells from horses, demonstrated their regulatory capability and showed both their expansion and induction in vitro. Insect bite hypersensitivity (IBH is a common allergy in horses resembling atopic dermatitis and studies have shown that first exposure to allergens in adult life results in an increased incidence of IBH. The aim of the present study was to characterize circulating CD4+CD25highFoxP3+cells in foals, evaluate their suppressive capability and their in vitro induction compared to adult horses. 19 foals (age range, 1-5 months, their adult mothers and six one-year-old horses (yearlings were included in the study. The proportion of FoxP3+ cells within the circulating CD4+CD25high population was significantly higher in foals (47% compared to their mothers (18% and to yearlings (26%. Treg cells from foals also displayed a higher suppressive capability. Furthermore, CD4+CD25high cells in foals could be induced in vitro from CD4+CD25- cells in a significantly higher proportion compared to mares. These cells also displayed a significantly enhanced suppressive capability. In summary these findings support the notion that exposure of horses to allergens during maturation of the immune system assists the establishment of induced (iTreg driven tolerance.

  7. Regulatory T cells in early life: comparative study of CD4+CD25high T cells from foals and adult horses.

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    Hamza, Eman; Mirkovitch, Jelena; Steinbach, Falko; Marti, Eliane

    2015-01-01

    The immune system of mammals is subject to continuous development during the postnatal phase of life. Studies following the longitudinal development of the immune system in healthy children are limited both by ethical considerations and sample volumes. Horses represent a particular valuable large animal model for T regulatory (Treg) cells and allergy research. We have recently characterised Treg cells from horses, demonstrated their regulatory capability and showed both their expansion and induction in vitro. Insect bite hypersensitivity (IBH) is a common allergy in horses resembling atopic dermatitis and studies have shown that first exposure to allergens in adult life results in an increased incidence of IBH. The aim of the present study was to characterize circulating CD4+CD25highFoxP3+cells in foals, evaluate their suppressive capability and their in vitro induction compared to adult horses. 19 foals (age range, 1-5 months), their adult mothers and six one-year-old horses (yearlings) were included in the study. The proportion of FoxP3+ cells within the circulating CD4+CD25high population was significantly higher in foals (47%) compared to their mothers (18%) and to yearlings (26%). Treg cells from foals also displayed a higher suppressive capability. Furthermore, CD4+CD25high cells in foals could be induced in vitro from CD4+CD25- cells in a significantly higher proportion compared to mares. These cells also displayed a significantly enhanced suppressive capability. In summary these findings support the notion that exposure of horses to allergens during maturation of the immune system assists the establishment of induced (i)Treg driven tolerance.

  8. Percentage of CD4+, CD8+, and CD25+ T lymphocytes in peripheral blood of pigs in the course of experimental burns and necrectomy

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    Aleksiewicz Roman

    2015-09-01

    Full Text Available The aim of the study was the evaluation of changes in the percentage profile of CD4+, CD8+, and CD25+ T lymphocytes, and their predictive value with respect to the course of experimental skin burns and early necrectomy in pigs. Thirty Large White Landrace pigs of both genders, weighing 50 kg (±2 kg, were used. Burns to their skin were performed with the use of a computer-controlled heating plate, applied to the animal’s body and heated to 2000°C, using 2.5 kg pressure for 10 s. It produced a burn of 30% (±2% of body surface with a range of damage between II b° and III°. In animals of each experimental group fascial necrectomy was performed, according to the testing module. Blood from experimental and non-treated control animals was collected from the external jugular vein before the beginning of the experiment (hour 0 and at 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, and 180 h of the experiment. An immune response profile was evaluated using flow cytometry analysis of the level and expression dynamics of CD4+, CD8+, and CD25+ particles on the surface of T lymphocytes. The study demonstrated that experimentally-induced burns in pigs caused cell-mediated immune response reflected in the changes in the percentage of CD4+, CD8+, and CD25+ T lymphocytes, and that early necrectomy in burnt pigs acted in a protective manner for the organism, based on the immunological index values. The study also proved that the dynamics of cell-mediated immunological response intensification determined on the basis of the percentage of CD4+, CD8+, and CD25+ T lymphocytes is conditioned by the size of the burnt surface and the time of necrectomy procedure.

  9. Abnormally high levels of virus-infected IFN-gamma+ CCR4+ CD4+ CD25+ T cells in a retrovirus-associated neuroinflammatory disorder.

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    Yoshihisa Yamano

    Full Text Available BACKGROUND: Human T-lymphotropic virus type 1 (HTLV-1 is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP, which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL. The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified. PRINCIPAL FINDINGS: Here, we demonstrate that CD4(+CD25(+CCR4(+ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2, Th17, and regulatory T (Treg cells in healthy individuals, we demonstrate that IFN-gamma production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4(+CD25(+CCR4(+ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-gamma-producing CD4(+CD25(+CCR4(+Foxp3(- T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity. CONCLUSIONS: We have defined a unique T cell subset--IFN-gamma(+CCR4(+CD4(+CD25(+ T cells--that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.

  10. Loss of regulatory characteristics in CD4+CD25+/hiT cells induced by impaired transforming growth factor beta secretion in pneumoconiosis.

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    Bian, Lu-Qin; Mao, Ling; Bi, Ying; Zhou, Shao-Wei; Chen, Zi-Dan; Wen, Jun; Shi, Jin; Wang, Ling

    2017-12-01

    Pneumoconiosis is caused by the accumulation of airborne dust in the lung, which stimulates a progressive inflammatory response that ultimately results in lung fibrosis and respiratory failure. It is possible that regulatory cells in the immune system could function to suppress inflammation and possibly slow or reverse disease progression. However, results in this study suggest that in pneumoconiosis patients, the regulatory T cells (Tregs) and B cells are functionally impaired. First, we found that pneumoconiosis patients presented an upregulation of CD4 + CD25 + T cells compared to controls, whereas the CD4 + CD25 + and CD4 + CD25 hi T cells were enriched with Th1- and Th17-like cells but not Foxp3-expressing Treg cells and evidenced by significantly higher T-bet, interferon (IFN)-γ, and interleukin (IL)-17 expression but lower Foxp3 and transforming growth factor (TGF)-β expression. Regarding the CD4 + CD25 hi T-cell subset, the frequency of this cell type in pneumoconiosis patients was significantly reduced compared to controls, together with a reduction in Foxp3 and TGF-β and an enrichment in T-bet, RORγt, IFN-γ, and IL-17. This skewing toward Th1 and Th17 types of inflammation could be driven by monocytes and B cells, since after depleting CD14 + monocytes and CD19 + B cells, the levels of IFN-γ and IL-17 were significantly decreased. Whole peripheral blood mononuclear cells and isolated monocytes and B cells in pneumoconiosis patients also presented reduced capacity of TGF-β secretion. Furthermore, monocytes and B cells from pneumoconiosis patients presented reduced capacity in inducing Foxp3 upregulation, a function that could be rescued by exogenous TGF-β. Together, these data indicated a potential pathway for the progression of pneumoconiosis through a loss of Foxp3 + Treg cells associated with impaired TGF-β secretion. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  11. Alternative Th17 and CD4+ CD25+ FoxP3+ cell frequencies increase and correlate with worse cardiac function in Chagas cardiomyopathy.

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    Almeida, M S; Lorena, V M B; Medeiros, C de A; Junior, W O; Cavalcanti, M da G A M; Martins, S M; de Morais, C N L

    2018-04-01

    Immune homeostasis has been suggested to play an important role in the clinical evolution of chronic Chagas disease; however, the immunopathologic factors involved have not been fully elucidated. Therefore, our study aimed to analyse the frequency of CD4 + CD25 + FoxP3 + cells, classic Th17 cells, alternative Th17 cells and IL-17 + B cells from peripheral blood of chronic cardiac patients after in vitro stimulation with Trypanosoma cruzi soluble EPI antigen. Patients were selected and classified according to clinical evaluation of cardiac involvement: mild, B1 (CARD1) (n = 20) and severe, C (CARD2) (n = 11). Patients with the indeterminate form of CD were included as the control group A (IND) (n = 17). Blood samples were collected and cultured in the presence of EPI antigen. Cells frequency and median fluorescence intensity (MFI) were obtained by flow cytometry. Our results showed that only CD4 + CD25 + FoxP3 + , CD4 + CD25 high FoxP3 + , CD4 + IL-17 + IFN-γ - and CD4 + IL-17 + IFN-γ + cells are more frequent in patients with severe cardiac disease and correlate with worse global cardiac function. However, while indeterminate patients demonstrated a positive correlation between CD4 + CD25 + FoxP3 + and CD4 + IL-17 + IFN-γ - Th17 cells, this relationship was not observed in cardiac patients. IL-17 expression by Th17 cells and B cells correlated with disease progression. Altogether our results suggest that the clinical progression of Chagas cardiomyopathy involves worsening of inflammation and impairment of immunoregulatory mechanisms. © 2018 The Foundation for the Scandinavian Journal of Immunology.

  12. Monomeric allergoid intragastric administration induces local and systemic tolerogenic response involving IL-10-producing CD4(+)CD25(+) T regulatory cells in mice.

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    Petrarca, C; Lazzarin, F; Pannellini, T; Iezzi, M; Braga, M; Mistrello, G; Falagiani, P; Di Giampaolo, L; Di Gioacchino, M

    2010-01-01

    The efficacy of sublingual immunotherapy, at present one of the treatments of choice for respiratory allergy, relies on the tolerance induced by oral mucosa-associated immune system; however, the gut-associated lymphoid tissue (GALT: Peyers patches and isolated lymphoid follicles) and mesenteric lymph nodes could also be involved, being stimulated by the ingested part of the allergen extract. The aim of the present study is to assess whether the exposure of the allergen exclusively to the GALT induces a tolerogenic response. For this purpose, mice were sensitized with ovalbumin or Par j 1 allergens. The corresponding gastric-resistant monomeric allergoids were then administered via orogastric gavage. After treatment, all mice were tested for: serum IgE, in vitro Th1 and Th2 cytokine release by allergen-stimulated peripheral blood lymphocytes, CD4(+)CD25(+) and CD4(+)CD25(+)IL-10(+) T cells in Peyers patches, mesenteric lymph nodes and spleen. Compared to the control, sensitized groups showed higher levels of serum IgE, lower frequency of CD4+CD25+IL-10+ T cells, at all sites, and higher amounts of in vitroreleased IL-4, IL-6 and TNF-alpha. Compared to the sensitized groups, higher frequency of CD4(+)CD25(+)IL-10(+) T cells was observed in the spleen of both Par-j 1 and OVA sensitized/treated groups and, only for ovalbumin-treated mice, in the Peyers patches and mesenteric lymph nodes, IgE and in vitro cytokines were significantly lower and equivalent to the control group. The results give the first evidence that the intragastric-restricted administration of gastric-resistant allergens restores local and peripheral tolerance in allergen-sensitized mice.

  13. Significance of the frequency of CD4+CD25+CD127- T-cells in patients with pulmonary tuberculosis and diabetes mellitus.

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    Sun, Qin; Zhang, Qing; Xiao, Heping; Cui, Haiyan; Su, Bo

    2012-07-01

    Pulmonary tuberculosis and diabetes mellitus (DM) are closely associated. The objective of this study was to determine whether the expression of CD4+CD25+CD127- T-cells (regulatory T-cells (Treg)) is associated with diabetic pulmonary tuberculosis. Flow cytometry was used to determine the frequencies of CD4+CD25+ and CD4+CD25+CD127- T-cells in peripheral blood, bronchoalveolar lavage fluid (BALF) and pleural effusions from 120 patients (30 with pulmonary tuberculosis and DM (TBDM), 30 with pulmonary tuberculosis without DM (TB), 30 with tuberculous pleurisy without DM (TBP) and 30 healthy volunteers). The concentrations of interferon (IFN)-γ and interleukin (IL)-10 in BALF and pleural effusions were determined by enzyme-linked immunosorbent assay. Treg frequencies in peripheral blood were significantly higher in patients with TBDM, TB and TBP than in the control group, with the frequency in TBDM being the highest (P tuberculosis and DM, the imbalance between Treg and effector T-cells at pathological sites may be associated with weakened immunity and clinical manifestations of TB. © 2012 The Authors. Respirology © 2012 Asian Pacific Society of Respirology.

  14. Sublingual Immunotherapy Induces Regulatory Function of IL-10-Expressing CD4(+)CD25(+)Foxp3(+) T Cells of Cervical Lymph Nodes in Murine Allergic Rhinitis Model.

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    Yamada, Takaya; Tongu, Miki; Goda, Kaoru; Aoi, Noriaki; Morikura, Ichiro; Fuchiwaki, Takafumi; Kawauchi, Hideyuki

    2012-01-01

    Sublingual immunotherapy (SLIT) has been considered to be a painless and efficacious therapeutic treatment of allergic rhinitis which is known as type I allergy of nasal mucosa. Nevertheless, its mechanisms need to be further investigated. In this study, we constructed an effective murine model of sublingual immunotherapy in allergic rhinitis, in which mice were sublingually administered with ovalbumin (OVA) followed by intraperitoneal sensitization and nasal challenge of OVA. Sublingually treated mice showed significantly decreased specific IgE responses as well as suppressed Th2 immune responses. Sublingual administration of OVA did not alter the frequency of CD4(+)CD25(+) regulatory T cells (Tregs), but led to upregulation of Foxp3- and IL-10-specific mRNAs in the Tregs of cervical lymph nodes (CLN), which strongly suppressed Th2 cytokine production from CD4(+)CD25(-) effector T cells in vitro. Furthermore, sublingual administration of plasmids encoding the lymphoid chemokines CCL19 and CCL21-Ser DNA together with OVA suppressed allergic responses. These results suggest that IL-10-expressing CD4(+)CD25(+)Foxp3(+) Tregs in CLN are involved in the suppression of allergic responses and that CCL19/CCL21 may contribute to it in mice that received SLIT.

  15. Expression of Th17 and CD4+ CD25+ T regulatory cells in peripheral blood of acute leukemia patients and their prognostic significance.

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    Xiang, Mingli; Guo, Li; Ma, Yan; Li, Yi

    2016-11-01

    To discuss the expression of T helper cell 17 (Th17) cells and CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in peripheral blood (PB) of patients with acute leukemia (AL), and to explore the relationship between them and disease prognosis. 40 patients diagnosed with acute leukemia in The First Affiliated Hospital of Zhengzhou University from July 2012 to August 2014 were selected as the observation group. Meanwhile, 40 healthy people were taken as the control group. Flow Cytometry Method (FCM) was used to detect the level of Th17 cells and CD4 + CD25 + Foxp3 + cells in peripheral blood of the two groups, and enzyme-linked immuno sorbent assay (ELISA) method was used to test the level of IL17 and TGF-β in peripheral blood of two groups; reverse transcription-polymerase chain reaction (RT-PCR) was adopted to analyze the mRNA levels of RORγT and Foxp3 in peripheral blood. In addition, we examined the levels of Th17 and CD4 + CD25 + Foxp3 + cells and associated factor levels in patients with remission after AL chemotherapy. the Th17 cells (CD3 + CD4 + IL-17 + ) in acute leukemia patients accounted for (1.51±0.27)%, which was significantly higher than that of control group (0.36±0.23)%, with statistical significance (t=20.51, Pcells in AL patients was (3.37±0.48)%, which was significantly higher than that of control group of (1.26±0.27)%, with statistical significance (t=24.23, Pt=7.83, Pt=7.83, Pt=12.27, Pt=7.89, Pcells and CD4 + CD25 + Foxp3 + cells, and the serum levels of IL-17 and TGF-β in acute leukemia patients all decreased significantly after 6 months of chemotherapy, and the difference was statistically significant (Pcells, CD4+ CD25+ Foxp3 + cells and their secretory proteins IL-17, TGF-β and transcription factors were significantly increased in AL patients. Therefore, regular detection of peripheral blood Th17 and Treg cells, as well as their secretory proteins are useful for monitoring the immune status and prognosis of patients.

  16. TLR2-dependent induction of IL-10 and Foxp3+ CD25+ CD4+ regulatory T cells prevents effective anti-tumor immunity induced by Pam2 lipopeptides in vivo.

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    Sayuri Yamazaki

    Full Text Available 16 S-[2,3-bis(palmitoylpropyl]cysteine (Pam2 lipopeptides act as toll-like receptor (TLR2/6 ligands and activate natural killer (NK cells and dendritic cells (DCs to produce inflammatory cytokines and cytotoxic NK activity in vitro. However, in this study, we found that systemic injection of Pam2 lipopeptides was not effective for the suppression of NK-sensitive B16 melanomas in vivo. When we investigated the immune suppressive mechanisms, systemic injection of Pam2 lipopeptides induced IL-10 in a TLR2-dependent manner. The Pam2 lipopeptides increased the frequencies of Foxp3(+CD4(+ regulatory T (T reg cells in a TLR2- and IL-10- dependent manner. The T reg cells from Pam2-lipopeptide injected mice maintained suppressor activity. Pam2 lipopeptides, plus the depletion of T reg with an anti-CD25 monoclonal antibody, improved tumor growth compared with Pam2 lipopeptides alone. In conclusion, our data suggested that systemic treatment of Pam2 lipopeptides promoted IL-10 production and T reg function, which suppressed the effective induction of anti-tumor immunity in vivo. It is necessary to develop an adjuvant that does not promote IL-10 and T reg function in vivo for the future establishment of an anti-cancer vaccine.

  17. The prognostic value of peripheral CD4+CD25+ T lymphocytes among early stage and triple negative breast cancer patients receiving dendritic cells-cytokine induced killer cells infusion.

    Science.gov (United States)

    Song, Qing-Kun; Ren, Jun; Zhou, Xin-Na; Wang, Xiao-Li; Song, Guo-Hong; Di, Li-Jun; Yu, Jing; Hobeika, Amy; Morse, Michael A; Yuan, Yan-Hua; Yang, Hua-Bing; Lyerly, Herbert Kim

    2015-12-01

    This study aimed to assess the prognostic value of CD4+CD25+ T lymphocyte in peripheral blood among breast cancer patients treated with adoptive T lymphocytes immunotherapy. 217 patients participated in the follow-up study. CD4+CD25+ proportion was measured by flow cytometry in peripheral T cells. The median survival was estimated by Kaplan-Meier curve, Log-rank test and Cox hazard proportion regression model, between groups of CD4+CD25+ proportion more than 5% and less than or equal to 5% in peripheral T cells. Peripheral CD4+CD25+ T lymphocytes had not a relationship with progression-free survival. It was featured that above 5% peripheral CD4+CD25+ proportion of T cells was related with the median overall survival by a shorten of 51 months (p < 0.05) with the HR 1.65 (95%CI 1.04, 2.62). Above 5% CD4+CD25+proportion of T cells produced the HR to be 1.76 (95%CI 1.07, 2.87) In stage 0-II patients, and 3.59 (95%CI 1.05, 12.29) in triple negative breast cancer patients. Cellular immunity restoration recovered by adoptive T cell infusions which resulted in less proportion of peripheral CD4+CD25+T lymphocytes could be a potential prognostic indicator among early stage and triple negative patients.

  18. In-vitro inhibition of IFNγ+ iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function

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    Daniel Volker

    2012-08-01

    Full Text Available Abstract Background IFNγ-producing CD4+CD25+Foxp3+ PBL represent a subtype of iTreg that are associated with good long-term graft outcome in renal transplant recipients and suppress alloresponses in-vitro. To study the mechanism of immunosuppression, we attempted to block cell surface receptors and thereby inhibited the function of this iTreg subset in-vitro using monoclonal antibodies and recombinant proteins. Methods PBL of healthy control individuals were stimulated polyclonally in-vitro in the presence of monoclonal antibodies or recombinant proteins against/of CD178, CD152, CD279, CD28, CD95, and HLA-DR. Induction of IFNγ+ iTreg and proliferation of effector cells was determined using four-color fluorescence flow cytometry. Blockade of iTreg function was analyzed using polyclonally stimulated co-cultures with separated CD4+CD25+CD127-IFNγ+ PBL. Results High monoclonal antibody concentrations inhibited the induction of CD4+CD25+Foxp3+IFNγ+ PBL (anti-CD152, anti-CD279, anti-CD95: p +CD25+CD127-IFNγ+ PBL (anti-CD178, anti-CD152, anti-CD279, anti-CD95: p +CD25+Foxp3+IFNγ+ PBL (rCD152 and rCD95: p +CD25+CD127-IFNγ+ PBL showed lower cell proliferation than co-cultures with CD4+CD25+CD127-IFNγ- PBL (p +CD25+CD127-IFNγ- PBL-containing co-cultures in the presence of monoclonal antibody (anti-CD28, anti-CD152, anti-CD279: p +CD25+CD127-IFNγ+ PBL (with the exception anti-CD28 monoclonal antibody: p +CD25+CD127-IFNγ- PBL but do not efficiently block suppressive iTreg function in co-cultures with CD4+CD25+CD127-IFNγ+ PBL. Conclusions CD178, CD152, CD279, CD28, CD95, and HLA-DR determinants are important for induction and suppressive function of IFNγ+ iTreg.

  19. Glycan microarray reveal induced IgGs repertoire shift against a dietary carbohydrate in response to rabbit anti-human thymocyte therapy

    Science.gov (United States)

    Amon, Ron; Ben-Arye, Shani Leviatan; Engler, Limor; Yu, Hai; Lim, Noha; Berre, Ludmilla Le; Harris, Kristina M.; Ehlers, Mario R.; Gitelman, Stephen E.; Chen, Xi; Soulillou, Jean-Paul; Padler-Karavani, Vered

    2017-01-01

    Humans have circulating antibodies against diverse glycans containing N-glycolylneuraminic acid (Neu5Gc) due to function-loss mutation of the CMAH gene. This xenogenic non-human carbohydrate is abundant in red meat, xenografts and biotherapeutics. Low levels of diet-derived Neu5Gc is also present on normal human endothelial cells, and together with anti-Neu5Gc antibodies could potentially mediate “xenosialitis” chronic-inflammation. Rabbit anti-human thymocyte globulin (ATG) is a drug containing polyclonal IgG glycoproteins commonly used as an immunosuppressant in human transplantation and autoimmune diseases. In type-1 diabetes patients, infusion of Neu5Gc-glycosylated ATG caused increased global anti-Neu5Gc response. Here, for the first time we explore changes in anti-Neu5Gc IgG repertoire following the immunization elicited by ATG, compared with the basal antibodies repertoire that reflect exposure to dietary-Neu5Gc. We used glycan microarrays with multiple Neu5Gc-glycans and controls to elucidate eventual differences in ATG-elicited repertoire, before/after ATG administration and track their kinetics (0, 1, 18 and 24 months). Response of all basal-pre-existing Neu5Gc-specific antibodies rapidly increased. This response peaked at one month post-ATG, with enhanced affinity, then resolved at 18–24 months. Induced-antibodies showed expanded diversity and de-novo recognition of different Neu5Gc-glycans, including endogenous glycolipids, that was further validated by affinity-purified anti-Neu5Gc antibodies from patients’ sera. These findings strongly suggest that ATG-induced anti-Neu5Gc IgGs represent a secondary exposure to this dietary carbohydrate-antigen in humans, with immune memory. Given their modified recognition patterns, ATG-evoked anti-Neu5Gc antibodies could potentially mediate biological effects different from pre-existing antibodies. PMID:29348821

  20. Characterization of CD4+ subpopulations and CD25+ cells in ileal lymphatic tissue of weaned piglets infected with Salmonella Typhimurium with or without Enterococus faecium feeding.

    Science.gov (United States)

    Kreuzer, S; Rieger, J; Strucken, E M; Thaben, N; Hünigen, H; Nöckler, K; Janczyk, P; Plendl, J; Brockmann, Gudrun A

    2014-04-15

    The aim of the present study was to test the effect of Enterococcus faecium NCIMB 10415 (E. faecium) on CD4+ T helper immune cell subpopulations and CD25+ cells in ileal lymphatic tissue after challenge with Salmonella (S.) Typhimurium DT 104. German Landrace piglets treated with E. faecium (n=16) as a feed additive and untreated controls (n=16) were challenged with S. Typhimurium 10 days after weaning. The expression of lineage specific T helper cell subtype master transcription factors on mRNA level was measured in the whole tissue of the gut associated lymphoid tissues (ileocecal mesenteric lymph node, ileum with Peyer's patches and papilla ilealis) and in magnetically sorted T helper cells from blood and ileocecal mesenteric lymph nodes at two and 28 days post infection. CD25 protein expression of T helper cells was studied by flow cytometry in ileal Peyer's patches, lymph nodes and blood. Distribution and morphology of CD25+ cells was demonstrated in situ by immunohistochemistry in paraffin embedded specimens of the ileum and the ileocecal mesenteric lymph nodes. The data provide evidence for a higher T helper 2 cell driven immune response in the control group compared to the E. faecium treated group (Pfaecium fed pigs and the control group, but provided a detailed description of the occurrence and morphology of these cells in the gut associate lymphoid tissues of piglets. In conclusion we suggest that (i) prolonged feeding with E. faecium can result in changes of the T helper cell response leading to a stronger infection with S. Typhimurium and (ii) that it is important to examine purified immune cells to be able to detect effects on T helper cell subpopulations. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. CD4+ CD25+ FOXP3+ T cell frequency in the peripheral blood is a biomarker that distinguishes intestinal tuberculosis from Crohn's disease.

    Science.gov (United States)

    Tiwari, Veena; Kedia, Saurabh; Garg, Sushil Kumar; Rampal, Ritika; Mouli, V Pratap; Purwar, Anuja; Mitra, D K; Das, Prasenjit; Dattagupta, S; Makharia, Govind; Acharya, S K; Ahuja, Vineet

    2018-01-01

    Distinguishing between Crohn's Disease (CD) and Intestinal Tuberculosis (ITB) has been a challenging task for clinicians due to their similar presentation. CD4+FOXP3+ T regulatory cells (Tregs) have been reported to be increased in patients with pulmonary tuberculosis. However, there is no such data available in ITB. The aim of this study was to investigate the differential expression of FOXP3+ T cells in patients with ITB and CD and its utility as a biomarker. The study prospectively recruited 124 patients with CD, ITB and controls: ulcerative colitis (UC) and patients with only haemorrhoidal bleed. Frequency of CD4+CD25+FOXP3+ Tregs in peripheral blood (flow cytometry), FOXP3 mRNA expression in blood and colonic mucosa (qPCR) and FOXP3+ T cells in colonic mucosa (immunohistochemistry) were compared between controls, CD and ITB patients. Frequency of CD4+CD25+FOXP3+ Treg cells in peripheral blood was significantly increased in ITB as compared to CD. Similarly, significant increase in FOXP3+ T cells and FOXP3 mRNA expression was observed in colonic mucosa of ITB as compared to CD. ROC curve showed that a value of >32.5% for FOXP3+ cells in peripheral blood could differentiate between CD and ITB with a sensitivity of 75% and a specificity of 90.6%. Phenotypic enumeration of peripheral CD4+CD25+FOXP3+ Treg cells can be used as a non-invasive biomarker in clinics with a high diagnostic accuracy to differentiate between ITB and CD in regions where TB is endemic.

  2. Sublingual tolerance induction with antigen conjugated to cholera toxin B subunit induces Foxp3+CD25+CD4+ regulatory T cells and suppresses delayed-type hypersensitivity reactions.

    Science.gov (United States)

    Sun, J-B; Cuburu, N; Blomquist, M; Li, B-L; Czerkinsky, C; Holmgren, J

    2006-09-01

    Although sublingual (s.l.) immunotherapy with selected allergens is safe and often effective for treating patients with allergies, knowledge of the immunological mechanisms involved remains limited. Can s.l. administration of antigen (Ag) induce peripheral immunological tolerance and also suppress delayed-type hypersensitivity (DTH) responses? To what extent can s.l.-induced tolerance be explained by the generation of Foxp3+CD25+CD4+ regulatory T cells (T(reg))? This study addressed these questions in mice and compared the relative efficacy of administering ovalbumin (OVA) conjugated to cholera toxin B (CTB) subunit with administration of the same Ag alone. We found that s.l. administration of a single or even more efficiently three repeated 40-mug doses of OVA/CTB conjugate suppressed T-cell proliferative responses to OVA by cervical lymph node (CLN), mesenteric lymph node (MLN) and spleen cells and concurrently strongly increased the frequency of Ag-specific T(reg) in CLN, MLN and spleen and also transforming growth factor-beta (TGF-beta) levels in serum. The CLN and splenic cells from OVA/CTB-treated BALB/c mice efficiently suppressed OVA-specific T-cell receptor (TCR) transgenic (DO11.10) CD25-CD4+ effector T-cell proliferation in vitro. Further, s.l. treatment with OVA/CTB completely suppressed OVA-specific DTH responses in vivo and T-cell proliferative responses in mice immunized subcutaneously with OVA in Freund's complete adjuvant. The intracellular expression of Foxp3 was strongly increased in OVA-specific (KJ1-26+) CD4+ T cells from OVA/CTB-treated mice. Thus, s.l. administration of CTB-conjugated Ag can efficiently induce peripheral T-cell tolerance associated with strong increases in serum TGF-beta levels and in Ag-specific Foxp3+CD25+CD4+ T(reg) cells.

  3. Dysregulation of CD4+CD25+CD127lowFOXP3+ regulatory T cells in HIV-infected pregnant women

    DEFF Research Database (Denmark)

    Kolte, Lilian; Gaardbo, Julie C; Karlsson, Ingrid

    2010-01-01

    prospectively during pregnancy and postpartum. A significant expansion of CD4(+)CD25(+)CD127(low)FoxP3(+) regulatory T cells indicating alteration of peripheral tolerance was seen during second trimester, but only in HIV-negative women. HIV-infected women had lower CD4 counts, lower thymic output and Th-2...... course of HIV infection. However, despite HAART during pregnancy, HIV-infected women display different immunologic profiles from HIV-negative women, which may have importance for the induction of fetal-maternal tolerance and in part explain the increased risk of abortion in HIV-infected women....

  4. Neonatal respiratory syncytial virus infection has an effect on lung inflammation and the CD4(+) CD25(+) T cell subpopulation during ovalbumin sensitization in adult mice.

    Science.gov (United States)

    Comas-García, A; López-Pacheco, C P; García-Zepeda, E A; Soldevila, G; Ramos-Martínez, P; Ramos-Castañeda, J

    2016-08-01

    In BALB/c adult mice, respiratory syncytial virus (RSV) infection enhances the degree of lung inflammation before and/or after ovalbumin (OVA) respiratory sensitization. However, it is unclear whether RSV infection in newborn mice has an effect on the immune response to OVA respiratory sensitization in adult mice. The aim of this study was to determine if RSV neonatal infection alters T CD4(+) population and lung inflammation during OVA respiratory sensitization in adult mice. BALB/c mice were infected with RSV on the fourth day of life and challenged by OVA 4 weeks later. We found that in adult mice, RSV neonatal infection prior to OVA sensitization reduces the CD4(+) CD25(+) and CD4(+) CD25(+) forkhead protein 3 (FoxP3)(+) cell populations in the lungs and bronchoalveolar lavage. Furthermore, it also attenuates the inflammatory infiltrate and cytokine/chemokine expression levels in the mouse airways. In conclusion, the magnitude of the immune response to a non-viral respiratory perturbation in adult mice is not enhanced by a neonatal RSV infection. © 2016 British Society for Immunology.

  5. IL-15 augments TCR-induced CD4+ T cell expansion in vitro by inhibiting the suppressive function of CD25 High CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Tom L Van Belle

    Full Text Available Due to its critical role in NK cell differentiation and CD8(+ T cell homeostasis, the importance of IL-15 is more firmly established for cytolytic effectors of the immune system than for CD4(+ T cells. The increased levels of IL-15 found in several CD4(+ T cell-driven (auto- immune diseases prompted us to examine how IL-15 influences murine CD4(+ T cell responses to low dose TCR-stimulation in vitro. We show that IL-15 exerts growth factor activity on both CD4(+ and CD8(+ T cells in a TCR-dependent and Cyclosporin A-sensitive manner. In CD4(+ T cells, IL-15 augmented initial IL-2-dependent expansion and once IL-15Rα was upregulated, IL-15 sustained the TCR-induced expression of IL-2/15Rβ, supporting proliferation independently of secreted IL-2. Moreover, IL-15 counteracts CD4(+ T cell suppression by a gradually expanding CD25(HighCD4(+ T cell subset that expresses Foxp3 and originates from CD4(+CD25(+ Tregs. These in vitro data suggest that IL-15 may dramatically strengthen the T cell response to suboptimal TCR-triggering by overcoming an activation threshold set by Treg that might create a risk for autoimmune pathology.

  6. Tryptophan deprivation induces inhibitory receptors ILT3 and ILT4 on dendritic cells favoring the induction of human CD4+CD25+ Foxp3+ T regulatory cells.

    Science.gov (United States)

    Brenk, Manuela; Scheler, Marina; Koch, Susanne; Neumann, Jürgen; Takikawa, Osamu; Häcker, Georg; Bieber, Thomas; von Bubnoff, Dagmar

    2009-07-01

    Tryptophan catabolism through IDO activity can cause nonresponsiveness and tolerance acting on T cells. Given the crucial importance of dendritic cells (DCs) in the initiation of a T cell response, surprisingly little is known about the impact of IDO activity and tryptophan deprivation on DCs themselves. In the present study, we show that human DCs differentiated under low-tryptophan conditions acquire strong tolerogenic capacity. This effect is associated with a markedly decreased Ag uptake as well as the down-regulation of costimulatory molecules (CD40, CD80). In contrast, the inhibitory receptors ILT3 and ILT4 are significantly increased. Functionally, tryptophan-deprived DCs show a reduced capacity to stimulate T cells, which can be restored by blockade of ILT3. Moreover, ILT3(high)ILT4(high) DCs lead to the induction of CD4(+)CD25(+) Foxp3(+) T regulatory cells with suppressive activity from CD4(+)CD25(-) T cells. The generation of ILT3(high)ILT4(high) DCs with tolerogenic properties by tryptophan deprivation is linked to a stress response pathway mediated by the GCN2 kinase. These results demonstrate that tryptophan degradation establishes a regulatory microenvironment for DCs, enabling these cells to induce T regulatory cells. The impact of IDO thus extends beyond local immune suppression to a systemic control of the immune response.

  7. LAP TGF-Beta Subset of CD4+CD25+CD127− Treg Cells is Increased and Overexpresses LAP TGF-Beta in Lung Adenocarcinoma Patients

    Science.gov (United States)

    Islas-Vazquez, Lorenzo; Aguilar-Cazares, Dolores; Meneses-Flores, Manuel; Galicia-Velasco, Miriam; Romero-Garcia, Susana; Camacho-Mendoza, Catalina; Lopez-Gonzalez, Jose Sullivan

    2015-01-01

    Lung cancer is the leading cause of cancer death worldwide. Adenocarcinoma, the most commonly diagnosed histologic type of lung cancer, is associated with smoking. Cigarette smoke promotes inflammation on the airways, which might be mediated by Th17 cells. This inflammatory environment may contribute to tumor development. In contrast, some reports indicate that tumors may induce immunosuppressive Treg cells to dampen immune reactivity, supporting tumor growth and progression. Thus, we aimed to analyze whether chronic inflammation or immunosuppression predominates at the systemic level in lung adenocarcinoma patients, and several cytokines and Th17 and Treg cells were studied. Higher proportions of IL-17-producing CD4+ T-cells were found in smoking control subjects and in lung adenocarcinoma patients compared to nonsmoking control subjects. In addition, lung adenocarcinoma patients increased both plasma concentrations of IL-2, IL-4, IL-6, and IL-10, and proportions of Latency Associated Peptide (LAP) TGF-β subset of CD4+CD25+CD127− Treg cells, which overexpressed LAP TGF-β. This knowledge may lead to the development of immunotherapies that could inhibit the suppressor activity mediated by the LAP TGF-β subset of CD4+CD25+CD127− Treg cells to promote reactivity of immune cells against lung adenocarcinoma cells. PMID:26582240

  8. LAP TGF-Beta Subset of CD4+CD25+CD127− Treg Cells is Increased and Overexpresses LAP TGF-Beta in Lung Adenocarcinoma Patients

    Directory of Open Access Journals (Sweden)

    Lorenzo Islas-Vazquez

    2015-01-01

    Full Text Available Lung cancer is the leading cause of cancer death worldwide. Adenocarcinoma, the most commonly diagnosed histologic type of lung cancer, is associated with smoking. Cigarette smoke promotes inflammation on the airways, which might be mediated by Th17 cells. This inflammatory environment may contribute to tumor development. In contrast, some reports indicate that tumors may induce immunosuppressive Treg cells to dampen immune reactivity, supporting tumor growth and progression. Thus, we aimed to analyze whether chronic inflammation or immunosuppression predominates at the systemic level in lung adenocarcinoma patients, and several cytokines and Th17 and Treg cells were studied. Higher proportions of IL-17-producing CD4+ T-cells were found in smoking control subjects and in lung adenocarcinoma patients compared to nonsmoking control subjects. In addition, lung adenocarcinoma patients increased both plasma concentrations of IL-2, IL-4, IL-6, and IL-10, and proportions of Latency Associated Peptide (LAP TGF-β subset of CD4+CD25+CD127− Treg cells, which overexpressed LAP TGF-β. This knowledge may lead to the development of immunotherapies that could inhibit the suppressor activity mediated by the LAP TGF-β subset of CD4+CD25+CD127− Treg cells to promote reactivity of immune cells against lung adenocarcinoma cells.

  9. Effects of sublingual immunotherapy for Dermatophagoides farinae on Th17 cells and CD4(+) CD25(+) regulatory T cells in peripheral blood of children with allergic asthma.

    Science.gov (United States)

    Tian, Man; Wang, Yu; Lu, Yueqing; Jiang, Yan-he; Zhao, De-yu

    2014-05-01

    Sublingual immunotherapy is becoming a more common treatment for allergic diseases, particularly in pediatric clinics. This type of treatment is highly effective for Dermatophagoides farinae allergy, but the mechanisms resulting in immune tolerance have not been investigated. We explored the effects of sublingual immunotherapy with D. farinae drops on populations of subsets of T immune cells, specifically Th17 cells and CD4(+) CD25(+) regulatory T cells (Treg cells), in peripheral blood of children with allergic asthma. We assessed immune cell populations in 60 patients allergic to D. farinae who were randomly divided into 2 groups: a treatment group (n = 30) and a control group (n = 30), treated with sublingual administration of D. farinae drops or placebo, respectively, for 48 weeks. Clinical symptoms of asthma were scored for each individual before and after treatment, and the percentages of Th17 cells and CD4(+) CD25(+) Treg cells in the peripheral blood were evaluated by flow cytometry at 12-week intervals beginning at baseline. Both the mean daily symptom scores and percentages of Th17 cells significantly declined in the treatment group throughout the study period (p Sublingual administration of D. farinae drops alters T immune cell profiles and reduces asthma symptoms, likely resulting in enhanced immunosuppression in children with asthma. © 2014 ARS-AAOA, LLC.

  10. Expression of CD4+CD25+Foxp3+ Regulatory T Cells, Interleukin 10 and Transforming Growth Factor β in Newly Diagnosed Type 2 Diabetic Patients.

    Science.gov (United States)

    Yuan, Ning; Zhang, Hai-Feng; Wei, Qi; Wang, Ping; Guo, Wei-Ying

    2018-02-01

    Recent studies have shown that dysfunction and decrease of regulatory T cells (Tregs) correlates with insulin resistance (IR), one of the most significant mechanisms for type 2 diabetes mellitus (T2DM). To examine potential relationships among Tregs, IR, blood lipid content, and related cytokines, we investigated the frequency of CD4+CD25+Foxp3+ Tregs, as well as expression levels of interleukin 10 (IL-10) and transforming growth factor-β (TGF-β) in newly diagnosed T2DM patients. Fifty-one newly diagnosed T2DM patients and 55 control individuals were enrolled. According to body mass index (BMI), the T2DM patients were grouped into non-obese and obese groups. Blood was collected in ethylene diamine tetraacetic acid (EDTA) anticoagulant tubes for detection of CD4+CD25+Foxp3+ Tregs by flow cytometry. Serum was collected to quantify IL-10 and TGF-β levels by enzyme-linked immunosorbent assay (ELISA). By comparing percentages of Tregs between non-obese and obese groups, correlation with Treg frequency, homeostasis model assessment of insulin resistance (HOMA-IR), IL-10 and TGF-β was examined. The percentage of CD4+CD25+Foxp3+ Tregs in the newly diagnosed T2DM group was significantly lower than in the control group (P<0.01). Further, levels of IL-10 and TGF-β were also lower in the T2DM group (P<0.05). The level of IL-10 was remarkably lower in the obese group than in the non-obese and the control groups (P<0.01), but there was no significant difference between non-obese group and the control group. The level of TGF-β was lower in obese group than in the control group (P<0.05). There was no significant difference between non-obese group and the control group. The frequency of CD4+CD25+Foxp3+ Tregs in the obese group was significantly lower than in the non-obese group (P<0.05). In the obese group, the percentage of Tregs negatively correlated with HOMA-IR and positively correlated with TGF-β (P<0.05). There was no obvious correlation between Treg and HOMA-IR in the

  11. Depletion of CD4+CD25+ regulatory T cells exacerbates sodium iodide-induced experimental autoimmune thyroiditis in human leucocyte antigen DR3 (DRB1*0301) transgenic class II-knock-out non-obese diabetic mice.

    Science.gov (United States)

    Flynn, J C; Meroueh, C; Snower, D P; David, C S; Kong, Y M

    2007-03-01

    Both genetic and environmental factors contribute to autoimmune disease development. Previously, we evaluated genetic factors in a humanized mouse model of Hashimoto's thyroiditis (HT) by immunizing human leucocyte antigen DR3 (HLA-DR3) and HLA-DQ8 transgenic class II-knock-out non-obese diabetic (NOD) mice. DR3+ mice were susceptible to experimental autoimmune thyroiditis (EAT) induction by both mouse thyroglobulin (mTg) and human (h) Tg, while DQ8+ mice were weakly susceptible only to hTg. As one environmental factor associated with HT and tested in non-transgenic models is increased sodium iodide (NaI) intake, we examined the susceptibility of DR3+ and/or DQ8+ mice to NaI-induced disease. Mice were treated for 8 weeks with NaI in the drinking water. At 0 x 05% NaI, 23% of DR3+, 0% of DQ8+ and 20% of DR3+DQ8+ mice had thyroid destruction. No spleen cell proliferation to mTg was observed. Most mice had undetectable anti-mTg antibodies, but those with low antibody levels usually had thyroiditis. At 0.3% NaI, a higher percentage of DR3+ and DR3+DQ8+ mice developed destructive thyroiditis, but it was not statistically significant. However, when DR3+ mice had been depleted of CD4+CD25+ regulatory T cells prior to NaI treatment, destructive thyroiditis (68%) and serum anti-mTg antibodies were exacerbated further. The presence of DQ8 molecules does not alter the susceptibility of DR3+DQ8+ mice to NaI-induced thyroiditis, similar to earlier findings with mTg-induced EAT. Susceptibility of DR3+ mice to NaI-induced EAT, in both the presence and absence of regulatory T cells, demonstrates the usefulness of HLA class II transgenic mice in evaluating the roles of environmental factors and immune dysregulation in autoimmune thyroid disease.

  12. Auditory stimulation of opera music induced prolongation of murine cardiac allograft survival and maintained generation of regulatory CD4+CD25+ cells

    Directory of Open Access Journals (Sweden)

    Uchiyama Masateru

    2012-03-01

    Full Text Available Abstract Background Interactions between the immune response and brain functions such as olfactory, auditory, and visual sensations are likely. This study investigated the effect of sounds on alloimmune responses in a murine model of cardiac allograft transplantation. Methods Naïve CBA mice (H2k underwent transplantation of a C57BL/6 (B6, H2b heart and were exposed to one of three types of music--opera (La Traviata, classical (Mozart, and New Age (Enya--or one of six different single sound frequencies, for 7 days. Additionally, we prepared two groups of CBA recipients with tympanic membrane perforation exposed to opera for 7 days and CBA recipients exposed to opera for 7 days before transplantation (pre-treatment. An adoptive transfer study was performed to determine whether regulatory cells were generated in allograft recipients. Immunohistochemical, cell-proliferation, cytokine, and flow cytometry assessments were also performed. Results CBA recipients of a B6 cardiac graft that were exposed to opera music and Mozart had significantly prolonged allograft survival (median survival times [MSTs], 26.5 and 20 days, respectively, whereas those exposed to a single sound frequency (100, 500, 1000, 5000, 10,000, or 20,000 Hz or Enya did not (MSTs, 7.5, 8, 9, 8, 7.5, 8.5 and 11 days, respectively. Untreated, CBA mice with tympanic membrane perforations and CBA recipients exposed to opera for 7 days before transplantation (pre-treatment rejected B6 cardiac grafts acutely (MSTs, 7, 8 and 8 days, respectively. Adoptive transfer of whole splenocytes, CD4+ cells, or CD4+CD25+ cells from opera-exposed primary allograft recipients resulted in significantly prolonged allograft survival in naive secondary recipients (MSTs, 36, 68, and > 100 days, respectively. Proliferation of splenocytes, interleukin (IL-2 and interferon (IFN-γ production was suppressed in opera-exposed mice, and production of IL-4 and IL-10 from opera-exposed transplant recipients increased

  13. Dysregulation of CD4+CD25+CD127lowFOXP3+ regulatory T cells in HIV-infected pregnant women

    DEFF Research Database (Denmark)

    Kolte, Lilian; Gaardbo, Julie C; Karlsson, Ingrid

    2010-01-01

    Pregnancy represents a major challenge to immunologic tolerance. How the fetal "semiallograft" evades maternal immune attack is unknown. Pregnancy success may involve alteration of both central (thymic) and peripheral tolerance mechanisms. HIV infection is characterized by CD4(+) T-cell depletion......, chronic immune activation, and altered lymphocyte subsets. We studied immunologic consequences of pregnancy in 20 HIV-infected women receiving highly active antiretroviral therapy (HAART), and for comparison in 16 HIV-negative women. Lymphocyte subsets, thymic output, and cytokine profiles were measured...... prospectively during pregnancy and postpartum. A significant expansion of CD4(+)CD25(+)CD127(low)FoxP3(+) regulatory T cells indicating alteration of peripheral tolerance was seen during second trimester, but only in HIV-negative women. HIV-infected women had lower CD4 counts, lower thymic output and Th-2...

  14. Inhibition of Allograft Inflammatory Factor-1 in Dendritic Cells Restrains CD4+ T Cell Effector Responses and Induces CD25+Foxp3+ T Regulatory Subsets

    Directory of Open Access Journals (Sweden)

    Diana M. Elizondo

    2017-11-01

    Full Text Available Allograft inflammatory factor-1 (AIF1 is a cytoplasmic scaffold protein shown to influence immune responses in macrophages and microglial cells. The protein contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes. This study now reports that AIF1 is expressed in CD11c+ dendritic cells (DC and silencing of expression restrains induction of antigen-specific CD4+ T cell effector responses. AIF1 knockdown in murine DC resulted in impaired T cell proliferation and skewed polarization away from T helper type 1 and 17 fates. In turn, there was a parallel expansion of IL-10-producing and CD25+Foxp3+ T regulatory subsets. These studies are the first to demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and presents a novel target for engineering tolerogenic DC-based immunotherapies.

  15. Dysregulation of CD4+CD25+CD127lowFOXP3+ regulatory T cells in HIV-infected pregnant women

    DEFF Research Database (Denmark)

    Kolte, Lilian; Gaardbo, Julie C; Karlsson, Ingrid

    2010-01-01

    Pregnancy represents a major challenge to immunologic tolerance. How the fetal "semiallograft" evades maternal immune attack is unknown. Pregnancy success may involve alteration of both central (thymic) and peripheral tolerance mechanisms. HIV infection is characterized by CD4(+) T-cell depletion...... prospectively during pregnancy and postpartum. A significant expansion of CD4(+)CD25(+)CD127(low)FoxP3(+) regulatory T cells indicating alteration of peripheral tolerance was seen during second trimester, but only in HIV-negative women. HIV-infected women had lower CD4 counts, lower thymic output and Th-2...... cytokines, and more immune activation at all time points compared with controls. Immune activation was decreased in HIV-infected patients during pregnancy. In contrast, CD4 counts were increased in both groups. In conclusion, the study does not indicate that pregnancy adversely affects the immunologic...

  16. Inhibition of Allograft Inflammatory Factor-1 in Dendritic Cells Restrains CD4+ T Cell Effector Responses and Induces CD25+Foxp3+ T Regulatory Subsets.

    Science.gov (United States)

    Elizondo, Diana M; Andargie, Temesgen E; Yang, Dazhi; Kacsinta, Apollo D; Lipscomb, Michael W

    2017-01-01

    Allograft inflammatory factor-1 (AIF1) is a cytoplasmic scaffold protein shown to influence immune responses in macrophages and microglial cells. The protein contains Ca 2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes. This study now reports that AIF1 is expressed in CD11c + dendritic cells (DC) and silencing of expression restrains induction of antigen-specific CD4 + T cell effector responses. AIF1 knockdown in murine DC resulted in impaired T cell proliferation and skewed polarization away from T helper type 1 and 17 fates. In turn, there was a parallel expansion of IL-10-producing and CD25 + Foxp3 + T regulatory subsets. These studies are the first to demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and presents a novel target for engineering tolerogenic DC-based immunotherapies.

  17. Decrease in circulating CD25(hi)Foxp3(+) regulatory T cells following vaccination with the candidate malaria vaccine RTS,S.

    Science.gov (United States)

    Parsons, Emily; Epstein, Judith; Sedegah, Martha; Villasante, Eileen; Stewart, Ann

    2016-08-31

    Regulatory T (Treg) cells have been shown in some cases to limit vaccine-specific immune responses and impact efficacy. Very little is known about the regulatory responses to the leading malaria vaccine candidate, RTS,S. The goal of this study was to begin to characterize the regulatory responses to the RTS,S vaccine. Using multi-parameter flow cytometry, we examined responses in 13 malaria naïve adult volunteers who received 2 doses of RTS,S given eight weeks apart. Five of these volunteers had previously received 3 doses of a candidate DNA-CSP vaccine, with the final dose given approximately one year prior to the first dose of the RTS,S vaccine. We found that the frequency of CD25(hi)Foxp3(+) Treg cells decreased following administration of RTS,S (p=0.0195), with no differences based on vaccine regimen. There was a concomitant decrease in CTLA-4 expression on CD25(hi)Foxp3(+) Treg cells (p=0.0093) and PD-1 levels on CD8(+) T cells (p=0.0002). Additionally, the frequency of anergic CTLA-4(+)CCR7(+) T cells decreased following vaccination. An inverse correlation was observed between the frequency of Plasmodium falciparum circumsporozoite protein (PfCSP)-specific IFN-γ and PfCSP-specific IL-10, as well as an inverse correlation between IL-10 induced by Hepatitis B surface antigen, the carrier of RTS,S, and PfCSP-specific IFN-γ, suggesting that immunity against the vaccine backbone could impact vaccine immunogenicity. These results have implications for future malaria vaccine design. Copyright © 2016. Published by Elsevier Ltd.

  18. Assessment of the frequency of regulatory T cells (CD4+CD25+CD127-) in children with hemophilia A: relation to factor VIII inhibitors and disease severity.

    Science.gov (United States)

    El-Asrar, Mohamed Abo; Hamed, Ahmed El-Saeed; Darwish, Yasser Wagih; Ismail, Eman Abdel Rahman; Ismail, Noha Ali

    2016-01-01

    A rapidly growing evidence showed that regulatory T cells (Tregs) play a crucial role in tolerance to coagulation factors and may be involved in the pathogenesis of inhibitor formation in patients with hemophilia. We determined the percentage of Tregs (CD4CD25CD127) in 45 children with hemophilia A compared with 45 healthy controls, and assessed their relation to the clinical characteristics of patients and factor VIII (FVIII) inhibitors. Patients were studied stressing on frequency of bleeding attacks, joint pain, history of viral hepatitis, and the received therapy (FVIII precipitate/cryotherapy). FVIII activity and FVIII inhibitors were assessed with flow cytometric analysis of CD4CD25CD127 Tregs. According to residual FVIII activity levels, 30 patients (66.7%) had mild/moderate hemophilia A, whereas 15 (33.3%) patients had severe hemophilia A. The frequency of Tregs was significantly lower among all patients with hemophilia A compared with controls (2.59 ± 1.1 versus 3.73 ± 1.12%; P = 0.002). Tregs were significantly decreased among patients with FVIII inhibitors compared with the inhibitor-negative group (P hemophilia A had lower Tregs levels than those without (P = 0.34 and P = 0.011, respectively). A significant positive correlation was found between the percentage of Tregs and FVIII among hemophilia A patients. ROC curve analysis revealed that the cut-off value of Tregs at 1.91% could differentiate patients with and without FVIII inhibitors, with a sensitivity of 100% and a specificity of 91.3%. We suggest that alteration in the frequency of Tregs in young patients with hemophilia A may contribute to inhibitor formation and disease severity.

  19. An increase in CD3+CD4+CD25+ regulatory T cells after administration of umbilical cord-derived mesenchymal stem cells during sepsis.

    Directory of Open Access Journals (Sweden)

    Yu-Hua Chao

    Full Text Available Sepsis remains an important cause of death worldwide, and vigorous immune responses during sepsis could be beneficial for bacterial clearance but at the price of collateral damage to self tissues. Mesenchymal stem cells (MSCs have been found to modulate the immune system and attenuate sepsis. In the present study, MSCs derived from bone marrow and umbilical cord were used and compared. With a cecal ligation and puncture (CLP model, the mechanisms of MSC-mediated immunoregulation during sepsis were studied by determining the changes of circulating inflammation-associated cytokine profiles and peripheral blood mononuclear cells 18 hours after CLP-induced sepsis. In vitro, bone marrow-derived MSCs (BMMSCs and umbilical cord-derived MSCs (UCMSCs showed a similar morphology and surface marker expression. UCMSCs had stronger potential for osteogenesis but lower for adipogenesis than BMMSCs. Compared with rats receiving PBS only after CLP, the percentage of circulating CD3+CD4+CD25+ regulatory T (Treg cells and the ratio of Treg cells/T cells were elevated significantly in rats receiving MSCs. Further experiment regarding Treg cell function demonstrated that the immunosuppressive capacity of Treg cells from rats with CLP-induced sepsis was decreased, but could be restored by administration of MSCs. Compared with rats receiving PBS only after CLP, serum levels of interleukin-6 and tumor necrosis factor-α were significantly lower in rats receiving MSCs after CLP. There were no differences between BMMSCs and UCMSCs. In summary, this work provides the first in vivo evidence that administering BMMSCs or UCMSCs to rats with CLP-induced sepsis could increase circulating CD3+CD4+CD25+ Treg cells and Treg cells/T cells ratio, enhance Treg cell suppressive function, and decrease serum levels of interleukin-6 and tumor necrosis factor-α, suggesting the immunomodulatory association of Treg cells and MSCs during sepsis.

  20. Naturally occurring CD4+ CD25+ FOXP3+ T-regulatory cells are increased in chronic myeloid leukemia patients not in complete cytogenetic remission and can be immunosuppressive.

    Science.gov (United States)

    Rojas, Jose M; Wang, Lihui; Owen, Sally; Knight, Katy; Watmough, Sarah J; Clark, Richard E

    2010-12-01

    Clinical presentation of chronic myeloid leukemia (CML) requires not only the deregulated tyrosine kinase BCR-ABL, but also the failure of an immune response against BCR-ABL-expressing cells. T-cell responses against BCR-ABL and other antigens are well-described, but their relevance to the in vivo control of CML is unclear. The suppressive role of naturally occurring T regulatory (T-reg) cells in antitumor immunity is well-established, although little is known about their role in modulating the T-cell response to BCR-ABL. Naturally occurring T-reg cells were characterized and quantified by flow cytometry in 39 CML patients and 10 healthy donors. Their function was studied by observing their effect on responses to purified protein derivative, a recall antigen, and on the response of an autologous T-cell line recognizing BCR-ABL. T-reg cells were CD4(+), CD25(+), FOXP3(+), CD127(low), and CD62L(high). T-reg numbers in patients in complete cytogenetic remission were significantly lower than in patients not in complete cytogenetic remission (p T-reg cell depletion using anti-CD25 selection enhanced proliferative responses to purified protein derivative. Furthermore, the interferon-γ and/or granzyme-B production of effector cells specific for viral peptides or a BCR-ABL HLA-A3-restricted peptide was inhibited when autologous T-reg cells were present. Taken together, these data suggest a role for T-reg cells in limiting immune responses in CML patients and this may include immune responses to BCR-ABL. The increased frequency of T-reg cells in patients with high levels of BCR-ABL transcripts indicates that an immune mechanism may be important in the control of CML. Copyright © 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  1. The flame retardants tetrabromobisphenol A and tetrabromobisphenol A-bisallylether suppress the induction of interleukin-2 receptor α chain (CD25) in murine splenocytes

    International Nuclear Information System (INIS)

    Pullen, Sabine; Boecker, Ronald; Tiegs, Gisa

    2003-01-01

    Polybrominated flame retardants (PBF) are frequently used additives in electronical equipment. They are ubiquitous environmental contaminants which bioaccumulate with several health effects for humans and the environment. This study investigated immunotoxic effects of the PBF tetrabromobisphenol A (TBBP A), tetrabromobisphenol A-bisallylether (TBBP A-AE), tetrabromobisphenol A-bis-(2,3-dibromopropyl-ether) (TBBP A-PE), decabromodiphenylether (DBDE), and 2,4,6-tribromophenol (TBP) in vitro. The structurally related polychlorinated aromatic hydrocarbon 3,4,3',4'-tetrachlorobiphenyl (PCB77) and dioxins mediate their immunotoxicity via the Ah-receptor gene complex. A highly relevant function of the Ah receptor, the induction of CYP 1A1 in hepatocytes of C57BL/6 mice by the established inducers 3-methylcholanthrene (MC) and PCB77 was compared to the effect of PBF by measurement of ethoxyresorufin-o-deethylase (EROD) activity. The PBF did not show any induction of CYP 1A1, while EROD activity of hepatocytes exposed to MC and PCB77 was induced 10.8- and 8.7-fold, respectively. To investigate immunotoxic effects of the flame retardants, splenocytes of C57BL/6 mice were incubated with subtoxic doses of the flame retardants and PCB77 and activated by concanavalin A (Con A). The flame retardants TBBP A and TBBP A-AE significantly inhibited the expression of interleukin-2 receptor α chain (CD25) in contrast to TBBP A-PE, DBDE, TBP, and PCB77 as shown by immunohistochemistry and quantitative analysis by laser scanning cytometry. None of the substances had any effect on the Con A-induced production of cytokines. Hence, TBBP A and TBBP A-AE may act as immunotoxic compounds by specifically inhibiting the expression of CD25

  2. Interplay of T Helper 17 Cells with CD4+CD25high FOXP3+ Tregs in Regulation of Allergic Asthma in Pediatric Patients

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    Amit Agarwal

    2014-01-01

    Full Text Available Background. There is evidence that Tregs are important to prevent allergic diseases like asthma but limited literature exists on role of TH17 cells in allergic diseases. Methods. Fifty children with asthma and respiratory allergy (study group and twenty healthy children (control group were recruited in this study. Total IgE levels and pulmonary function tests were assessed. The expression of Tregs and cytokines was determined by flow cytometry. Results. The average level of total IgE in study group (316.8 ± 189.8 IU/mL was significantly higher than controls (50 ± 17.5 IU/mL, P<0.0001. The frequency of TH17 cells and culture supernatant level of IL-17 in study group (12.09 ± 8.67 pg/mL was significantly higher than control group (2.01 ± 1.27 pg/mL, P<0.001. Alternatively, the frequency of FOXP3 level was significantly lower in study group [(49.00 ± 13.47%] than in control group [(95.91 ± 2.63%] and CD4+CD25+FOXP3+ to CD4+CD25+ ratio was also significantly decreased in study group [(6.33 ± 2.18%] compared to control group [(38.61 ± 11.04%]. The total serum IgE level is negatively correlated with FOXP3 level (r=-0.5273, P<0.0001. The FOXP3 expression is negatively correlated with the IL-17 levels (r=-0.5631, P<0.0001 and IL-4 levels (r=-0.2836, P=0.0460. Conclusions. Imbalance in TH17/Tregs, elevated IL-17, and IL-4 response and downregulation of FOXP3 were associated with allergic asthma.

  3. A stable reagent system for screening and identifying red blood cell irregular antibodies: application to commercial antibodies.

    Science.gov (United States)

    Million, L; Pellerin, C; Marchand-Arvier, M; Vigneron, C

    1998-01-01

    Development of a new solid-phase system for screening and identifying irregular red cell antibodies. Red blood cell membranes were prepared by a semi-automated procedure in which the hemolysate solution was passed through a hollow-fiber system. The membranes were fixed to the solid phase (microtiter plates) by centrifugation and incubated with 8% fat-free milk. Antibodies added to the microtiter plate were detected by anti-human antibodies adsorbed onto yellow latex particles. The system had good sensitivity (titer antibodies that are important in transfusion.

  4. Increase of Circulating CD4(+)CD25(high)Foxp3(+) Regulatory T Cells in Patients With Metastatic Renal Cell Carcinoma During Treatment With Dendritic Cell Vaccination and Low-Dose Interleukin-2

    DEFF Research Database (Denmark)

    Berntsen, Annika; Brimnes, M.K.; Straten, P.T.

    2010-01-01

    in patients with metastatic renal cell carcinoma on the frequency of CD4(+) CD25(high)Foxp3(+) Treg cells in peripheral blood. We found that the treatment increased the frequency of Treg cells more than 7-fold compared with pretreatment levels (P ... to an increase in the number of Treg cells whereas IL-21 does not stimulate the induction of Treg cells. These findings demonstrate that even low doses of IL-2 in combination with DC vaccination are able to expand CD4(+)CD25(+)Foxp3(+) Treg cells in vivo in metastatic renal cell carcinoma patients. Further...

  5. Shen-Qi-Jie-Yu-Fang exerts effects on a rat model of postpartum depression by regulating inflammatory cytokines and CD4+CD25+ regulatory T cells

    Directory of Open Access Journals (Sweden)

    Li JY

    2016-04-01

    Full Text Available Jingya Li,1,* Ruizhen Zhao,1,* Xiaoli Li,1 Wenjun Sun,1 Miao Qu,1 Qisheng Tang,1 Xinke Yang,1 Shujing Zhang2 1Third Affiliated Hospital, 2School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, People’s Republic of China *These authors contributed equally to this work Background: Shen-Qi-Jie-Yu-Fang (SJF is composed of eight Chinese medicinal herbs. It is widely used in traditional Chinese medicine for treating postpartum depression (PPD. Previous studies have shown that SJF treats PPD through the neuroendocrine mechanism. Aim: To further investigate the effect of SJF on the immune system, including the inflammatory response system and CD4+CD25+ regulatory T (Treg cells. Materials and methods: Sprague Dawley rats were used to create an animal model of PPD by inducing hormone-simulated pregnancy followed by hormone withdrawal. After hormone withdrawal, the PPD rats were treated with SJF or fluoxetine for 1, 2, and 4 weeks. Levels of Treg cells in peripheral blood were measured by flow cytometry analysis. Serum interleukin (IL-1β and IL-6 were evaluated by enzyme-linked immunosorbent assay, and gene and protein expressions of IL-1RI, IL-6Rα, and gp130 in the hippocampus were observed by reverse-transcription polymerase chain reaction and Western blot. Results: Serum IL-1β in PPD rats increased at 2 weeks and declined from then on, while serum IL-6 increased at 1, 2, and 4 weeks. Both IL-1β and IL-6 were downregulated by SJF and fluoxetine. Changes in gene and protein expressions of IL-1RI and gp130 in PPD rats were consistent with changes in serum IL-1β, and were able to be regulated by SJF and fluoxetine. The levels of Treg cells were negatively correlated with serum IL-1β and IL-6, and were decreased in PPD rats. The levels of Treg cells were increased by SJF and fluoxetine. Conclusion: Dysfunction of proinflammatory cytokines and Tregs in different stages of PPD was attenuated by SJF and fluoxetine through

  6. Identification of key genes implicated in the suppressive function of human FOXP3+CD25+CD4+ regulatory T cells through the analysis of time‑series data.

    Science.gov (United States)

    Bai, Xiaofeng; Shi, Hua; Yang, Mingxi; Wang, Yuanlin; Sun, Zhaolin; Xu, Shuxiong

    2018-03-01

    Human forkhead box P3 (FOXP3)+ cluster of differentiation (CD)25+CD4+ regulatory T cells (Tregs) are a type of T cell that express CD4, CD25 and FOXP3, which are critical for maintaining immune homeostasis. The present study aimed to determine the mechanisms underlying Treg function. The GSE11292 dataset was downloaded from the Gene Expression Omnibus, which included data from Treg cells at 19 time points (0‑360 min) with an equal interval of 20 min, and corresponding repeated samples. However, data for Treg cells at time point 120 min were missing. Using the Mfuzz package, the key genes were identified by clustering analysis. Subsequently, regulatory networks and protein‑protein interaction (PPI) networks were constructed and merged into integrated networks using Cytoscape software. Using Database for Annotation, Visualization and Integrated Discover software, enrichment analyses were performed for the genes involved in the PPI networks. Cluster 1 (including 292 genes), cluster 2 (including 111 genes), cluster 3 (including 194 genes) and cluster 4 (including 103 genes) were obtained from the clustering analysis. GAPDH (degree, 40) in cluster 1, Janus kinase 2 (JAK2) (degree, 10) and signal transducer and activator of transcription 5A (STAT5A) (degree, 9) in cluster 3, and tumor necrosis factor (TNF) (degree, 26) and interleukin 2 (IL2) (degree, 22) in cluster 4 had higher degrees in the PPI networks. In addition, it was indicated that several genes may have a role in Treg function by targeting other genes [e.g. microRNA (miR)‑146b‑3p→TNF, miR‑146b‑5p→TNF, miR‑142‑5p→TNF and tripartite motif containing 28 (TRIM28)→GAPDH]. Enrichment analyses indicated that IL2 and TNF were enriched in the immune response and T cell receptor signaling pathway. In conclusion, GAPDH targeted by TRIM28, TNF targeted by miR‑146b‑3p, miR‑146b‑5p and miR‑142‑5p, in addition to JAK2, IL2, and STAT5A may serve important roles in Treg function.

  7. [Level of Th17 cell and CD4(+);CD25(+); Foxp3(+); regulatory T cell in peripheral blood mononuclear cells of primary nephrotic syndrome in children].

    Science.gov (United States)

    Wang, Li; Li, Qiu; Wang, Li-jia; Li, Xin

    2010-08-01

    To investigate the levels and functions of Th17 cells and CD4(+); CD25(+); Foxp3(+); regulate cells(Treg) and explore their role in pathogenesis of primary nephrotic syndrome (PNS) in children. Children with PNS were divided into simple type nephritic syndrome group (SNS) (n = 20), and nephritic type nephritic syndrome group (NNS) (n = 15). 20 healthy subjects were selected as control group. The circulating frequencies of Th17 cells and Treg were measured by FCM. Real-time PCR were used to analyze the mRNA expressions of RORC, IL-23p19 and Foxp3 in peripheral blood mononuclear cells. The serum of IL-1ß, IL-6, TGF-ß1 were measured by ELISA. Circulating frequencies of Th17 cells, the mRNA levels of RORC, IL-23p19 and the serum of IL-1ß, IL-6 were higher in SNS and NNS groups than control group (P 0.05). Imbalance of Th17 and Treg cells might contribute to the pathogenesis of PNS in children and have associated with clinical presentation, pathological type, glucocorticoid sensitivity and prognosis of the disease.

  8. 4-1BB Signaling in Conventional T Cells Drives IL-2 Production That Overcomes CD4+CD25+FoxP3+ T Regulatory Cell Suppression.

    Directory of Open Access Journals (Sweden)

    Hampartsoum B Barsoumian

    Full Text Available Costimulation with the recombinant SA-4-1BBL agonist of 4-1BB receptor on conventional CD4+ T cells (Tconvs overcomes the suppression mediated by naturally occurring CD4+CD25+FoxP3+ T regulatory cells (Tregs. The mechanistic basis of this observation has remained largely unknown. Herein we show that Tconvs, but not Tregs, are the direct target of SA-4-1BBL-mediated evasion of Treg suppression. IL-2 produced by Tconvs in response to 4-1BB signaling is both necessary and sufficient for overcoming Treg suppression. Supernatant from Tconvs stimulated with SA-4-1BBL contains high levels of IL-2 and overcomes Treg suppression in ex vivo Tconv:Treg cocultures. Removal of IL-2 from such supernatant restores Treg suppression and repletion of Tconv:Treg cocultures with exogenous recombinant IL-2 overcomes suppression. This study establishes 4-1BB signaling as a key circuit that regulates physical and functional equilibrium between Tregs and Tconvs with important implications for immunotherapy for indications where a fine balance between Tregs and Teffs plays a decisive role.

  9. Frequency of CD4+CD25+Foxp3+ cells in peripheral blood in relation to urinary bladder cancer malignancy indicators before and after surgical removal.

    Science.gov (United States)

    Jóźwicki, Wojciech; Brożyna, Anna A; Siekiera, Jerzy; Slominski, Andrzej T

    2016-03-08

    Tumor cells communicate with stromal cells, including cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs), to form microenvironment inhibiting immune responses. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) stimulate immune tolerance and facilitate tumor progression. We analyzed the changes in Treg frequencies assessed using flow cytometry in the peripheral blood of patients with urothelial bladder cancer before and after tumor-removal. Changes in Treg frequency were investigated in relation to clinicopathomorphological indicators of tumor malignancy and expression of RCAS1 on CAFs and TAMs. Higher Treg frequencies were observed in early phase of tumor growth (pTa-pT2), in larger tumors, with more aggressive type of invasion, and with expression of RCAS1. The later phase of tumor development, accompanied by a nonclassic differentiations and pT3-pT4 advancement, had lower number of tumor infiltrating lymphocytes (TILs) and lower Treg frequency. Furthermore, in pT2-pT4 tumors, a decreased post-surgery Treg frequency was associated with poorer prognosis: patients with the lowest frequency of Tregs died first. These findings strongly suggest that the Treg frequencies at later phase of tumor growth, associated with a low anti-tumor response, represent a new and important prognostic indicator in urinary bladder cancer.

  10. Foxp3+ CD25+ regulatory T cells specific for a neo-self-antigen develop at the double-positive thymic stage

    Science.gov (United States)

    Cabarrocas, Julie; Cassan, Cécile; Magnusson, Fay; Piaggio, Eliane; Mars, Lennart; Derbinski, Jens; Kyewski, Bruno; Gross, David-Alexandre; Salomon, Benoit L.; Khazaie, Khashayarsha; Saoudi, Abdelhadi; Liblau, Roland S.

    2006-01-01

    Thymus-derived regulatory T cells (Tregs) expressing CD4, CD25, and the transcription factor Foxp3 play major roles in preventing autoimmunity. The Treg population is enriched in T cells expressing high-avidity self-reactive T cell receptors, and thymic epithelial cells expressing self-antigens (Ag) have been implicated in their induction and/or selection. However, the thymic selection events leading to Treg lineage commitment remain unclear. We followed the thymic development of self-Ag-specific Tregs in double-transgenic mice coexpressing a neo-self-Ag, hemagglutinin (HA) under the control of a neural tissue-specific promoter, and a transgenic class II-restricted T cell antigen receptor specific for HA111-119. Our data show that the promiscuous expression of the HA transgene in thymic epithelial cells is involved in the selective induction and/or expansion of HA-specific Foxp3+ Treg thymic precursors as early as the double-positive stage. PMID:16709665

  11. Orally-Induced Intestinal CD4+ CD25+ FoxP3+ Treg Controlled Undesired Responses towards Oral Antigens and Effectively Dampened Food Allergic Reactions.

    Directory of Open Access Journals (Sweden)

    Paola Lorena Smaldini

    Full Text Available The induction of peripheral tolerance may constitute a disease-modifying treatment for allergic patients. We studied how oral immunotherapy (OIT with milk proteins controlled allergy in sensitized mice (cholera toxin plus milk proteins upon exposure to the allergen. Symptoms were alleviated, skin test was negativized, serum specific IgE and IgG1 were abrogated, a substantial reduction in the secretion of IL-5 and IL-13 by antigen-stimulated spleen cells was observed, while IL-13 gene expression in jejunum was down-regulated, and IL-10 and TGF-β were increased. In addition, we observed an induction of CD4+CD25+FoxP3+ cells and IL-10- and TGF-β-producing regulatory T cells in the lamina propria. Finally, transfer experiments confirmed the central role of these cells in tolerance induction. We demonstrated that the oral administration of milk proteins pre- or post-sensitization controlled the Th2-immune response through the elicitation of mucosal IL-10- and TGF-β-producing Tregs that inhibited hypersensitivity symptoms and the allergic response.

  12. Clinical Implications of CD4+CD25+Foxp3+Regulatory T Cell Frequencies After CHP-MAGE-A4 Cancer Vaccination.

    Science.gov (United States)

    Wada, Masataka; Tsuchikawa, Takahiro; Kyogoku, Noriaki; Abiko, Takehiro; Miyauchi, Kengo; Takeuchi, Shintaro; Kuwatani, Toshihiko; Shichinohe, Toshiaki; Miyahara, Yoshihiro; Kageyama, Shinichi; Ikeda, Hiroaki; Shiku, Hiroshi; Hirano, Satoshi

    2018-03-01

    The aim of this study was to explore whether the treatment effect or immune response to a cancer vaccine can be predicted by the percentage of CD4 + CD25 + Foxp3 + regulatory T cells (Tregs) in peripheral blood mononuclear cells (PBMCs) after vaccination. Sixteen patients (9 men, 7 women; median age 61.5 years) enrolled in the CHP-MAGE-A4 cancer vaccine clinical trial who had a fixed dose (300 μg of CHP-MAGE-A4 cancer vaccine and 0.5 Klinische Einheit (KE) of OK432 and received at least four vaccinations were investigated. Safety, immune response, and clinical effects were assessed before and after the cancer vaccination. Treg ratios that remained low both before and after vaccination were associated with a good prognosis, and a low Treg/CD4 lymphocyte ratio 7-weeks after the initial vaccination was correlated with a better prognosis. The Treg ratio following vaccination appears to have some utility for predicting patient prognosis. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  13. c-Jun N-terminal kinase 1 defective CD4+CD25+FoxP3+ cells prolong islet allograft survival in diabetic mice.

    Science.gov (United States)

    Tripathi, Deepak; Cheekatla, Satyanarayana S; Paidipally, Padmaja; Radhakrishnan, Rajesh Kumar; Welch, Elwyn; Thandi, Ramya Sivangala; Tvinnereim, Amy R; Vankayalapati, Ramakrishna

    2018-02-19

    CD4+CD25+FoxP3+ cells (Tregs) inhibit inflammatory immune responses to allografts. Here, we found that co-transplantation of allogeneic pancreatic islets with Tregs that are defective in c-Jun N-terminal kinase 1 (JNK1) signaling prolongs islet allograft survival in the liver parenchyma of chemically induced diabetic mice (CDM). Adoptively transferred JNK1 -/- but not wild-type (WT) Tregs survive longer in the liver parenchyma of CDM. JNK1 -/- Tregs are resistant to apoptosis and express anti-apoptotic molecules. JNK1 -/- Tregs express higher levels of lymphocyte activation gene-3 molecule (LAG-3) on their surface and produce higher amounts of the anti-inflammatory cytokine interleukin (IL)-10 compared with WT Tregs. JNK1 -/- Tregs inhibit liver alloimmune responses more efficiently than WT Tregs. JNK1 -/- but not WT Tregs are able to inhibit IL-17 and IL-21 production through enhanced LAG-3 expression and IL-10 production. Our study identifies a novel role of JNK1 signaling in Tregs that enhances islet allograft survival in the liver parenchyma of CDM.

  14. The presence of B7-H4+ macrophages and CD25+CD4+ and FOXP3+ regulatory T cells in the microenvironment of nasal polyps - a preliminary report.

    Directory of Open Access Journals (Sweden)

    P Strek

    2011-04-01

    Full Text Available The nasal polyp (NP seems to represent the end-stage of longstanding inflammation in patients with chronic rhinosinusitis. The aim of our study has been to evaluate the presence of two regulatory cell populations in the microenvironment of NP: CD4+CD25high Foxp3+ (Treg cells and B7-H4-expressing macrophages. Treg cells are actively able to inhibit T lymphocytes, while the population of B7-H4-expressing macrophages has recently been described as characterized by a regulatory function similar to that of Treg cells. For our study, we evaluated 14 NP tissue samples. The samples were divided into two main groups, eosinophilic (NP and lymphocytic (NP, according to the predominant type of immune cell infiltration. The presence of Treg cells and B7-H4 positive macrophages in the samples was analyzed by FACS. Treg cells and B7-H4-expressing macrophages were identified in all the examined nasal polyps. The percentages of both Treg cells and of B7H4 positive cells found in the eosinophilic nasal polyps were higher than those found in the lymphocytic nasal polyps. Treg cells and B7H4+ macrophage subpopulations were present in the NP microenvironment and the alterations in their percentages were related to a distinct pattern of immune cell infiltration.

  15. CD4+CD25+CD127 regulatory cells play multiple roles in maintaining HIV-1 p24 production in patients on long-term treatment: HIV-1 p24-producing cells and suppression of anti-HIV immunity

    Directory of Open Access Journals (Sweden)

    Yan-Mei Jiao

    2015-08-01

    Conclusions: CD4+CD25+CD127 regulatory cells play multiple roles in maintaining HIV-1 p24 production in long-term ART patients. Treg cells may be a target for eliminating the latent HIV reservoir after effective long-term ART.

  16. Tr-1-like CD4+CD25-CD127-/lowFOXP3- cells are the main source of interleukin 10 in patients with cutaneous leishmaniasis due to Leishmania braziliensis.

    Science.gov (United States)

    Costa, Diego L; Cardoso, Tiago M; Queiroz, Adriano; Milanezi, Cristiane M; Bacellar, Olívia; Carvalho, Edgar M; Silva, João S

    2015-03-01

    CD4(+)CD25(+)FOXP3(+) regulatory T cells have long been shown to mediate susceptibility to Leishmania infection, mainly via interleukin 10 production. In this work, we showed that the main sources of interleukin 10 in peripheral blood mononuclear cells (PBMCs) from patients with cutaneous leishmaniasis due to Leishmania braziliensis are CD4(+)CD25(-)CD127(-/low)FOXP3(-) cells. Compared with uninfected controls, patients with CL had increased frequencies of circulating interleukin 10-producing CD4(+)CD25(-)CD127(-/low) cells, which efficiently suppressed tumor necrosis factor α production by the total PBMC population. Also, in CL lesions, interleukin 10 was mainly produced by CD4(+)CD25(-) cells, and interleukin 10 messenger RNA expression was associated with interleukin 27, interleukin 21, and interferon γ expression, rather than with FOXP3 or transforming growth factor β expressions. Active production of both interleukin 27 and interleukin 21, together with production of interferon γ and interleukin 10, was also detected in the lesions. Since these cytokines are associated with the differentiation and activity of Tr-1 cells, our results suggest that this cell population may play an important role in the immunomodulation of CL. Therefore, development of treatments that interfere with this pathway may lead to faster parasite elimination. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Immunosuppression during active tuberculosis is characterized by decreased interferon- gamma production and CD25 expression with elevated forkhead box P3, transforming growth factor- beta , and interleukin-4 mRNA levels.

    Science.gov (United States)

    Roberts, Teri; Beyers, Nulda; Aguirre, Ana; Walzl, Gerhard

    2007-03-15

    The balance between effector and regulatory responses after Mycobacterium tuberculosis infection may dictate outcome and progression to active disease. We investigated effector and regulatory T cell responses in bacille Calmette-Guerin (BCG)-stimulated peripheral blood mononuclear cells and whole blood cultures from persons with active tuberculosis (TB), persons with TB at the end of 6 months of treatment, and healthy control subjects with latent TB infection. All 3 groups displayed BCG-induced increases in effector and regulatory T cell phenotypes as defined by CD4(+)CD25(lo) and CD4(+)CD25(hi) T cells, respectively. In case patients with active disease, BCG stimulation induced the lowest increase of CD25, CD4(+)CD25(hi), CTLA-4, and interferon- gamma . However, these case patients expressed the highest mRNA levels of forkhead box P3, transforming growth factor (TGF)- beta , and interleukin (IL)-4 and a lower T-bet : GATA-3 ratio. There were no significant differences in IL-4 delta 2, IL-10, or TGF- beta receptor-II mRNA expression between groups. Together, these results suggest that immunosuppression seen after mycobacterial stimulation in case patients with active TB is associated with naturally occurring regulatory T cells.

  18. Relationship of the Content of Systemic and Endobronchial Soluble Molecules of CD25, CD38, CD8, and HLA-I-CD8 and Lung Function Parameters in COPD Patients

    Directory of Open Access Journals (Sweden)

    Nailya Kubysheva

    2017-01-01

    Full Text Available The definition of new markers of local and systemic inflammation of chronic obstructive pulmonary disease (COPD is one of the priority directions in the study of pathogenesis and diagnostic methods improvement for this disease. We investigated 91 patients with COPD and 21 healthy nonsmokers. The levels of soluble CD25, CD38, CD8, and HLA-I-CD8 molecules in the blood serum and exhaled breath condensate (EBC in moderate-to-severe COPD patients during exacerbation and stable phase were studied. An unidirectional change in the content of sCD25, sCD38, and sCD8 molecules with increasing severity of COPD was detected. The correlations between the parameters of lung function and sCD8, sCD25, and sHLA-I-CD8 levels in the blood serum and EBC were discovered in patients with severe COPD. The findings suggest a pathogenetic role of the investigated soluble molecules of the COPD development and allow considering the content of sCD8, sCD25, and sHLA-I-CD8 molecules as additional novel systemic and endobronchial markers of the progression of chronic inflammation of this disease.

  19. Plasmodium falciparum-mediated induction of human CD25Foxp3 CD4 T cells is independent of direct TCR stimulation and requires IL-2, IL-10 and TGFbeta.

    Directory of Open Access Journals (Sweden)

    Anja Scholzen

    2009-08-01

    Full Text Available CD4(+CD25(+Foxp3(+ regulatory T cells (Tregs regulate disease-associated immunity and excessive inflammatory responses, and numbers of CD4(+CD25(+Foxp3(+ Tregs are increased during malaria infection. The mechanisms governing their generation, however, remain to be elucidated. In this study we investigated the role of commonly accepted factors for Foxp3 induction, TCR stimulation and cytokines such as IL-2, TGFbeta and IL-10, in the generation of human CD4(+CD25(+Foxp3(+ T cells by the malaria parasite Plasmodium falciparum. Using a co-culture system of malaria-infected red blood cells (iRBCs and peripheral blood mononuclear cells from healthy individuals, we found that two populations of Foxp3(hi and Foxp3(int CD4(+CD25(hi T cells with a typical Treg phenotype (CTLA-4(+, CD127(low, CD39(+, ICOS(+, TNFRII(+ were induced. Pro-inflammatory cytokine production was confined to the Foxp3(int subset (IFNgamma, IL-4 and IL-17 and inversely correlated with high relative levels of Foxp3(hi cells, consistent with Foxp3(hi CD4 T cell-mediated inhibition of parasite-induced effector cytokine T cell responses. Both Foxp3(hi and Foxp3(int cells were derived primarily from proliferating CD4(+CD25(- T cells with a further significant contribution from CD25(+Foxp3(+ natural Treg cells to the generation of the Foxp3(hi subset. Generation of Foxp3(hi, but not Foxp3(int, cells specifically required TGFbeta1 and IL-10. Add-back experiments showed that monocytes expressing increased levels of co-stimulatory molecules were sufficient for iRBC-mediated induction of Foxp3 in CD4 T cells. Foxp3 induction was driven by IL-2 from CD4 T cells stimulated in an MHC class II-dependent manner. However, transwell separation experiments showed that direct contact of monocytes with the cells that acquire Foxp3 expression was not required. This novel TCR-independent and therefore antigen-non specific mechanism for by-stander CD4(+CD25(hiFoxp3(+ cell induction is likely to reflect a

  20. A Model System for Concurrent Detection of Antigen and Antibody Based on Immunological Fluorescent Method

    Directory of Open Access Journals (Sweden)

    Yuan-Cheng Cao

    2015-01-01

    Full Text Available This paper describes a combined antigen/antibody immunoassay implemented in a 96-well plate using fluorescent spectroscopic method. First, goat anti-human IgG was used to capture human IgG (model antigen; goat anti-human IgG (Cy3 or FITC was used to detect the model antigen; a saturating level of model antigen was then added followed by unlabelled goat anti-human IgG (model antibody; finally, Cy3 labelled rabbit anti-goat IgG was used to detect the model antibody. Two approaches were applied to the concomitant assay to analyze the feasibility. The first approach applied FITC and Cy3 when both targets were present at the same time, resulting in 50 ng/mL of the antibody detection limit and 10 ng/mL of antigen detection limit in the quantitative measurements of target concentration, taking the consideration of FRET efficiency of 68% between donor and acceptor. The sequential approach tended to lower the signal/noise (S/N ratio and the detection of the model antigen (lower than 1 ng/mL had better sensitivity than the model antibody (lower than 50 ng/mL. This combined antigen/antibody method might be useful for combined detection of antigens and antibodies. It will be helpful to screen for both antigen and antibody particularly in the situations of the multiserotype and high-frequency mutant virus infections.

  1. Combined effect of ultrasound/SonoVue microbubble on CD4(+)CD25(+) regulatory T cells viability and optimized parameters for its transfection.

    Science.gov (United States)

    Shi, Chunying; Zhang, Yu; Yang, Haichao; Dong, Tianxiu; Chen, Yaodong; Xu, Yutong; Yang, Xiuhua

    2015-09-01

    The purpose of this study was to investigate the combined effect of ultrasound and SonoVue microbubble on CD4(+)CD25(+) regulatory T cells (Tregs) viability and to explore the appropriate parameters for Tregs transfection. Tregs were separated from peripheral venous blood of patients with hepatocellular carcinoma and seeded in 96-well plates. The optimal ultrasound exposure time and optimal SonoVue microbubble concentration for Tregs were measured by mechanical index (MI) of 1.2 or 1.4, exposure time of 0, 30, 60, 90, 120, 150, 180s, and 0, 10, 20, 30, 40, 50μL/100μL microbubble per well, respectively. In addition, the combined effect of ultrasound and microbubble on Tregs viability was evaluated according to the following parameters: MI 1.2/1.4+exposure time of 120, 150, 180s+0, 10, 20, 30, 40, 50μL/100μL microbubble per well. Tregs viability investigations were performed in order to explore the optimal transfection condition. The efficiency of plasmid transfer was determined by detection of luciferase activity on the microscopic examinations. The proliferation of Tregs could be promoted by ultrasound exposures, while being decreased with the increasing concentration of microbubbles. Under the current experimental conditions, the optimal ultrasound parameters were MI=1.4 and exposure time=150/180s. The optimal microbubble concentration was 10μL/100μL. Compared with treatment with ultrasound or microbubbles alone, the transfection efficiency of Tregs improved 50% by combining ultrasound and microbubble. The results indicate that both ultrasound and microbubble could affect the Tregs proliferation and the optimal Treg transfection rate was obtained by treating with 10% microbubbles and ultrasound exposure for 150/180s under ultrasound MI of 1.4. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Low-dose temozolomide before dendritic-cell vaccination reduces (specifically) CD4+CD25++Foxp3+ regulatory T-cells in advanced melanoma patients.

    Science.gov (United States)

    Ridolfi, Laura; Petrini, Massimiliano; Granato, Anna Maria; Gentilcore, Giusy; Simeone, Ester; Ascierto, Paolo Antonio; Pancisi, Elena; Ancarani, Valentina; Fiammenghi, Laura; Guidoboni, Massimo; de Rosa, Francesco; Valmorri, Linda; Scarpi, Emanuela; Nicoletti, Stefania Vittoria Luisa; Baravelli, Stefano; Riccobon, Angela; Ridolfi, Ruggero

    2013-05-31

    In cancer immunotherapy, dendritic cells (DCs) play a fundamental role in the dialog between innate and adaptive immune response, but several immunosuppressive mechanisms remain to be overcome. For example, a high number of CD4+CD25++Foxp3+ regulatory T-cells (Foxp3+Tregs) have been observed in the peripheral blood and tumor microenvironment of cancer patients. On the basis of this, we conducted a study on DC-based vaccination in advanced melanoma, adding low-dose temozolomide to obtain lymphodepletion. Twenty-one patients were entered onto our vaccination protocol using autologous DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin. Patients received low-dose temozolomide before vaccination and 5 days of low-dose interleukin-2 (IL-2) after vaccination. Circulating Foxp3+Tregs were evaluated before and after temozolomide, and after IL-2. Among the 17 evaluable patients we observed 1 partial response (PR), 6 stable disease (SD) and 10 progressive disease (PD). The disease control rate (PR+SD = DCR) was 41% and median overall survival was 10 months. Temozolomide reduced circulating Foxp3+Treg cells in all patients. A statistically significant reduction of 60% was observed in Foxp3+Tregs after the first cycle, whereas the absolute lymphocyte count decreased by only 14%. Conversely, IL-2 increased Foxp3+Treg cell count by 75.4%. Of note the effect of this cytokine, albeit not statistically significant, on the DCR subgroup led to a further 33.8% reduction in Foxp3+Treg cells. Our results suggest that the combined immunological therapy, at least as far as the DCR subgroup is concerned, effectively reduced the number of Foxp3+Treg cells, which exerted a blunting effect on the growth-stimulating effect of IL-2. However, this regimen, with its current modality, would not seem to be capable of improving clinical outcome.

  3. Phenotypical analysis of ectoenzymes CD39/CD73 and adenosine receptor 2A in CD4+ CD25high Foxp3+ regulatory T-cells in psoriasis.

    Science.gov (United States)

    Han, Ling; Sugiyama, Hideaki; Zhang, Qi; Yan, Kexiang; Fang, Xu; McCormick, Thomas S; Cooper, Kevin D; Huang, Qiong

    2018-02-01

    CD39 and CD73 are two novel cell surface markers of CD25 high Foxp3 + regulatory T-cells (Tregs). Concordant expression of these two ectoenzymes not only discriminate Tregs from other cell populations, but also generates pericellular adenosine, which has been reported to suppress proliferation of activated T effector (Teff) cells. Because it is currently unclear whether human ectoenzymes (CD39/CD73) are involved in the impaired suppressive activity of Tregs in psoriasis, we examined the frequencies and phenotypes of CD39/CD73-expressing Tregs and related receptor adenosine receptor 2A (A 2A R) in peripheral blood of patients with different types of psoriasis. Peripheral blood mononuclear cells (PMBC) were prepared from patients with three different types of psoriasis (psoriasis vulgaris, pustular psoriasis and erythrodermic psoriasis). CD4 + cells were separated from PBMC by negative selection on midiMACS columns, and the frequencies and phenotypes of CD39 and CD73 expressing Tregs, and A 2A R expressing Teff were all determined by flow cytometry analysis. Blood from healthy volunteers served as controls. The expression of single CD73 + Tregs was markedly reduced (approximately 50%) in psoriasis vulgaris, compared to normal controls. In pustular psoriasis, the mean numbers of CD39 + Tregs and A 2A R + Teff was significantly lower than in normal controls. Among three different types of psoriasis, CD39 expression was strikingly reduced in the blood Treg population of pustular psoriasis patients. Decreased CD73 + Tregs levels were observed in psoriasis vulgaris compared to pustular psoriasis and erythrodermic psoriasis. The differences in the expression of CD39 - and CD73 - Tregs may be a factor in the pathogenesis of psoriasis. © 2017 The Authors. Australasian Journal of Dermatology published by Wiley Publishing Asia Pty Ltd on behalf of The Australasian College of Dermatologists.

  4. CD4+CD25+Foxp3+ regulatory T cells depletion may attenuate the development of silica-induced lung fibrosis in mice.

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    Fangwei Liu

    Full Text Available BACKGROUND: Silicosis is an occupational lung disease caused by inhalation of silica dust characterized by lung inflammation and fibrosis. Previous study showed that Th1 and Th2 cytokines are involved in silicosis, but Th1/Th2 polarization during the development of silicosis is still a matter of debate. Regulatory T cells (Treg cells represent a crucial role in modulation of immune homeostasis by regulating Th1/Th2 polarization, but their possible implication in silicosis remains to be explored. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the implication of Treg cells in the development of silicosis, we generated the Treg-depleted mice model by administration of anti-CD25 mAbs and mice were exposed to silica by intratracheal instillation to establish experimental model of silica-induced lung fibrosis. The pathologic examinations show that the Treg-depleted mice are susceptive to severer inflammation in the early stage, with enhanced infiltration of inflammatory cells. Also, depletion of Treg cells causes a delay of the progress of silica-induced lung fibrosis in mice model. Further study of mRNA expression of cytokines reveals that depletion of Tregs leads to the increased production of Th1-cytokines and decreased production of Th2-cytokine. The Flow Cytometry and realtime PCR study show that Treg cells exert the modulation function both directly by expressing CTLA-4 at the inflammatory stage, and indirectly by secreting increasing amount of IL-10 and TGF-β during the fibrotic stage in silica-induced lung fibrosis. CONCLUSION/SIGNIFICANCE: Our study suggests that depletion of Tregs may attenuate the progress of silica-induced lung fibrosis and enhance Th1 response and decelerate Th1/Th2 balance toward a Th2 phenotype in silica-induced lung fibrosis. The regulatory function of Treg cells may depend on direct mechanism and indirect mechanism during the inflammatory stage of silicosis.

  5. QSAR modeling for anti-human African trypanosomiasis activity of substituted 2-Phenylimidazopyridines

    Science.gov (United States)

    Masand, Vijay H.; El-Sayed, Nahed N. E.; Mahajan, Devidas T.; Mercader, Andrew G.; Alafeefy, Ahmed M.; Shibi, I. G.

    2017-02-01

    In the present work, sixty substituted 2-Phenylimidazopyridines previously reported with potent anti-human African trypanosomiasis (HAT) activity were selected to build genetic algorithm (GA) based QSAR models to determine the structural features that have significant correlation with the activity. Multiple QSAR models were built using easily interpretable descriptors that are directly associated with the presence or the absence of a structural scaffold, or a specific atom. All the QSAR models have been thoroughly validated according to the OECD principles. All the QSAR models are statistically very robust (R2 = 0.80-0.87) with high external predictive ability (CCCex = 0.81-0.92). The QSAR analysis reveals that the HAT activity has good correlation with the presence of five membered rings in the molecule.

  6. Anti-IL-2 receptor antibody decreases cytokine-induced apoptosis of human renal tubular epithelial cells (TEC).

    Science.gov (United States)

    Wang, Shuang; Zhang, Zhu-Xu; Yin, Ziqin; Liu, Weihua; Garcia, Bertha; Huang, Xuyan; Acott, Philip; Jevnikar, Anthony M

    2011-07-01

    Transplant rejection is mediated by T-cell activation which is modulated by interleukin-2 (IL-2) binding to IL-2R (CD25). Monoclonal anti-IL-2 receptor antibody is used in renal transplantation to reduce rejection. Interestingly, proximal tubular epithelial cells (TEC) express CD25, similar to T cells. We have demonstrated that IL-2 induces murine TEC apoptosis through down-regulation of the caspase-8 inhibitor protein c-FLIP. Anti-CD25 antibody may be useful clinically to limit renal injury, but this has not been tested in human TEC. Human PT-2 TEC were isolated and cloned from the urine of transplant patients. Apoptosis was determined by FACS with Annexin-V FITC. Protein expression was studied using western blot, and mRNA levels by quantitative real-time (PR-PCR). We demonstrated that the morphology of a human kidney cell line (PT-2) cloned from urine was consistent with proximal TEC and expresses alkaline phosphatase, cytokeratin, vimentin, CD13, CD26, and low levels of E-cadherin. Basal IL-2 receptor (CD25) was up-regulated by IL-2/IFN-γ stimulation, and cytokine exposure induced apoptosis in a dose-dependent manner. Apoptosis with IL-2/IFN-γ was associated with increased caspase-8 activity and decreased endogenous caspase-8 inhibitor c-FLIP mRNA and protein expression. IL-2/IFN-γ-induced apoptosis could be blocked by pre-treatment of PT-2 with anti IL-2R antibody (basiliximab) but not control IgG antibody. These data demonstrate for the first time in human TEC that IL-2 and IFN-γ can induce TEC apoptosis which can be blocked by CD25 blockade antibody. These data suggest that anti-CD25 mAb might similarly attenuate inflammation-induced TEC injury in vivo. Kidney-expressed CD25 may represent a clinically important new target for attenuating early inflammatory injury in donor kidneys and preserving renal function during anti-rejection therapy.

  7. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    International Nuclear Information System (INIS)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M.

    1982-01-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys. (Auth.)

  8. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M. (National Inst. for Biological Standards and Control, London (UK))

    1982-10-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

  9. Isolation of highly suppressive CD25+FoxP3+ T regulatory cells from G-CSF-mobilized donors with retention of cytotoxic anti-viral CTLs: application for multi-functional immunotherapy post stem cell transplantation.

    Directory of Open Access Journals (Sweden)

    Edward R Samuel

    Full Text Available Previous studies have demonstrated the effective control of cytomegalovirus (CMV infections post haematopoietic stem cell transplant through the adoptive transfer of donor derived CMV-specific T cells (CMV-T. Strategies for manufacturing CMV immunotherapies has involved a second leukapheresis or blood draw from the donor, which in the unrelated donor setting is not always possible. We have investigated the feasibility of using an aliquot of the original G-CSF-mobilized graft as a starting material for manufacture of CMV-T and examined the activation marker CD25 as a targeted approach for identification and isolation following CMVpp65 peptide stimulation. CD25+ cells isolated from G-CSF-mobilized apheresis revealed a significant increase in the proportion of FoxP3 expression when compared with conventional non-mobilized CD25+ cells and showed a superior suppressive capacity in a T cell proliferation assay, demonstrating the emergence of a population of Tregs not present in non-mobilized apheresis collections. The expansion of CD25+ CMV-T in short-term culture resulted in a mixed population of CD4+ and CD8+ T cells with CMV-specificity that secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. Furthermore CD25 expanded cells retained their suppressive capacity but did not maintain FoxP3 expression or secrete IL-10. In summary our data indicates that CD25 enrichment post CMV stimulation in G-CSF-mobilized PBMCs results in the simultaneous generation of both a functional population of anti-viral T cells and Tregs thus illustrating a potential single therapeutic strategy for the treatment of both GvHD and CMV reactivation following allogeneic haematopoietic stem cell transplantation. The use of G-CSF-mobilized cells as a starting material for cell therapy manufacture represents a feasible approach to alleviating the many problems incurred with successive donations and procurement of cells from

  10. Prognostic Relevance of Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor α-Chain (CD25 Expression Predicts a Poor Prognosis.

    Directory of Open Access Journals (Sweden)

    Kazunori Nakase

    Full Text Available A variety of cytokine/cytokine receptor systems affect the biological behavior of acute leukemia cells. However, little is known about the clinical relevance of cytokine receptor expression in acute myeloid leukemia (AML. We quantitatively examined the expression of interleukin-2 receptor α-chain (IL-2Rα, also known as CD25, IL-2Rβ, IL-3Rα, IL-4Rα, IL-5Rα, IL-6Rα, IL-7Rα, the common β-chain (βc, γc, granulocyte-macrophage colony-stimulating factor (GM-CSFRα, G-CSFR, c-fms, c-mpl, c-kit, FLT3, and GP130 in leukemia cells from 767 adult patients with AML by flow cytometry and determined their prevalence and clinical significance. All cytokine receptors examined were expressed at varying levels, whereas the levels of IL-3Rα, GM-CSFRα, IL-2Rα, γc, c-kit, and G-CSFR exhibited a wide spectrum of ≥10,000 sites/cell. In terms of their French-American-British classification types, GM-CSFRα and c-fms were preferentially expressed in M4/M5 patients, G-CSF in M3 patients, and IL-2Rα in non-M3 patients. Elevated levels of IL-3Rα, GM-CSFRα, and IL-2Rα correlated with leukocytosis. In patients ≤60 years old, higher levels of these 3 receptors correlated with poor responses to conventional chemotherapy, but only IL-2Rα was associated with a shorter overall survival. By incorporating IL-2Rα status into cytogenetic risk stratification, we could sort out a significantly adverse-risk cohort from the cytogenetically intermediate-risk group. Analyses with various phenotypical risk markers revealed the expression of IL-2Rα as an independent prognostic indicator in patients with intermediate-risk cytogenetics. These findings were not observed in patients >60 years old. Our results indicate that several cytokine receptors were associated with certain cellular and clinical features, but IL-2Rα alone had prognostic value that provides an additional marker to improve current risk evaluation in AML patients ≤60 years old.

  11. Allergy-related changes in levels of CD8+CD25+FoxP3(bright) Treg cells and FoxP3 mRNA expression in peripheral blood: the role of IL-10 or TGF-beta.

    Science.gov (United States)

    Eusebio, M; Kuna, P; Kraszula, L; Kupczyk, M; Pietruczuk, M

    2014-01-01

    There is an increasing body of evidence that alterations of regulatory T (Treg) cell numbers and functions lead to immune disorders. Accordingly, understanding the regulatory mechanisms that maintain peripheral regulatory T (Treg) cell homeostasis is key to the development of effective targeted biologic therapies. We previously demonstrated the effects of IL-10 or TGF-beta on distinct CD8+CD28- T cell subsets in vitro. Allergy-related changes of CD8+CD25+FoxP3(bright)Treg cells and FoxP3 mRNA expression in peripheral blood were assessed by means of multicolor flow cytometry and real-time polymerase chain reaction (RT-PCR). Co-stimulation of CD8+CD25+ T cells with anti-CD3/CD28 in the presence of either IL-10 or TGF-beta increased the frequency of CD8+CD25+FoxP3(bright)Treg cells in patients with asthma and controls. Likewise, CD8+CD25+ T cell activation with anti-CD3/CD28 and TGF-beta increased FoxP3mRNA expression in all groups. Anti-CD3/CD28 and IL-10 appeared to regulate FoxP3 mRNA expression in a phenotype-dependent manner. Specifically, co-stimulation by anti-CD3/CD28 and IL-10 markedly increased FoxP3 mRNA expression in the severe asthma group whereas it had opposite effects on this value in other groups. Taken altogether, these data suggest that IL-10 and TGF-beta may modulate FoxP3 expressions at the protein and mRNA levels in respect to their need for peripheral tolerance.

  12. Identification of compounds with anti-human cytomegalovirus activity that inhibit production of IE2 proteins.

    Science.gov (United States)

    Beelontally, Rooksarr; Wilkie, Gavin S; Lau, Betty; Goodmaker, Charles J; Ho, Catherine M K; Swanson, Chad M; Deng, Xianming; Wang, Jinhua; Gray, Nathanael S; Davison, Andrew J; Strang, Blair L

    2017-02-01

    Using a high throughput screening methodology we surveyed a collection of largely uncharacterized validated or suspected kinase inhibitors for anti-human cytomegalovirus (HCMV) activity. From this screen we identified three structurally related 5-aminopyrazine compounds (XMD7-1, -2 and -27) that inhibited HCMV replication in virus yield reduction assays at low micromolar concentrations. Kinase selectivity assays indicated that each compound was a kinase inhibitor capable of inhibiting a range of cellular protein kinases. Western blotting and RNA sequencing demonstrated that treatment of infected cells with XMD7 compounds resulted in a defect in the production of the major HCMV transcriptional transactivator IE2 proteins (IE2-86, IE2-60 and IE2-40) and an overall reduction in transcription from the viral genome. However, production of certain viral proteins was not compromised by treatment with XMD7 compounds. Thus, these novel anti-HCMV compounds likely inhibited transcription from the viral genome and suppressed production of a subset of viral proteins by inhibiting IE2 protein production. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Diphtheria toxin-based anti-human CD19 immunotoxin for targeting human CD19+tumors.

    Science.gov (United States)

    Zheng, Qian; Wang, Zhaohui; Zhang, Huiping; Huang, Qi; Madsen, Joren C; Sachs, David H; Huang, Christene A; Wang, Zhirui

    2017-05-01

    CD19 is expressed on normal and neoplastic B cells and is a promising target for immunotherapy. However, there is still an unmet need to further develop novel therapeutic drugs for the treatment of the refractory/relapsing human CD19 + tumors. We have developed a diphtheria toxin-based anti-human CD19 immunotoxin for targeting human CD19 + tumors. We have constructed three isoforms of the CD19 immunotoxin: monovalent, bivalent, and foldback diabody. In vitro binding affinity and efficacy analysis demonstrated that the bivalent isoform had the highest binding affinity and in vitro efficacy. The in vivo efficacy of the CD19 immunotoxins was assessed using human CD19 + JeKo-1 tumor-bearing NOD/SCID IL-2 receptor γ -/- (NSG) mouse model. In these animals, CD19 immunotoxins significantly prolonged the median survival from 31 days in controls to 34, 36, and 40 days in animals receiving the monovalent isoform, foldback diabody isoform, and bivalent isoform, respectively. The bivalent CD19 immunotoxin is a promising therapeutic drug candidate for targeting relapsing/refractory human CD19 + tumors. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  14. Administration of anti-CD25 mAb leads to impaired alpha-galactosylceramide-mediated induction of IFN-gamma production in a murine model

    Czech Academy of Sciences Publication Activity Database

    Rosalia, Rodney Alexander; Štěpánek, Ivan; Polláková, Veronika; Šímová, Jana; Bieblová, Jana; Indrová, Marie; Moravcová, Simona; Přibylová, Hana; Bontkes, H.; Bubeník, Jan; Sparwasser, T.; Reiniš, Milan

    2013-01-01

    Roč. 218, č. 6 (2013), s. 851-859 ISSN 0171-2985 R&D Projects: GA ČR GA301/07/1410; GA ČR GAP301/10/2174 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : cancer immunotherapy * DEREG mice * interferon γ * natural killer T cells * PC61 monoclonal antibody * T regulatory cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.180, year: 2013

  15. Anthocyanin contents in the seed coat of black soya bean and their anti-human tyrosinase activity and antioxidative activity.

    Science.gov (United States)

    Jhan, J-K; Chung, Y-C; Chen, G-H; Chang, C-H; Lu, Y-C; Hsu, C-K

    2016-06-01

    The seed coat of black soya bean (SCBS) contains high amount of anthocyanins and shows antioxidant and anti-mushroom tyrosinase activities. The objectives of this study were to analyse the anthocyanins in SCBS with different solvents and to find the relationship between anthocyanin profile with anti-human and anti-mushroom tyrosinase activities. SCBS was extracted with hot water, 50 and 80% ethanol, 50 and 80% acetone and 50 and 80% acidified acetone. Total phenol and total flavonoid contents in the extracts were determined. Anthocyanins in the extracts were analysed using HPLC and LC/MS/MS. A genetically engineered human tyrosinase was used to evaluate the anti-tyrosinase potential of the extracts from SCBS. 80% acetone extract from SCBS obtained the highest total phenol, total flavonoid and cyanidin-3-O-glucoside (C3G) contents among all the extracts, whereas the hot water extract showed the lowest antioxidant contents. Three anthocyanin compounds were found in all the extracts from SCBS, and the analysis of HPLC and LC/MS/MS indicated that they were C3G, delphinidin-3-O-glucoside (D3G) and peonidin-3-O-glucoside (P3G). The ratios of C3G (2.84 mg g(-1) ), D3G (0.34 mg g(-1) ) and P3G (0.35 mg g(-1) ) in 80% acidified acetone extract were 76.6, 9.1 and 9.3%, respectively. All the extracts from SCBS possessed anti-human tyrosinase activity. Moreover, a good correlation was found between the anti-human tyrosinase activities and C3G contents in the extracts. Antioxidants in SCBS also possess anti-human and anti-mushroom tyrosinase activities. © 2015 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  16. Immunoreactivity of adsorbed anti human chorionic gonadotropin studied with an optical waveguide interferometric sensor

    NARCIS (Netherlands)

    Heideman, Rene; Kooyman, R.P.H.; Greve, Jan

    1994-01-01

    A study on the immunoreactivity of adsorbed αhCG molecules as a function of substrate hydrophobicity and protein coverage is presented. The experiments were performed with a planar waveguide interferometrical immunosensor. The substrate hydrophobicity and the antibody density were found to be of

  17. Anti-human-cytomegalovirus immunoglobulin G levels in glioma risk and prognosis

    International Nuclear Information System (INIS)

    Susan Amirian, E.; Marquez-Do, Deborah; Bondy, Melissa L.; Scheurer, Michael E.

    2013-01-01

    The role of human cytomegalovirus (HCMV) in glioma development and progression remains controversial. The purpose of our study was to assess the potential associations between anti-HCMV antibodies (immunoglobulin G [IgG] and immunoglobulin M [IgM]) and glioma risk and prognosis using data from the Harris County Case–Control Study. Multivariable logistic regression models were utilized to estimate odds ratios and 95% confidence intervals (CI) for the associations between glioma status and antibody levels among glioma cases (n = 362) and cancer-free controls (n = 462). Hazard ratios and 95% CIs were calculated using Cox proportional hazards regression, adjusting for age, race, and sex, to determine if antibody levels were associated with survival over time among cases. Among IgG-positive participants, increasing anti-HCMV IgG levels were associated with decreasing glioma risk (P for trend = 0.0008), and those with the lowest level of anti-HCMV IgG (<10 U/mL) had the highest glioma risk, controlling for age, sex, and race/ethnicity (OR: 2.51, 95% CI: 1.42–4.43). Antibody levels were not associated with survival among glioma cases. Our study contributes new evidence toward the potential importance of the direct and indirect effects of HCMV infection in gliomagenesis

  18. A general approach to standardization of the solid-phase radioimmunoassay for quantitation of class-specific antibodies

    International Nuclear Information System (INIS)

    Zollinger, W.D.; Boslego, J.W.

    1981-01-01

    The feasibility of using an anti-human immunoglobulin/human immunoglobulin/[ 125 I]anti-human immunoglobulin 'sandwich' in a solid-phase radioimmunoassay to produce a standard curve which could be used to quantitate antigen-specific antibody of a particular immunoglobulin class was investigated. The amount of secondary antibody (SAb) bound was determined as a function of whether the primary antibody (PAb) was bound to its specific solid-phase antigen or by a solid-phase anti-human immunoglobulin. No significant difference between the two values was observed. Quantitation of anti-tetanus toxoid antibody by this method was in a good agreement with quantitative precipitin tests. Comparison of SAb binding as a function of the way the PAb is bound was extended to class-specific PAb by use of murine monoclonal antibodies to meningococcal antigens. In most cases somewhat greater binding of SAb occurred when PAb was bound to antigen, but in several cases where low avidity antibody and/or poor quality antigens were used, greater SAb binding occurred when PAb was bound by anti-mouse immunoglobulin. The results indicate that this approach may be useful as a general method for standardizing the SPRIA and other solid-phase immunoassays such as the ELISA to measure class-specific antibody. (Auth.)

  19. Effect of IC14, an anti-CD14 antibody, on plasma and cell-associated chemokines during human endotoxemia

    NARCIS (Netherlands)

    Olszyna, Dariusz P.; Verbon, Annelies; Pribble, John P.; Turner, Terence; Axtelle, Tim; van Deventer, Sander J. H.; van der Poll, Tom

    2003-01-01

    To determine the role of CD14 in lipopolysaccharide (LPS)-induced release of chemokines, 16 humans were injected with LPS (4 ng/kg) preceded (-2 h) by intravenous IC14, an anti-human CD14 monoclonal antibody, or placebo. LPS elicited increases in interleukin (IL)-8 concentrations in plasma and in

  20. [Effects of transcription factor T-bet, GATA-3, FoxP3 and CD4(+)CD25(+)regulatory T cells in pathogenesis of child Hench-Schonlein purpura].

    Science.gov (United States)

    Wang, Qiang; Ren, Shu-Hong; Dong, Wei; Fang, Jun

    2012-02-01

    The aim of this study was to investigate the effects of transcription factors T-bet, GATA-3 in the pathogenesis of Hench-Schonlein purpura (HSP) in children, the relationship between CD4(+)CD25(+)regulatory T cells, transcription factor FoxP3 and the development of child HSP, and the molecular mechanisms of Th1/Th2 imbalance of child HSP at acute phase, so as to may provide a new approach and strategy for the treatment of HSP at the molecular levels. The expression of T-bet, GATA-3 and FoxP3 mRNA were detected by real time PCR using SYBR Green I in 46 patients with HSP at acute phase and 30 healthy children as controls. The expression of T lymphocyte subsets CD4(+)CD25(+) in peripheral blood mononuclear cells was detected by flow cytometry. The results showed that the relative level of GATA-3 mRNA in peripheral blood mononuclear cells of patients with HSP was significantly higher than those of the control group (964.30 ± 655.18 vs 78.09 ± 57.20, P child HSP, especially predominant activation of Th2, which correlates with the abnormal expression of transcription factor T-bet and GATA-3 mRNA. At acute phase of child HSP, the expression of CD4(+)CD25(+)Treg and its special transcription factor FoxP3 mRNA are down-regulated. Treg cells decreases, which indicates that insufficient immunosuppressive effects resulting from the reduction of Treg cells may be one of the important reason in the immune imbalance of HSP acute phase. This study provides experimental evidence for illustrating the pathogenesis of HSP from the molecular mechanism of Treg cells and its regulation, and also provides a new thinking and new strategies for the treatment of HSP at molecular levels.

  1. Fermented fish oil suppresses T helper 1/2 cell response in a mouse model of atopic dermatitis via generation of CD4+CD25+Foxp3+ T cells

    Directory of Open Access Journals (Sweden)

    Han Sang-Chul

    2012-08-01

    Full Text Available Abstract Background Allergic skin inflammation such as atopic dermatitis (AD, which is characterized by pruritus and inflammation, is regulated partly through the activity of regulatory T cells (Tregs. Tregs play key roles in the immune response by preventing or suppressing the differentiation, proliferation and function of various immune cells, including CD4+ T cells. Recent studies report that fermentation has a tremendous capacity to transform chemical structures or create new substances, and the omega-3 polyunsaturated fatty acids (n-3 PUFAs in fish oil can reduce inflammation in allergic patients. The beneficial effects of natural fish oil (NFO have been described in many diseases, but the mechanism by which fermented fish oil (FFO modulates the immune system and the allergic response is poorly understood. In this study, we produced FFO and tested its ability to suppress the allergic inflammatory response and to activate CD4+CD25+Foxp3+ Tregs. Results The ability of FFO and NFO to modulate the immune system was investigated using a mouse model of AD. Administration of FFO or NFO in the drinking water alleviated the allergic inflammation in the skin, and FFO was more effective than NFO. FFO treatment did increase the expression of the immune-suppressive cytokines TGF-β and IL-10. In addition, ingestion of FFO increased Foxp3 expression and the number of CD4+CD25+Foxp3+ Tregs compared with NFO. Conclusions These results suggest that the anti-allergic effect of FFO is associated with enrichment of CD4+CD25+ Foxp3+ T cells at the inflamed sites and that FFO may be effective in treating the allergic symptoms of AD.

  2. INFα-2b inhibitory effects on CD4+CD25+FOXP3+ regulatory T cells in the tumor microenvironment of C57BL/6 J mice with melanoma xenografts

    International Nuclear Information System (INIS)

    Yu, Yang; Huang, Run; Zong, Xiangyun; He, Xiangming; Mo, Wenju

    2016-01-01

    Regulatory T cells (Treg s ), particularly the CD4 + CD25 + Foxp3 + Treg s , down regulate immunity and promote tumor cell growth by directly suppressing CD8 + and CD4 + T cells. Alternatively they can promote tumor growth by generating interleukin-10 (IL-10) and transforming growth factor β (TGFβ) in situ, which help tumor cells to evade the immune system. In vivo tumor models were prepared via subcutaneous injection with a suspension of B16 melanoma cells into the left upper flank of C57BL/6 J mice. The mice were randomized into five groups: radiotherapy (RT), chemotherapy (CT), radiochemotherapy (RCT), Inteferon α (INFα) groups, and a control group. Flow cytometry was used to determine the Treg s levels in the spleen and peripheral blood, and immunohistochemistry was performed to determine the expression levels of TGFβ and IL-10 in the tumor microenvironment. Tumor weight was significantly reduced in the CT or RCT groups (40.91 % and 41.83 %, respectively), while the reduction in tumor weight was relatively lower for the RT and IFNα groups (15.10 % and 13.15 %, respectively). The flow cytometry results showed that the ratios of CD4 + CD25 + Foxp3 + Treg s to lymphocytes and CD4 + cells in the spleen and in peripheral blood were significantly decreased after treatment with IFNα (P < 0.05). Expression of TGFβ and IL-10 in the tumor microenvironment in the CT and RT groups was higher compared with the control group (P < 0.01), while the expression of TGFβ and IL-10 in the INFα group was not significantly different (P > 0.05). The results show that INFα-2b inhibits cancer cell immune evasion by decreasing the levels of CD4 + CD25 + Foxp3 + Treg s and suppressing the expression of TGFβ and IL-10 in the tumor microenvironment

  3. Increase of circulating CD4+CD25highFoxp3+ regulatory T cells in patients with metastatic renal cell carcinoma during treatment with dendritic cell vaccination and low-dose interleukin-2

    DEFF Research Database (Denmark)

    Berntsen, Annika; Brimnes, Marie Klinge; thor Straten, Per

    2010-01-01

    Regulatory T cells (Treg) play an important role in the maintenance of immune tolerance and may be one of the obstacles of successful tumor immunotherapy. In this study, we analyzed the impact of administration of dendritic cell (DC) vaccination in combination with low-dose interleukin (IL)-2 in ...... in patients with metastatic renal cell carcinoma on the frequency of CD4+CD25highFoxp3+ Treg cells in peripheral blood. We found that the treatment increased the frequency of Treg cells more than 7-fold compared with pretreatment levels (P...

  4. 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

  5. Technetium-99m labelled anti-human chorionic gonadotropin, technetium-99m labelled anti-human chorionic gonadotropin-beta or its combinations along with a diagnostic kit for forming a composition useful in identifying cancer cell and/or a malignant tumour

    International Nuclear Information System (INIS)

    Crockford, D.R.; Rhodes, B.A.

    1979-01-01

    Anti-human chorionic gonadotropin and anti-human chorionic gonadotropin-beta have been labelled with technetium 99m, and applied separately or in mixture for scintiscanning of malignant tumors. Besides the labelled compound, the diagnostic kit includes an anionic exchange resin capable of selectively removing pertechnetate ion from aqueous solution from a protein labelled with Tcsup(99m). (E.G.)

  6. The Second Extracellular Loop of CCR5 Contains the Dominant Epitopes for Highly Potent Anti-Human Immunodeficiency Virus Monoclonal Antibodies▿

    Science.gov (United States)

    Zhang, Jun; Rao, Eileen; Dioszegi, Marianna; Kondru, Rama; DeRosier, Andre; Chan, Eva; Schwoerer, Stephan; Cammack, Nick; Brandt, Michael; Sankuratri, Surya; Ji, Changhua

    2007-01-01

    Six mouse anti-human CCR5 monoclonal antibodies (mAbs) that showed potent antiviral activities were identified from over 26,000 mouse hybridomas. The epitopes for these mAbs were determined by using various CCR5 mutants, including CCR5/CCR2B chimeras. One mAb, ROAb13, was found to bind to a linear epitope in the N terminus of CCR5. Strikingly, the other five mAbs bind to epitopes derived from extracellular loop 2 (ECL2). The three most potent mAbs, ROAb12, ROAb14, and ROAb18, require residues from both the N-terminal (Lys171 and Glu172) and C-terminal (Trp190) halves of ECL2 for binding; two other mAbs, ROAb10 and ROAb51, which also showed potent antiviral activities, require Lys171 and Glu172 but not Trp190 for binding. Binding of the control mAb 2D7 completely relies on Lys171 and Glu172. Unlike 2D7, the novel mAbs ROAb12, ROAb14, and ROAb18 do not bind to the linear peptide 2D7-2SK. In addition, all three mAbs bind to monkey CCR5 (with Arg at position 171 instead of Lys); however, 2D7 does not. Since five of the six most potent CCR5 mAbs derived from the same pool of immunized mice require ECL2 as epitopes, we hypothesize that CCR5 ECL2 contains the dominant epitopes for mAbs with potent antiviral activities. These dominant epitopes were found in CCR5 from multiple species and were detected in large proportions of the total cell surface CCR5. mAbs recognizing these epitopes also showed high binding affinity. A homology model of CCR5 was generated to aid in the interpretation of these dominant epitopes in ECL2. PMID:17242138

  7. Kinetics of T cell receptor β, γ, and δ rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+CD25+ Pro-T thymocytes

    Science.gov (United States)

    Capone, Myriam; Hockett, Richard D.; Zlotnik, Albert

    1998-01-01

    We performed a comprehensive analysis of T cell receptor (TCR) γ rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRγ rearrangements are present in CD44+CD25+ Pro-T thymocytes much earlier than expected. TCRγ rearrangements increase significantly from the Pro-T to the CD44−CD25+ Pre-T cell transition, and follow different patterns depending on each Vγ gene segment, suggesting that ordered waves of TCRγ rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRγ genes occur concurrently with TCRδ and D-Jβ rearrangements, but before Vβ gene assembly. Productive TCRγ rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4+CD8+ double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRδ−/− mice display random proportions of TCRγ rearranged alleles, supporting a role for functional TCRγ/δ rearrangements in the γδ divergence process. PMID:9770518

  8. Effects of oral Lactobacillus administration on antioxidant activities and CD4+CD25+forkhead box P3 (FoxP3)+ T cells in NZB/W F1 mice.

    Science.gov (United States)

    Tzang, Bor-Show; Liu, Chung-Hsien; Hsu, Kuo-Ching; Chen, Yi-Hsing; Huang, Chih-Yang; Hsu, Tsai-Ching

    2017-09-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterised by a dysregulation of the immune system, which causes inflammation responses, excessive oxidative stress and a reduction in the number of cluster of differentiation (CD)4+CD25+forkhead box P3 (FoxP3)+ T cells. Supplementation with certain Lactobacillus strains has been suggested to be beneficial in the comprehensive treatment of SLE. However, little is known about the effect and mechanism of certain Lactobacillus strains on SLE. To investigate the effects of Lactobacillus on SLE, NZB/W F1 mice were orally gavaged with Lactobacillus paracasei GMNL-32 (GMNL-32), Lactobacillus reuteri GMNL-89 (GMNL-89) and L. reuteri GMNL-263 (GMNL-263). Supplementation with GMNL-32, GMNL-89 and GMNL-263 significantly increased antioxidant activity, reduced IL-6 and TNF-α levels and significantly decreased the toll-like receptors/myeloid differentiation primary response gene 88 signalling in NZB/W F1 mice. Notably, supplementation with GMNL-263, but not GMNL-32 and GMNL-89, in NZB/W F1 mice significantly increased the differentiation of CD4+CD25+FoxP3+ T cells. These findings reveal beneficial effects of GMNL-32, GMNL-89 and GMNL-263 on NZB/W F1 mice and suggest that these specific Lactobacillus strains can be used as part of a comprehensive treatment of SLE patients.

  9. An Imbalance between Frequency of CD4+CD25+FOXP3+ Regulatory T Cells and CCR4+ and CCR9+ Circulating Helper T Cells Is Associated with Active Perennial Allergic Conjunctivitis

    Directory of Open Access Journals (Sweden)

    J. Galicia-Carreón

    2013-01-01

    Full Text Available Allergic conjunctivitis (AC is one of the most common eye disorders in ophthalmology. In mice models, it has been suggested that control of allergic conjunctivitis is a delicate balance between Tregs and inflammatory migrating effector cells. Our aim was to evaluate the frequency of Tregs and the frequency of homing receptors expressing cells in peripheral blood mononuclear cells (PBMC from patients with perennial allergic conjunctivitis (PAC. The analyses of phenotypic markers on CD4+ T cells and both soluble or intracellular cytokines were performed by flow cytometry. CD4+CD25+ cells were 15 times more frequent in PBMC from patients than HC; the vast majority of these CD4+CD25+ cells were FOXP3−, and most of CD4+ T cells were CCR4+ and CCR9+ cells. Upon allergen-stimulation, no significant changes were observed in frequency of Treg; however, an increased frequency of CD4+CCR4+CCR9+ cells, CD4+CD103+ cells and CD4+CD108+ cells with increased IL-5, IL-6, and IL-8 production was observed. These findings suggest an immune dysregulation in PAC, characterized by diminished frequency of Tregs and increased frequency of circulating activated CD4+ T cells; upon allergen-stimulation, these cells were expressing cell-surface molecules related to mucosa homing and were able to trigger an inflammatory microenvironment.

  10. The effect of interleukin (IL)-21 and CD4+CD25++T cells on cytokine production of CD4+responder T cells in patients with myasthenia gravis.

    Science.gov (United States)

    Alahgholi-Hajibehzad, M; Durmuş, H; Aysal, F; Gülşen-Parman, Y; Oflazer, P; Deymeer, F; Saruhan-Direskeneli, G

    2017-11-01

    Impairment of the suppressive function of regulatory T (T reg ) cells has been reported in myasthenia gravis (MG). In this study, cytokine-related mechanisms that may lead to the defect of T reg were investigated in patients with anti-acetylcholine receptor antibody-positive MG (AChR + MG). Proliferation and cytokine production of responder T (T resp ) cells in response to polyclonal activation were measured in a suppression assay. The effect of interleukin (IL)-21 on suppression was evaluated in vitro in co-culture. IL-21 increased the proliferation of T resp cells in T resp /T reg co-cultures. T resp cells from patients with MG secreted significantly lower levels of IL-2. In patients with MG, IL-2 levels did not change with the addition of T reg to cultures, whereas it decreased significantly in controls. In T resp /T reg co-cultures, IL-4, IL-6 and IL-10 production increased in the presence of T reg in patients. Interferon (IFN)-γ was decreased, whereas IL-17A was increased in both patient and control groups. IL-21 inhibited the secretion of IL-4 in MG and healthy controls (HC), and IL-17A in HC only. The results demonstrated that IL-21 enhances the proliferation of T resp cells in the presence of T reg . An effect of IL-21 mainly on T resp cells through IL-2 is implicated. © 2017 British Society for Immunology.

  11. 2-Substituted benzoxazinone analogues as anti-human coronavirus (anti-HCoV) and ICAM-1 expression inhibition agents.

    Science.gov (United States)

    Hsieh, Pei-Wen; Chang, Fang-Rong; Chang, Cheng-Hsien; Cheng, Pei-Wen; Chiang, Lien-Chai; Zeng, Fu-Long; Lin, Kuei-Hsiang; Wu, Yang-Chang

    2004-09-20

    A series of 2-substituted benzoxazinones were synthesized and subjected to anti-human coronavirus and ICAM-1 expression inhibition assays. Among them, compounds 1, 3, 4, 5, 6, and 7 exhibited significant anti-HCoV activities, and the IC(50) value of these compounds are 6.08, 5.06, 6.83, 1.92, 7.59, and 5.79 microg/mL, respectively. Furthermore, compounds 1 and 6 showed significant inhibitory effect on ICAM-1 expression, the ED(50) values of 1 and 6 are 1.00 and 0.50 microg/mL, respectively.

  12. Screening of multiple myeloma by polyclonal rabbit anti-human plasmacytoma cell immunoglobulin.

    Directory of Open Access Journals (Sweden)

    Bo Mu

    Full Text Available Antibody-based immunotherapy has been effectively used for tumor treatment. However, to date, only a few tumor-associated antigens (TAAs or therapeutic targets have been identified. Identification of more immunogenic antigens is essential for improvements in multiple myeloma (MM diagnosis and therapy. In this study, we synthesized a polyclonal antibody (PAb by immunizing rabbits with whole human plasmacytoma ARH-77 cells and identified MM-associated antigens, including enlonase, adipophilin, and HSP90s, among others, via proteomic technologies. 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay showed that 200 µg/mL PAb inhibits the proliferation of ARH-77 cells by over 50% within 48 h. Flow cytometric assay indicated that PAb treatment significantly increases the number of apoptotic cells compared with other treatments (52.1% vs. NS, 7.3% or control rabbit IgG, 9.9%. In vivo, PAb delayed tumor growth and prolonged the lifespan of mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that PAb also induces statistically significant changes in apoptosis compared with other treatments (P<0.05. We therefore conclude that PAb could be used for the effective screening and identification of TAA. PAb may have certain anti-tumor functions in vitro and in vivo. As such, its combination with proteomic technologies could be a promising approach for sieving TAA for the diagnosis and therapy of MM.

  13. Characterization of immobilization methods of antiviral antibodies in serum for electrochemical biosensors

    Science.gov (United States)

    Huy, Tran Quang; Hanh, Nguyen Thi Hong; Van Chung, Pham; Anh, Dang Duc; Nga, Phan Thi; Tuan, Mai Anh

    2011-06-01

    In this paper, we describes different methods to immobilize Japanese encephalitis virus (JEV) antibodies in human serum onto the interdigitated surface of a microelectrode sensor for optimizing electrochemical detection: (1) direct covalent binding to the silanized surface, (2) binding to the silanized surface via a cross-linker of glutaraldehyde (GA), (3) binding to glutaraldehyde/silanized surface via goat anti-human IgG polyclonal antibody and (4) binding to glutaraldehyde/silanized surface via protein A (PrA). Field emission scanning electron microscopy, Fourier transform infrared spectrometry, and fluorescence microscopy are used to verify the characteristics of antibodies on the interdigitated surface after the serum antibodies immobilization. The analyzed results indicate that the use of protein A is an effective choice for immobilization and orientation of antibodies in serum for electrochemical biosensors. This study provides an advantageous immobilization method of serum containing antiviral antibodies to develop electrochemical biosensors for preliminary screening of viruses in clinical samples from outbreaks.

  14. Determination of antibody levels to Candida albicans in healthy and hospitalised adults using a radioimmunoassay

    International Nuclear Information System (INIS)

    Cobb, S.J.; Parratt, D.

    1978-01-01

    A radioimmunoassay for antibody to Candida albicans is described. The test uses whole, killed of organisms as the antigen and radiolabelled sheep anti-human globulins to quantitate different classes of antibody to C. albicans. The assay has been compared with an Ouchterlony precipitin method and found to be simpler, more rapid, and more sensitive than the latter. Results obtained from two groups of symptomless adults indicated that the range of antibody level was wider for a hospitalised group than for a group of blood transfusion donors, particularly in respect of IgG and IgA antibody. The reason for the increase of antibody in hospital patients was not clear but may have been related to antibiotic therapy. The difficulties in interpretation of Candida serology have therefore been re-assessed in the light of more detailed knowledge of the range and type of antibody to be expected in normal individuals. (author)

  15. Impact of Uniform Methods on Interlaboratory Antibody Titration Variability: Antibody Titration and Uniform Methods.

    Science.gov (United States)

    Bachegowda, Lohith S; Cheng, Yan H; Long, Thomas; Shaz, Beth H

    2017-01-01

    -Substantial variability between different antibody titration methods prompted development and introduction of uniform methods in 2008. -To determine whether uniform methods consistently decrease interlaboratory variation in proficiency testing. -Proficiency testing data for antibody titration between 2009 and 2013 were obtained from the College of American Pathologists. Each laboratory was supplied plasma and red cells to determine anti-A and anti-D antibody titers by their standard method: gel or tube by uniform or other methods at different testing phases (immediate spin and/or room temperature [anti-A], and/or anti-human globulin [AHG: anti-A and anti-D]) with different additives. Interlaboratory variations were compared by analyzing the distribution of titer results by method and phase. -A median of 574 and 1100 responses were reported for anti-A and anti-D antibody titers, respectively, during a 5-year period. The 3 most frequent (median) methods performed for anti-A antibody were uniform tube room temperature (147.5; range, 119-159), uniform tube AHG (143.5; range, 134-150), and other tube AHG (97; range, 82-116); for anti-D antibody, the methods were other tube (451; range, 431-465), uniform tube (404; range, 382-462), and uniform gel (137; range, 121-153). Of the larger reported methods, uniform gel AHG phase for anti-A and anti-D antibodies had the most participants with the same result (mode). For anti-A antibody, 0 of 8 (uniform versus other tube room temperature) and 1 of 8 (uniform versus other tube AHG), and for anti-D antibody, 0 of 8 (uniform versus other tube) and 0 of 8 (uniform versus other gel) proficiency tests showed significant titer variability reduction. -Uniform methods harmonize laboratory techniques but rarely reduce interlaboratory titer variance in comparison with other methods.

  16. Over-expression of Stat5b confers protection against diabetes in the non-obese diabetic (NOD) mice via up-regulation of CD4{sup +}CD25{sup +} regulatory T cells

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Yulan; Purohit, Sharad [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); Department of Pathology, Medical College of Georgia, Georgia Health Sciences University, GA (United States); Chen, Xueqin; Yi, Bing [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); She, Jin-Xiong, E-mail: jshe@georgiahealth.edu [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); Department of Pathology, Medical College of Georgia, Georgia Health Sciences University, GA (United States)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer This is the first study to provide direct evidence of the role of Stat5b in NOD mice. Black-Right-Pointing-Pointer Over-expression of wild type Stat5b transgene protects NOD mice against diabetes. Black-Right-Pointing-Pointer This protection may be mediated by the up-regulation of CD4{sup +}CD25{sup +} Tregs. -- Abstract: The signal transducers and activators of transcription (STAT) family of proteins play a critical role in cytokine signaling required for fine tuning of immune regulation. Previous reports showed that a mutation (L327M) in the Stat5b protein leads to aberrant cytokine signaling in the NOD mice. To further elaborate the role of Stat5b in diabetes, we established a NOD transgenic mouse that over-expresses the wild type Stat5b gene. The incidences of spontaneous diabetes as well as cyclophosphamide-induced diabetes were significantly reduced and delayed in the Stat5b transgenic NOD mice compared to their littermate controls. The total cell numbers of CD4{sup +} T cells and especially CD8{sup +} T cells in the spleen and pancreatic lymph node were increased in the Stat5b transgenic NOD mice. Consistent with these findings, CD4{sup +} and CD8{sup +} T cells from the Stat5b transgenic NOD mice showed a higher proliferation capacity and up-regulation of multiple cytokines including IL-2, IFN-{gamma}, TNF-{alpha} and IL-10 as well as anti-apoptotic gene Bcl-xl. Furthermore, the number and proportion of CD4{sup +}CD25{sup +} regulatory T cells were significantly increased in transgenic mice although in vitro suppression ability of the regulatory T-cells was not affected by the transgene. Our results suggest that Stat5b confers protection against diabetes in the NOD mice by regulating the numbers and function of multiple immune cell types, especially by up-regulating CD4{sup +}CD25{sup +} regulatory T cells.

  17. Identification of CD4+CD25+CD127-regulatory T cells and CD14+HLA-DR-/low myeloid-derived suppressor cells and their roles in the prognosis of breast cancer.

    Science.gov (United States)

    Wang, Jinhu; Yang, Jianhong

    2016-08-01

    The aim of the present study was to identify cluster of differentiation 4 + (CD4 + )CD25 + CD127 - regulatory T cells (Tregs) and CD14 + human leukocyte antigen-antigen D-related (HLA - DR - )/low myeloid-derived suppressor cells (MDSCs) in patients with breast cancer of varying stages, and investigate their roles and the potential interactions in the prognosis of breast cancer. A total of 40 patients with breast cancer were included in the study. A total of 30 healthy individuals served as the healthy control. Flow cytometry was performed for the identification of biomarkers. Natural Tregs were characterized by the expression of CD4 + CD25 + CD127 - . The MDSC frequency was expressed as the percentage of CD33 + CD11b + HLA - DR - lineage markers (Lin) - . The absolute number of Tregs was higher in breast cancer patients compared to the healthy control. The absolute number of Tregs in the patients with stage III or IV breast cancer was higher than those of the stage I or II, respectively. The percentage showed a gradual increase in the patients with breast cancer compared with the normal control. No direct correlation was established between the number or percentage of Tregs and the patient survival. There was a higher percentage of circulating MDSCs in breast cancer patients compared with the normal individuals. A close correlation was established between clinical cancer stage and percentage and total number of circulating MDSCs. To be exact, a significant increase of MDSC percentage and total number was observed in patients with stage III-IV breast cancer compared with the other cancer patients (stage I-II) and the normal individuals. No statistical difference was observed in the 3- and 5-year survival rates in the breast cancer patients with enhanced expression of Tregs, compared with the normal individuals. In conclusion, enhanced expression of CD4 + CD25 + CD127 - Tregs cells and CD33 + CD11 + HLA - DR - LIN - MDSCs were identified from patients with breast

  18. Increase of Circulating CD4(+)CD25(high)Foxp3(+) Regulatory T Cells in Patients With Metastatic Renal Cell Carcinoma During Treatment With Dendritic Cell Vaccination and Low-Dose Interleukin-2

    DEFF Research Database (Denmark)

    Berntsen, Annika; Brimnes, M.K.; Straten, P.T.

    2010-01-01

    Regulatory T cells (Treg) play an important role in the maintenance of immune tolerance and may be one of the obstacles of successful tumor immunotherapy. In this study, we analyzed the impact of administration of dendritic cell (DC) vaccination in combination with low-dose interleukin (IL)-2...... to an increase in the number of Treg cells whereas IL-21 does not stimulate the induction of Treg cells. These findings demonstrate that even low doses of IL-2 in combination with DC vaccination are able to expand CD4(+)CD25(+)Foxp3(+) Treg cells in vivo in metastatic renal cell carcinoma patients. Further......, the results indicate that the IL-2-induced effect on Treg cells is reversible and declines shortly after termination of IL-2 treatment. Our data suggest that approaches combining DC-mediated immunotherapy and depletion of Treg cells may be necessary to enhance the ability of vaccination therapy to elicit...

  19. Design of indirect solid-phase immunosorbent methods for detecting arenavirus antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, A.P.; Rezapkin, G.V.; Dzagurova, T.K.; Tkachenko, E.A.

    1984-05-01

    Specifications have been elaborated for formulating indirect solid-phase enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (SPRIA) methods that employ anti-human and anti-mice G class immunoglobulin (IgG), conjugated with horseradish peroxidase and /sup 125/I for detecting the arenaviruses Junin, Machupo, Tacaribe, Amalpari, Tamiami, Lassa, and LCM (lymphocytic choriomeningitis). These methods make it possible to identify with a high degree of sensitivity arenavirus antigens and antibodies in various kinds of material.

  20. Antigen-specific over-expression of human cartilage glycoprotein 39 on CD4+ CD25+ forkhead box protein 3+ regulatory T cells in the generation of glucose-6-phosphate isomerase-induced arthritis.

    Science.gov (United States)

    Tanaka, Y; Matsumoto, I; Inoue, A; Umeda, N; Takai, C; Sumida, T

    2014-08-01

    Human cartilage gp-39 (HC gp-39) is a well-known autoantigen in rheumatoid arthritis (RA). However, the exact localization, fluctuation and function of HC gp-39 in RA are unknown. Therefore, using a glucose-6-phosphate isomerase (GPI)-induced model of arthritis, we investigated these aspects of HC gp-39 in arthritis. The rise in serum HC gp-39 levels was detected on the early phase of GPI-induced arthritis (day 7) and the HC gp-39 mRNA was increased significantly on splenic CD4(+) T cells on day7, but not on CD11b(+) cells. Moreover, to identify the characterization of HC gp-39(+) CD4(+) T cells, we assessed the analysis of T helper (Th) subsets. As a result, HC gp-39 was expressed dominantly in CD4(+) CD25(+) forkhead box protein 3 (FoxP3)(+) refulatory T cells (T(reg)), but not in Th1, Th2 or Th17 cells. Furthermore, to investigate the effect of HC gp-39 to CD4(+) T cells, T cell proliferation assay and cytokine production from CD4(+) T cells using recombinant HC gp-39 was assessed. We found that GPI-specific T cell proliferation and interferon (IFN)-γ or interleukin (IL)-17 production were clearly suppressed by addition of recombinant HC gp-39. Antigen-specific over-expression of HC gp-39 in splenic CD4(+) CD25(+) FoxP3(+) T(reg) cells occurs in the induction phase of GPI-induced arthritis, and addition of recombinant HC gp-39 suppresses antigen-specific T-cell proliferation and cytokine production, suggesting that HC gp-39 in CD4(+) T cells might play a regulatory role in arthritis. © 2014 British Society for Immunology.

  1. Antibody biotechnology

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... and automated, the hybrid cells can be stored for many years in liquid nitrogen and antibodies production is homogeneous. The hybridoma method .... they may be modified to vehicle active molecules such as radio-isotopes, toxins, cytokines, enzyme etc. In these cases, the therapeutic effect is due to ...

  2. Catalytic Antibodies

    Indian Academy of Sciences (India)

    The ability of the highly evolved machinery of immune system to produce structurally and functionally complex ... to Pauling, if the structure of the antigen binding site of antibodies were to be produced in a random ..... where the immune system of the body is destructive, as in autoimmune disorders or after organ transplant.

  3. Catalytic Antibodies

    Indian Academy of Sciences (India)

    While chemistry provides the framework for understanding the structure and function of biomolecules, the immune sys- tem provides a highly evolved natural process to generate one class of complex biomolecules – the antibodies. A combination of the two could be exploited to generate new classes of molecules with novel ...

  4. Evaluation of an immunoassay for human-specific quantitation of therapeutic antibodies in serum samples from non-human primates.

    Science.gov (United States)

    Stubenrauch, Kay; Wessels, Uwe; Lenz, Helmut

    2009-05-01

    Pharmacokinetic characterization of therapeutic antibodies plays an important role during preclinical and clinical development. However, accurate pharmacokinetic evaluation of therapeutic antibodies in serum samples from non-human primates is often complicated by insufficient specificity of the assays to measure drug levels. The present paper describes the use of a murine monoclonal antibody in an immunoassay format to specifically and quantitatively measure human therapeutic antibodies in serum from non-human primates. This murine antibody is directed against a unique epitope on the constant region CH2 domain of all isotypes of human immunoglobulin G (IgG). The antibody, designated anti-human Fcgamma-pan: R10Z8E9, does not cross-react with serum from mouse, rat, and the non-human primates marmoset, rhesus macaque, cynomolgus monkey and baboon when using an enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance technology (Biacore) format for measurement of the therapeutic antibody. Use of the antibody anti-human Fcgamma-pan: R10Z8E9 as capturing and detection reagent allowed human-specific quantitation of total therapeutic antibody anti-IGF-1R in spiked cynomolgus monkey serum via a Sandwich ELISA format. In contrast, a commercially available polyclonal antibody (PAB) directed to the Fcgamma fragment of human IgG only specifically measured the therapeutic antibody in buffer samples, but not in serum from cynomolgus monkeys. This generic human IgG assay was already applied in several pharmacokinetic studies in cynomolgus monkeys to determine serum levels of different therapeutic antibodies, including the anti-IGF-1R. Validation of the assay for a humanized IgG1 therapeutic antibody against a membrane protein revealed a lower limit of quantitation of 8 ng/mL in undiluted serum. Intra-assay and inter-assay precision was characterized by a coefficient of variation of less than 10% and accuracy was within 15%. Dilutional linearity was evidenced by a

  5. Cross-Linking GPVI-Fc by Anti-Fc Antibodies Potentiates Its Inhibition of Atherosclerotic Plaque- and Collagen-Induced Platelet Activation

    Directory of Open Access Journals (Sweden)

    Janina Jamasbi, RPh

    2016-04-01

    Full Text Available To enhance the antithrombotic properties of recombinant glycoprotein VI fragment crystallizable (GPVI-Fc, the authors incubated GPVI-Fc with anti-human Fc antibodies to cross-link the Fc tails of GPVI-Fc. Cross-linking potentiated the inhibition of human plaque- and collagen-induced platelet aggregation by GPVI-Fc under static and flow conditions without increasing bleeding time in vitro. Cross-linking with anti-human-Fc Fab2 was even superior to anti-human-Fc immunoglobulin G (IgG. Advanced optical imaging revealed a continuous sheath-like coverage of collagen fibers by cross-linked GPVI-Fc complexes. Cross-linking of GPVI into oligomeric complexes provides a new, highly effective, and probably safe antithrombotic treatment as it suppresses platelet GPVI-plaque interaction selectively at the site of acute atherothrombosis.

  6. NY-ESO-1-specific circulating CD4+ T cells in ovarian cancer patients are prevalently T(H)1 type cells undetectable in the CD25+ FOXP3+ Treg compartment.

    Science.gov (United States)

    Redjimi, Nassima; Duperrier-Amouriaux, Karine; Raimbaud, Isabelle; Luescher, Immanuel; Dojcinovic, Danijel; Classe, Jean-Marc; Berton-Rigaud, Dominique; Frenel, Jean-Sébastien; Bourbouloux, Emmanuelle; Valmori, Danila; Ayyoub, Maha

    2011-01-01

    Spontaneous CD4(+) T-cell responses to the tumor-specific antigen NY-ESO-1 (ESO) are frequently found in patients with epithelial ovarian cancer (EOC). If these responses are of effector or/and Treg type, however, has remained unclear. Here, we have used functional approaches together with recently developed MHC class II/ESO tetramers to assess the frequency, phenotype and function of ESO-specific cells in circulating lymphocytes from EOC patients. We found that circulating ESO-specific CD4(+) T cells in EOC patients with spontaneous immune responses to the antigen are prevalently T(H)1 type cells secreting IFN-γ but no IL-17 or IL-10 and are not suppressive. We detected tetramer(+) cells ex vivo, at an average frequency of 1:25,000 memory cells, that is, significantly lower than in patients immunized with an ESO vaccine. ESO tetramer(+) cells were mostly effector memory cells at advanced stages of differentiation and were not detected in circulating CD25(+)FOXP3(+)Treg. Thus, spontaneous CD4(+) T-cell responses to ESO in cancer patients are prevalently of T(H)1 type and not Treg. Their relatively low frequency and advanced differentiation stage, however, may limit their efficacy, that may be boosted by immunogenic ESO vaccines.

  7. NY-ESO-1-Specific Circulating CD4+ T Cells in Ovarian Cancer Patients Are Prevalently TH1 Type Cells Undetectable in the CD25+FOXP3+Treg Compartment

    Science.gov (United States)

    Redjimi, Nassima; Duperrier-Amouriaux, Karine; Raimbaud, Isabelle; Luescher, Immanuel; Dojcinovic, Danijel; Classe, Jean-Marc; Berton-Rigaud, Dominique; Frenel, Jean-Sébastien; Bourbouloux, Emmanuelle

    2011-01-01

    Spontaneous CD4+ T-cell responses to the tumor-specific antigen NY-ESO-1 (ESO) are frequently found in patients with epithelial ovarian cancer (EOC). If these responses are of effector or/and Treg type, however, has remained unclear. Here, we have used functional approaches together with recently developed MHC class II/ESO tetramers to assess the frequency, phenotype and function of ESO-specific cells in circulating lymphocytes from EOC patients. We found that circulating ESO-specific CD4+ T cells in EOC patients with spontaneous immune responses to the antigen are prevalently TH1 type cells secreting IFN-γ but no IL-17 or IL-10 and are not suppressive. We detected tetramer+ cells ex vivo, at an average frequency of 1∶25000 memory cells, that is, significantly lower than in patients immunized with an ESO vaccine. ESO tetramer+ cells were mostly effector memory cells at advanced stages of differentiation and were not detected in circulating CD25+FOXP3+Treg. Thus, spontaneous CD4+ T-cell responses to ESO in cancer patients are prevalently of TH1 type and not Treg. Their relatively low frequency and advanced differentiation stage, however, may limit their efficacy, that may be boosted by immunogenic ESO vaccines. PMID:21829534

  8. Uptake of donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light by recipient dendritic cells induces CD4{sup +}CD25{sup +}Foxp3{sup +} regulatory T cells and down-regulates cardiac allograft rejection

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, De-Hua [Organ Transplant Center, Chinese PLA 309th Hospital, No. 17A Hei-Shan-Hu Road, Beijing 100091 (China); Dou, Li-Ping [Department of Hematology, Chinese PLA General Hospital, No. 28 Fu-Xing Road, Beijing 100853 (China); Wei, Yu-Xiang; Du, Guo-Sheng; Zou, Yi-Ping; Song, Ji-Yong; Zhu, Zhi-Dong; Cai, Ming; Qian, Ye-Yong [Organ Transplant Center, Chinese PLA 309th Hospital, No. 17A Hei-Shan-Hu Road, Beijing 100091 (China); Shi, Bing-Yi, E-mail: shibingyi@medmail.com.cn [Organ Transplant Center, Chinese PLA 309th Hospital, No. 17A Hei-Shan-Hu Road, Beijing 100091 (China)

    2010-05-14

    Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy and has been demonstrated to be beneficial for graft-vs-host disease and solid-organ allograft rejection. ECP involves reinfusion of a patient's autologous peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen and UVA light radiation (PUVA). Previous studies focused only on ECP treatment of recipient immune cells. Our study is the first to extend the target of ECP treatment to donor immune cells. The results of in vitro co-culture experiments demonstrate uptake of donor PUVA-treated splenic lymphocytes (PUVA-SPs) by recipient immature dendritic cells (DCs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient DCs. In the same co-culture system, donor PUVA-SPs enhanced production of interleukin-10 and interferon-{gamma} by recipient DCs and impaired the subsequent capability of recipient DCs to stimulate recipient naive T cells. Phagocytosis of donor PUVA-SP (PUVA-SP DCs) by recipient DCs shifted T-cell responses in favor of T helper 2 cells. Infusion of PUVA-SP DCs inhibited cardiac allograft rejection in an antigen-specific manner and induced CD4{sup +}CD25{sup high}Foxp3{sup +} regulatory T cells. In conclusion, PUVA-SP DCs simultaneously deliver the donor antigen and the regulatory signal to the transplant recipient, and thus can be used to develop a novel DC vaccine for negative immune regulation and immune tolerance induction.

  9. CD4+CD25highCD127low Regulatory T Cells in Peripheral Blood Are Not an Independent Factor for Chronic Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Jolanta B. Perz

    2012-01-01

    Full Text Available Background. The therapeutic efficacy of allogeneic hemopoietic stem cell transplantation (HSCT largely relies on the graft-versus-leukemia (GVL effect. Uncontrolled graft-versus-host disease (GVHD is a feared complication of HSCT. Regulatory T cells (Treg are a subset of CD4+ T-helper cells believed to maintain tolerance after HSCT. It remains unclear whether low peripheral blood Treg have an impact on the risk for acute (aGVHD and chronic GVHD (cGVHD. Methods. In this paper we enumerated the CD4+CD25highCD127low Treg in the peripheral blood of 84 patients after at least 150 days from HSCT and in 20 healthy age-matched controls. Results. Although similar mean lymphocyte counts were found in patients and controls, CD3+CD4+ T-cell counts were significantly lower in patients. Patients also had significantly lower Treg percentages among lymphocytes as compared to controls. Patients with cGVHD had even higher percentages of Treg if compared to patients without cGVHD. In multivariate analysis, Treg percentages were not an independent factor for cGVHD. Conclusions. This paper did not show a relation between deficient peripheral blood Treg and cGVHD, therefore cGVHD does not seem to occur as a result of peripheral Treg paucity.

  10. Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease.

    Directory of Open Access Journals (Sweden)

    Mareike Florek

    Full Text Available The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs in murine models of allogeneic hematopoietic cell transplantation (HCT has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L, which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.

  11. Uptake of donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light by recipient dendritic cells induces CD4+CD25+Foxp3+ regulatory T cells and down-regulates cardiac allograft rejection

    International Nuclear Information System (INIS)

    Zheng, De-Hua; Dou, Li-Ping; Wei, Yu-Xiang; Du, Guo-Sheng; Zou, Yi-Ping; Song, Ji-Yong; Zhu, Zhi-Dong; Cai, Ming; Qian, Ye-Yong; Shi, Bing-Yi

    2010-01-01

    Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy and has been demonstrated to be beneficial for graft-vs-host disease and solid-organ allograft rejection. ECP involves reinfusion of a patient's autologous peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen and UVA light radiation (PUVA). Previous studies focused only on ECP treatment of recipient immune cells. Our study is the first to extend the target of ECP treatment to donor immune cells. The results of in vitro co-culture experiments demonstrate uptake of donor PUVA-treated splenic lymphocytes (PUVA-SPs) by recipient immature dendritic cells (DCs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient DCs. In the same co-culture system, donor PUVA-SPs enhanced production of interleukin-10 and interferon-γ by recipient DCs and impaired the subsequent capability of recipient DCs to stimulate recipient naive T cells. Phagocytosis of donor PUVA-SP (PUVA-SP DCs) by recipient DCs shifted T-cell responses in favor of T helper 2 cells. Infusion of PUVA-SP DCs inhibited cardiac allograft rejection in an antigen-specific manner and induced CD4 + CD25 high Foxp3 + regulatory T cells. In conclusion, PUVA-SP DCs simultaneously deliver the donor antigen and the regulatory signal to the transplant recipient, and thus can be used to develop a novel DC vaccine for negative immune regulation and immune tolerance induction.

  12. Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells.

    Science.gov (United States)

    Jin, Takashi; Tiwari, Dhermendra K; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M

    2010-11-01

    To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells.

  13. Serological analysis of human IgG and IgE anti-insulin antibodies by solid-phase radioimmunoassays

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, R.G.; Rendell, M.; Adkinson, N.F. Jr.

    1980-12-01

    A single solid-phase assay system which is useful for quantitative measurement of both IgG and IgE anti-insulin antibodies in human serum has been developed. Insulin-specific immunoglobulins are absorbed from human serum by excess quantities of insulin-agarose. After washes to remove unbound immunoglobulins, radioiodinated Staph A or rabbit anti-human IgE is added to detect bound IgG or IgE anbitodies, respectively.

  14. The anti-human trafficking collaboration model and serving victims: Providers' perspectives on the impact and experience.

    Science.gov (United States)

    Kim, Hea-Won; Park, Taekyung; Quiring, Stephanie; Barrett, Diana

    2018-01-01

    A coalition model is often used to serve victims of human trafficking but little is known about whether the model is adequately meeting the needs of the victims. The purpose of this study was to examine anti-human trafficking collaboration model in terms of its impact and the collaborative experience, including challenges and lessons learned from the service providers' perspective. Mixed methods study was conducted to evaluate the impact of a citywide anti-trafficking coalition model from the providers' perspectives. Web-based survey was administered with service providers (n = 32) and focus groups were conducted with Core Group members (n = 10). Providers reported the coalition model has made important impacts in the community by increasing coordination among the key agencies, law enforcement, and service providers and improving quality of service provision. Providers identified the improved and expanded partnerships among coalition members as the key contributing factor to the success of the coalition model. Several key strategies were suggested to improve the coalition model: improved referral tracking, key partner and protocol development, and information sharing.

  15. Hydrogen-Rich Saline Ameliorates Allergic Rhinitis by Reversing the Imbalance of Th1/Th2 and Up-Regulation of CD4+CD25+Foxp3+Regulatory T Cells, Interleukin-10, and Membrane-Bound Transforming Growth Factor-β in Guinea Pigs.

    Science.gov (United States)

    Xu, Feifei; Yu, Shaoqing; Qin, Mali; Mao, Yong; Jin, Ling; Che, Na; Liu, Shuangxi; Ge, Rongming

    2018-02-01

    It is well known that CD4+CD25+Foxp3+Treg cells play an important role in the development of allergic rhinitis (AR); the defect of cell numbers and functions contribute to AR. Hydrogen has been proven effective in alleviating symptoms of AR. We herein aim to verify the protective effects of hydrogen on CD4+CD25+Foxp3+Treg cells in guinea pigs with AR and to explore the effect of hydrogen-rich saline (HRS) on CD4+CD25+Foxp3+Treg cells in animals with AR and investigate the underlying anti-inflammatory mechanism. Eighteen guinea pigs were randomly divided into three groups (control group/AR group/AR-HRS group). The guinea pigs were injected with hydrogen-rich saline (AR-HRS group) for 10 days after sensitization. The control group was injected with an equal volume of normal saline. The number of sneezes, degree of runny nose, and nasal-rubbing movements were scored. Peripheral blood eosinophil count was recorded. The proportions of Th1/Th2 of the peripheral blood and the CD4+CD25+Foxp3+T cells in the CD4+T cells of the spleen and peripheral blood were determined by flow cytometry. The content of interleukin (IL)-10 and transforming growth factor (TGF)-β in the serum was detected by enzyme-linked immunosorbent assay (ELISA). The protein and mRNA expression of Foxp3, IL-10, and TGF-β were determined by Western blot, immunofluorescence, and real-time PCR analysis, respectively. Scores of symptoms, number of eosinophils,and nasal mucosa damage were dramatically reduced after HRS treatment. HRS increased the expression of Foxp3, IL-10, TGF-β, and number of CD4+CD25+Foxp3+Treg cells, which were reduced in AR. HRS also revised the dysregulation of Th1/Th2 balance. Both the number and biological activity of CD4+CD25+Foxp3+Treg cells increased with up-regulation of Th1/Th2 after HRS administration. HRS could play a protective role in attenuating AR through improving the proportion and functions of CD4+CD25+Foxp3+Treg cells.

  16. Detection of serum anti-sperm antibody in infertile couples with dot-immunogold filtration assay (DIGFA)

    International Nuclear Information System (INIS)

    Xie Xiaoxian

    2005-01-01

    Objective: To develop a new method for rapid detection of serum anti-sperm antibody in infertile couples. Methods: Human sperm antigen was prepared from pooled semen specimens of fertile males. Nitro-cellulose membrane was used as solid-phase carrier of the antigen. Colloidal gold pellet combined goat anti-human IgG was taken as labelled antibody. A dot-immunogold filtration assay system was established for test of serum anti-human sperm antibody. Serum specimens from 137 infertile couples were tested and the result compared with flat from ELISA. Results: The human sperm antigen would react with the anti-sperm antibody in the tested serum over the cellulose membrane through filtration and the result could be read with naked eye within 6 minutes. In this study of 137 infertile coupled, the anti-sperm antibody was positive in 21.9% of the female serum specimens and 13.19% of the males. Compared with the result from ELISA, the consistency rate was 96.1%. The sensitivity of the assay was 90.2% and specificity was 95.4%. The p reparation was stable after 6 months refrigerator storage. Conclusion: This newly developed DIGFA is very adequate for rap id detection of anti-sperm antibody and deserves popularization. (authors)

  17. B and T Cell Phenotypic Profiles of African HIV-Infected and HIV-Exposed Uninfected Infants: Associations with Antibody Responses to the Pentavalent Rotavirus Vaccine

    Directory of Open Access Journals (Sweden)

    Adriana Weinberg

    2018-01-01

    Full Text Available We examined associations between B and T cell phenotypic profiles and antibody responses to the pentavalent rotavirus vaccine (RV5 in perinatally HIV-infected (PHIV infants on antiretroviral therapy and in HIV-exposed uninfected (PHEU infants enrolled in International Maternal Pediatric Adolescent AIDS Clinical Trials P1072 study (NCT00880698. Of 17 B and T cell subsets analyzed, PHIV and PHEU differed only in the number of CD4+ T cells and frequency of naive B cells, which were higher in PHEU than in PHIV. In contrast, the B and T cell phenotypic profiles of PHIV and PHEU markedly differed from those of geographically matched contemporary HIV-unexposed infants. The frequency of regulatory T and B cells (Treg, Breg of PHIV and PHEU displayed two patterns of associations: FOXP3+ CD25+ Treg positively correlated with CD4+ T cell numbers; while TGFβ+ Treg and IL10+ Treg and Breg positively correlated with the frequencies of inflammatory and activated T cells. Moreover, the frequencies of activated and inflammatory T cells of PHIV and PHEU positively correlated with the frequency of immature B cells. Correlations were not affected by HIV status and persisted over time. PHIV and PHEU antibody responses to RV5 positively correlated with CD4+ T cell counts and negatively with the proportion of immature B cells, similarly to what has been previously described in chronic HIV infection. Unique to PHIV and PHEU, anti-RV5 antibodies positively correlated with CD4+/CD8+FOXP3+CD25+% and negatively with CD4+IL10+% Tregs. In conclusion, PHEU shared with PHIV abnormal B and T cell phenotypic profiles. PHIV and PHEU antibody responses to RV5 were modulated by typical HIV-associated immune response modifiers except for the association between CD4+/CD8+FOXP3+CD25+Treg and increased antibody production.

  18. Characterization of antibodies against ferret immunoglobulins, cytokines and CD markers

    DEFF Research Database (Denmark)

    Martel, Cyril Jean-Marie; Aasted, Bent

    2009-01-01

    Ferret IgG and IgM were purified from normal serum, while ferret IgA was purified from bile. The estimated molecular weights of the immunoglobulin gamma, alpha and mu heavy chains were found to be 54 kDa, 69 kDa and 83 kDa, respectively. For immunological (ELISA) quantification of ferret...... immunoglobulins, we identified and characterized polyclonal antibodies towards ferret IgG, IgM and IgA. We also identified 22 monoclonal antibodies (mAbs) raised mostly against human CD markers which cross-reacted with ferret leukocytes. These antibodies were originally specific against human CD8, CD9, CD14, CD18......, CD25, CD29, CD32, CD44, CD61, CD71, CD79b, CD88, CD104, CD172a and mink CD3. Finally, we identified 4 cross-reacting mAbs with specificities against ferret interferon-gamma, TNF-alpha, interleukin-4 and interleukin-8....

  19. Severe neonatal neutropenia due to anti-human leucocyte antigen B49 alloimmunization only: a case report.

    Science.gov (United States)

    Tomicić, M; Starcević, M; Bux, J; Zach, V; Hundrić-Haspl, Z; Drazić, V; Grahovac, B

    2003-08-01

    Alloimmune neonatal neutropenia (ANN) is a rare but potentially life-threatening disorder of neonates. Demonstration of alloantibodies against granulocyte-specific antigens shared by neonatal and paternal granulocytes in the maternal serum is essential in the diagnosis of ANN. In contrast to granulocyte-specific alloantibodies, the significance of human leucocyte antigen (HLA) class I antibodies for ANN is still a matter of debate. We report on a case of severe isolated and prolonged neutropenia due to anti-HLA B49 alloimmunization only. Immediately after birth, severe, isolated neutropenia was observed and lasted for up to 2 months. Results of serologic testing showed only anti-HLA B49 antibodies in the maternal and neonate's sera. HLA typing showed HLA class I (B49) incompatibility between the mother and the child. Granulocyte-specific antibodies were not detected. Adsorption of the maternal serum with HLA B49-bearing platelets removed serum reactivity with paternal neutrophils. Our results support the idea that certain HLA class I antibodies can induce ANN.

  20. Neutrophil attractant protein-1-immunoglobulin G immune complexes and free anti-NAP-1 antibody in normal human serum.

    OpenAIRE

    Sylvester, I; Yoshimura, T; Sticherling, M; Schröder, J M; Ceska, M; Peichl, P; Leonard, E J

    1992-01-01

    After obtaining data indicating the presence of a neutrophil attractant protein-1 (NAP-1)-IgG complex in normal human serum, we developed sandwich ELISAs that could quantify NAP-1 and NAP-1-IgG in mixtures of the two moieties. The ELISA for free NAP-1 used a monoclonal capture antibody that did not bind NAP-1-IgG. The ELISA for NAP-1-IgG was based on omission of the anti-NAP-1 detection antibody (required for the free NAP-1 ELISA) and on interaction of phosphatase-conjugated anti-human IgG wi...

  1. A radioimmunoassay for human antibody specific for microbial antigens

    International Nuclear Information System (INIS)

    Tew, J.G.; Burmeister, J.; Greene, E.J.; Pflaumer, S.K.; Goldstein, J.

    1977-01-01

    A simple and sensitive method for detecting and quantitating antibody specific or microbial antigens is described. Bacterial, fungal, parasitic or viral antigens attached to bromoacetyl cellulose or the intact cells themselves were added to a series of two-fold dilutions of human serum. After a short incubation period, which allowed human antibody to attach to the antigens, the complex was thoroughly washed and carbon-14 labeled anti-human light chain antibody was added to each dilution. The resulting complex was washed, collected on a filter pad, placed in a scintillation vial and radioassayed. The relationship between radioactivity bound and -log 2 of the serum dilution was linear. The endpoint for each assay and a confidence interval was calculated by doing inverse prediction from simple linear regression. Results obtained using this assay indicated the presence of antibody in a pool of normal human sera specific for herpes virus and for both cell surface and intracellular antigens of Streptococcus mutans, Naegleria fowleri and Cryptococcus neoformans. In general the dominant response was against the intracellular antigens rather than cell surface antigens

  2. Assessment of Spanish Panel Reactive Antibody Calculator and Potential Usefulness

    Directory of Open Access Journals (Sweden)

    Esther Asensio

    2017-05-01

    Full Text Available Background and objectivesThe calculated panel reactive of antibodies (cPRAs necessary for kidney donor-pair exchange and highly sensitized programs are estimated using different panel reactive antibody (PRA calculators based on big enough samples in Eurotransplant (EUTR, United Network for Organ Sharing (UNOS, and Canadian Transplant Registry (CTR websites. However, those calculators can vary depending on the ethnic they are applied. Here, we develop a PRA calculator used in the Spanish Program of Transplant Access for Highly Sensitized patients (PATHI and validate it with EUTR, UNOS, and CTR calculators.MethodsThe anti-human leukocyte antigen (HLA antibody profile of 42 sensitized patients on waiting list was defined, and cPRA was calculated with different PRA calculators.ResultsDespite different allelic frequencies derived from population differences in donor panel from each calculator, no differences in cPRA between the four calculators were observed. The PATHI calculator includes anti-DQA1 antibody profiles in cPRA calculation; however, no improvement in total cPRA calculation of highly sensitized patients was demonstrated.Interpretation and conclusionThe PATHI calculator provides cPRA results comparable with those from EUTR, UNOS, and CTR calculators and serves as a tool to develop valid calculators in geographical and ethnic areas different from Europe, USA, and Canada.

  3. A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

    Directory of Open Access Journals (Sweden)

    Leili Aghebati Maleki

    2013-08-01

    Full Text Available Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG. Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases.

  4. Human immune response to anti-carcinoembryonic antigen murine monoclonal antibodies.

    Science.gov (United States)

    Losman, M J; DeJager, R L; Monestier, M; Sharkey, R M; Goldenberg, D M

    1990-02-01

    We previously demonstrated that patients with carcinoembryonic antigen [CEA]-producing neoplastic tumors, treated with murine monoclonal antibody to CEA, produced antibodies directed against the constant regions [human anti-mouse antibody (HAMA)] and the idiotypes [anti-Id] of these murine immunoglobulins. In this study, we describe a method for analyzing the presence of such antibodies in the sera of these patients. The HAMAs were measured by enzyme immunoassay and removed by immunoadsorption on Affi-Gel mouse IgG. The unabsorbed fraction contained the anti-Id antibodies; their presence was demonstrated by binding to the CEA monoclonal antibody (Ab1). The specificity of the binding was assessed by preincubating the sera with Ab1 and measuring the residual nonspecific binding. When specific binding was detected, the anti-Id antibodies were isolated by adsorption and elution on Affi-Gel Ab1. The anti-Id antibodies were fixed on enzyme immunoassay plates and incubated with a panel of mouse anti-human immunoglobulin to determine their isotypes. In a first series of 24 patients, HAMAs were found in 20 cases and anti-Id antibodies in 19 cases. The isolation of a specific IgG to Ab1 was achieved in 2 cases. In an ongoing series, the HAMA and anti-Id antibodies were detected in all five patients given injections of another monoclonal antibody to CEA. In two patients an IgG1 kappa anti-Id was isolated from the serum. The potential therapeutic effect of these antibodies is under investigation.

  5. The effect of high antigen density on solid-phase radioimmunoassays for antibody regardless of immunoglobulin class

    International Nuclear Information System (INIS)

    Rubin, R.L.; Hardtke, M.A.; Carr, R.I.

    1980-01-01

    Human sera containing antibody to casein or to bovine serum albumin were used to assess the validity and utility of a solid-phase assay for quantitating antibody activity. Rabbit anti-human immunoglobulin radiolabeled with 125 I and capable of reacting with all human immunoglobulin classes was used to detect antibody bound to antigen immobilized to polystyrene tubes by a new covalent technique. This method results in very high antigen concentrations in highly stable association with polystyrene tubes. Kinetic and absorption studies demonstrated that low avidity antibodies are better detected when antigen is immobilized by the covalent method than when passively adsorbed. Conditions are described for minimizing artifactual interactions and for obtaining results similar to those obtained with conventional, liquid-phase assays. Failure to reach equilibrium in solid-phase assays and other problems are proposed to explain, in part, the inability to obtain a better correlation between solid- and liquid-phase immunoassays. (Auth.)

  6. Adoptive transfer of pregnancy-induced CD4+CD25+ regulatory T cells reverses the increase in abortion rate caused by interleukin 17 in the CBA/JxBALB/c mouse model.

    Science.gov (United States)

    Wang, Wen-Juan; Liu, Fu-Jun; Xin-Liu; Hao, Cui-Fang; Bao, Hong-Chu; Qu, Qing-Lan; Liu, Xue-Mei

    2014-05-01

    Could adoptive transfer of pregnancy-induced CD4+CD25+ regulatory T cells (Tregs) reverse the increase in abortion rate caused by interleukin 17 (IL-17) in the CBA/J × BALB/c mouse model? The effects of exogenous IL-17 on increased abortion rate, as well as decreased transforming growth factor (TGF)-β and IL-10 expression, are reversed by a pre-mating transfusion of Tregs in a mouse model of pregnancy. IL-17 is a pro-inflammatory cytokine mainly expressed by T helper 17 cells, and plays a pivotal role in the pathogenesis of endometriosis, miscarriage, preterm labor and pre-eclampsia. The activity of Th17 cells is attenuated by the anti-inflammatory action of Tregs. Fifty microliters of phosphate-buffered saline (PBS) (Group 1,) or recombinant IL-17 (rIL) (10 µg/mouse) supernatant (Group 2) was administered in the vaginal vaults of anesthetized pregnant CBA/J mice on Day 1 of pregnancy. Tregs (2 × 10(5) cells) purified from pregnant CBA/J × BALB/c mice were given i.v. via the tail vein 2 days before mating (Group 3) or on Day 7 of pregnancy (Group 4). Mice (n = 40) were randomly assigned to one of four experimental groups. The numbers of surviving and reabsorbed fetuses in each group were counted on Day 14 of pregnancy, and the expression of interferon (IFN)-γ, IL-4, TGF-β and IL-10 in the decidual tissue was assessed by real-time RT-PCR and western blotting. Normal pregnant CBA/J mice mated with BALB/c males which received transvaginal rIL-17 presented with a significantly increased abortion rate compared with the group which received PBS (27.7 versus 9.9%, respectively; P abortion rate caused by IL-17 (12.5 versus 27.7%, respectively; P effect. Transfusion of Tregs did not affect IFN-γ or IL-4 expression in the decidual tissue at either the mRNA or protein level. Administration of rIL-17 resulted in a decrease in production of TGF-β and IL-10 at both mRNA and protein levels (P effect on TGF-β or IL-10 expression. These data derive from only a small

  7. Acetylcholine receptor antibody

    Science.gov (United States)

    ... this page: //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 ...

  8. Platelet antibodies blood test

    Science.gov (United States)

    This blood test shows if you have antibodies against platelets in your blood. Platelets are a part of the blood ... Chernecky CC, Berger BJ. Platelet antibody - blood. In: Chernecky ... caused by platelet destruction, hypersplenism, or hemodilution. ...

  9. Optimization of ELISA conditions to quantify colorectal cancer antigen-antibody complex protein (GA733-FcK) expressed in transgenic plant.

    Science.gov (United States)

    Ahn, Junsik; Lee, Kyung Jin; Ko, Kisung

    2014-02-01

    The purpose of this study is to optimize ELISA conditions to quantify the colorectal cancer antigen GA733 linked to the Fc antibody fragment fused to KDEL, an ER retention motif (GA733-FcK) expressed in transgenic plant. Variable conditions of capture antibody, blocking buffer, and detection antibody for ELISA were optimized with application of leaf extracts from transgenic plant expressing GA733-FcK. In detection antibody, anti-EpCAM/CD362 IgG recognizing the GA733 did not detect any GA733-FcK whereas anti-human Fc IgG recognizing the human Fc existed in plant leaf extracts. For blocking buffer conditions, 3% BSA buffer clearly blocked the plate, compared to the 5% skim-milk buffer. For capture antibody, monoclonal antibody (MAb) CO17-1A was applied to coat the plate with different amounts (1, 0.5, and 0.25 μg/well). Among the amounts of the capture antibody, 1 and 0.5 μg/well (capture antibody) showed similar absorbance, whereas 0.25 μg/well of the capture antibody showed significantly less absorbance. Taken together, the optimized conditions to quantify plant-derived GA733-FcK were 0.5 μg/well of MAb CO17-1A per well for the capture antibody, 3% BSA for blocking buffer, and anti-human Fc conjugated HRP. To confirm the optimized ELISA conditions, correlation analysis was conducted between the quantified amount of GA733-FcK in ELISA and its protein density values of different leaf samples in Western blot. The co-efficient value R(2) between the ELISA quantified value and protein density was 0.85 (pFcK expression in transgenic plant.

  10. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    Haisma, H.J.

    1987-01-01

    The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111 In, 67 Ga and 131 I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

  11. Generic anti-drug antibody assay with drug tolerance in serum samples from mice exposed to human antibodies.

    Science.gov (United States)

    Stubenrauch, Kay; Mackeben, Klaus; Vogel, Rudolf; Heinrich, Julia

    2012-11-15

    Knowledge of the anti-drug antibody (ADA) status is necessary in early research studies. Because specific assay materials are sparse and time is pressing, a generic assay format with drug tolerance for detection of ADAs in serum samples from mice exposed to immunoglobulin G (IgG) or antigen-binding fragments (Fabs) is highly desirable. This article describes a generic immune complex assay in the sandwich enzyme-linked immunosorbent assay (ELISA) format based on (i) transformation of free ADAs to immune complexes by preincubation with excess drug, (ii) the use of a murine anti-human Fab constant domain Fab as capture reagent, (iii) detection of the immune complexes by a peroxidase-labeled rabbit anti-murine Fc antibody, and (iv) ADA-positive control conjugates consisting of human Fab and murine IgG. Results of the experiments suggest that the generic immune complex assay for mouse serum samples was at least equivalent to specific ADA immune assays and even superior regarding drug tolerance. The generic immune complex assay confers versatility as it detects ADAs in complex with full-length IgG as well as with Fabs independent of the target specificity in mouse serum samples. These features help to save the sparse amounts of specific antibodies available in early research and development and speed up drug candidate selection. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Radiolabeled antibody imaging

    International Nuclear Information System (INIS)

    Wahl, R.L.

    1987-01-01

    Radiolabeled antibodies, in particular monoclonal antibodies, offer the potential for the specific nuclear imaging of malignant and benign diseases in man. If this imaging potential is realized, they may also have a large role in cancer treatment. This paper reviews: (1) what monoclonal antibodies are and how they differ from polyclonal antibodies, (2) how they are produced and radiolabeled, (3) the results of preclinical and clinical trials in cancer imaging, including the utility of SPECT and antibody fragments, (4) the role of antibodies in the diagnosis of benign diseases, (5) alternate routes of antibody delivery, (6) the role of these agents in therapy, and (7) whether this technology ''revolutionizes'' the practice of nuclear radiology, or has a more limited complementary role in the imaging department

  13. Anti-Human Platelet Antigen-1a Immunoglobulin G Preparation Intended to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia.

    Directory of Open Access Journals (Sweden)

    Ying-Jan Weng

    Full Text Available Fetal and neonatal alloimmune thrombocytopenia (FNAIT is a severe disease that is caused by maternal alloantibodies generated during pregnancy or at delivery as a result of incompatibility between maternal and fetal human platelet antigens (HPAs inherited from the father. Antibody-mediated immune suppression using anti-HPA-1a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1a immunoglobulin (Ig G (IgG from human plasma was therefore developed. Anti-HPA-1a plasma was obtained from volunteer mothers who underwent alloimmunization against HPA-1a during a previous pregnancy. Plasma was cryoprecipitated and the supernatant treated with caprylic acid and solvent/detergent (S/D, purified by chromatography, nanofiltered, concentrated, and sterile-filtered. The anti-HPA-1a immunoglobulin fraction was characterized for purity and safety. PAK12 and quantitative monoclonal antibody immobilization of platelet antigen (MAIPA assays were used to detect anti-HPA-1a IgG. Hepatitis C virus (HCV removal during nanofiltration was assessed by spiking experiments, using cell culture-derived reporter HCV and luciferase analysis. The caprylic acid treatment precipitated non-Ig proteins yielding a 90% pure Ig supernatant. S-HyperCel chromatography of the S/D-treated supernatant followed by HyperCel STAR AX provided high IgG recovery (>80% and purity (>99.5%, and efficient IgA and IgM removal. Concentrations of complement factors C3 and C4 were < 0.5 and < 0.4 mg/dL, respectively. The final IgG could be nanofiltered on Planova 20N under conditions removing more than 3 log HCV infectivity to baseline mock infection level, and concentrated to ca. 30 g/L. Proteolytic activity and thrombin generation were low in the final fraction. The Pak12 and MAIPA assays showed good recovery of anti-HPA-1a throughout the process. Clinical-grade HPA-1a IgG can be prepared using a process compliant with current quality

  14. Anti-Human Platelet Antigen-1a Immunoglobulin G Preparation Intended to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia.

    Science.gov (United States)

    Weng, Ying-Jan; Husebekk, Anne; Skogen, Björn; Kjaer, Mette; Lin, Liang-Tzung; Burnouf, Thierry

    2016-01-01

    Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe disease that is caused by maternal alloantibodies generated during pregnancy or at delivery as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father. Antibody-mediated immune suppression using anti-HPA-1a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1a plasma was obtained from volunteer mothers who underwent alloimmunization against HPA-1a during a previous pregnancy. Plasma was cryoprecipitated and the supernatant treated with caprylic acid and solvent/detergent (S/D), purified by chromatography, nanofiltered, concentrated, and sterile-filtered. The anti-HPA-1a immunoglobulin fraction was characterized for purity and safety. PAK12 and quantitative monoclonal antibody immobilization of platelet antigen (MAIPA) assays were used to detect anti-HPA-1a IgG. Hepatitis C virus (HCV) removal during nanofiltration was assessed by spiking experiments, using cell culture-derived reporter HCV and luciferase analysis. The caprylic acid treatment precipitated non-Ig proteins yielding a 90% pure Ig supernatant. S-HyperCel chromatography of the S/D-treated supernatant followed by HyperCel STAR AX provided high IgG recovery (>80%) and purity (>99.5%), and efficient IgA and IgM removal. Concentrations of complement factors C3 and C4 were < 0.5 and < 0.4 mg/dL, respectively. The final IgG could be nanofiltered on Planova 20N under conditions removing more than 3 log HCV infectivity to baseline mock infection level, and concentrated to ca. 30 g/L. Proteolytic activity and thrombin generation were low in the final fraction. The Pak12 and MAIPA assays showed good recovery of anti-HPA-1a throughout the process. Clinical-grade HPA-1a IgG can be prepared using a process compliant with current quality requirements

  15. The frequency of CD127low expressing CD4+CD25high T regulatory cells is inversely correlated with human T lymphotrophic virus type-1 (HTLV-1 proviral load in HTLV-1-infection and HTLV-1-associated myelopathy/tropical spastic paraparesis

    Directory of Open Access Journals (Sweden)

    Chieia Marco

    2008-07-01

    Full Text Available Abstract Background CD4+CD25high regulatory T (TReg cells modulate antigen-specific T cell responses, and can suppress anti-viral immunity. In HTLV-1 infection, a selective decrease in the function of TReg cell mediated HTLV-1-tax inhibition of FOXP3 expression has been described. The purpose of this study was to assess the frequency and phenotype of TReg cells in HTLV-1 asymptomatic carriers and in HTLV-1-associated neurological disease (HAM/TSP patients, and to correlate with measures of T cell activation. Results We were able to confirm that HTLV-I drives activation, spontaneous IFNγ production, and proliferation of CD4+ T cells. We also observed a significantly lower proportion of CTLA-4+ TReg cells (CD4+CD25high T cells in subjects with HAM/TSP patients compared to healthy controls. Ki-67 expression was negatively correlated to the frequency of CTLA-4+ TReg cells in HAM/TSP only, although Ki-67 expression was inversely correlated with the percentage of CD127low TReg cells in healthy control subjects. Finally, the proportion of CD127low TReg cells correlated inversely with HTLV-1 proviral load. Conclusion Taken together, the results suggest that TReg cells may be subverted in HAM/TSP patients, which could explain the marked cellular activation, spontaneous cytokine production, and proliferation of CD4+ T cells, in particular those expressing the CD25highCD127low phenotype. TReg cells represent a potential target for therapeutic intervention for patients with HTLV-1-related neurological diseases.

  16. Human monoclonal antibody 99mTc-88BV59: detection of colorectal cancer, recurrent or metastatic disease and immunogenicity assessment.

    Science.gov (United States)

    Krause, B J; Baum, R P; Staib-Sebler, E; Lorenz, M; Niesen, A; Hör, G

    1997-01-01

    This study presents immunoscintigraphic results in 24 patients suffering from primary colorectal cancer, recurrent or metastatic disease after the injection of 1197-1351 MBq technetium-99m labelled totally human monoclonal antibody 88BV59. Labelling efficacy of 99mTc-88BV59 ranged from 97% to 99%. Immunoscintigraphy was performed 18-20 h after injection. Scintigraphic findings were compared with those of computed tomography (CT). Patients underwent surgery in order to evaluate immunoscintigraphic findings histologically. Sera of the patients (before injection and 1 and 3 months post infusion) were analysed for the presence of human anti-human antibodies (HAHA). None of the patients showed a HAHA response as assessed by a solid-phase ELISA assay. The antibody scan detected about 25% more lesions than CT. In the detection of extrahepatic disease, the sensitivity of the antibody scan proved to be 68%, whereas the sensitivity of CT was 41%.

  17. Human monoclonal antibody {sup 99m}Tc-88BV59: detection of colorectal cancer, recurrent or metastatic disease and immunogenicity assessment

    Energy Technology Data Exchange (ETDEWEB)

    Krause, B.J. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Baum, R.P. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Staib-Sebler, E. [Department of General and Abdominal Surgery, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Lorenz, M. [Department of General and Abdominal Surgery, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Niesen, A. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Hoer, G. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany)

    1997-01-01

    This study presents immunoscintigraphic results in 24 patients suffering from primary colorectal cancer, recurrent or metastatic disease after the injection of 1197-1351 MBq technetium-99m labelled totally human monoclonal antibody 88BV59. Labelling efficacy of {sup 99m}Tc-88BV59 ranged from 97% to 99%. Immunoscintigraphy was performed 18-20 h after injection. Scintigraphic findings were compared with those of computed tomography (CT). Patients underwent surgery in order to evaluate immunoscintigraphic findings histologically. Sera of the patients (before injection and 1 and 3 months post infusion) were analysed for the presence of human anti-human antibodies (HAHA). None of the patients showed a HAHA response as assessed by a solid-phase ELISA assay. The antibody scan detected about 25% more lesions than CT. In the detection of extrahepatic disease, the sensitivity of the antibody scan proved to be 68%, whereas the sensitivity of CT was 41%. (orig.). With 3 figs., 1 tab.

  18. Human monoclonal antibody 99mTc-88BV59: detection of colorectal cancer, recurrent or metastatic disease and immunogenicity assessment

    International Nuclear Information System (INIS)

    Krause, B.J.; Baum, R.P.; Staib-Sebler, E.; Lorenz, M.; Niesen, A.; Hoer, G.

    1997-01-01

    This study presents immunoscintigraphic results in 24 patients suffering from primary colorectal cancer, recurrent or metastatic disease after the injection of 1197-1351 MBq technetium-99m labelled totally human monoclonal antibody 88BV59. Labelling efficacy of 99m Tc-88BV59 ranged from 97% to 99%. Immunoscintigraphy was performed 18-20 h after injection. Scintigraphic findings were compared with those of computed tomography (CT). Patients underwent surgery in order to evaluate immunoscintigraphic findings histologically. Sera of the patients (before injection and 1 and 3 months post infusion) were analysed for the presence of human anti-human antibodies (HAHA). None of the patients showed a HAHA response as assessed by a solid-phase ELISA assay. The antibody scan detected about 25% more lesions than CT. In the detection of extrahepatic disease, the sensitivity of the antibody scan proved to be 68%, whereas the sensitivity of CT was 41%. (orig.). With 3 figs., 1 tab

  19. 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.

  20. Antibodies Against Melanin

    African Journals Online (AJOL)

    1973-01-06

    Jan 6, 1973 ... Departments of Internal Medicine and Anatomical Pathology, University of Stellenbosch and MRC. Pigment Metabolism Research Unit, ... at the production of antibodies against natural melanoprotein. and a consideration of our negative .... the random polymerization of several monomers, antibody formed ...

  1. Recombinant renewable polyclonal antibodies.

    Science.gov (United States)

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  2. Correlation between levels of human papillomavirus (HPV)-16 and 18 antibodies in serum and cervicovaginal secretions in girls and women vaccinated with the HPV-16/18 AS04-adjuvanted vaccine

    DEFF Research Database (Denmark)

    Schwarz, Tino F; Kocken, Mariëlle; Petäjä, Tiina

    2010-01-01

    This pooled analysis of data from four Phase III clinical trials was undertaken to assess the correlation between levels of anti-human papillomavirus (HPV)-16/18 antibodies in serum and cervicovaginal secretions (CVS) in girls and women vaccinated with the HPV-16/18 AS04-adjuvanted vaccine. Serum...... and CVS samples were collected from a subset of women aged 10-65 years (N=350) at pre-specified time-points from 7 to 36 months post-vaccination. Anti-HPV-16/18 antibody levels in serum and CVS were measured by enzyme-linked immunosorbent assay. Pearson correlation coefficients between serum and CVS...... antibody levels, standardized for total immunoglobulin G, were calculated at each time-point in women with detectable antibodies in both serum and CVS. All subjects had seroconverted at Month 7 and remained seropositive through Month 36 for both antigens. Geometric mean titers of anti-HPV-16/18 antibodies...

  3. Antibody engineering: methods and protocols

    National Research Council Canada - National Science Library

    Chames, Patrick

    2012-01-01

    "Antibody Engineering: Methods and Protocols, Second Edition was compiled to give complete and easy access to a variety of antibody engineering techniques, starting from the creation of antibody repertoires and efficient...

  4. Anti-insulin antibody test

    Science.gov (United States)

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... You appear to have an allergic response to insulin Insulin no longer seems to control your diabetes

  5. Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma

    Science.gov (United States)

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Krejci, Eric; Blake, Thomas A.; Johnson, Rudolph C.; Masson, Patrick; Lockridge, Oksana

    2016-01-01

    Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immunoextract BChE from the plasma of humans, monkey and other animals. PMID:26585590

  6. Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia

    Science.gov (United States)

    Shingai, Masashi; Nishimura, Yoshiaki; Klein, Florian; Mouquet, Hugo; Donau, Olivia K.; Plishka, Ronald; Buckler-White, Alicia; Seaman, Michael; Piatak, Michael; Lifson, Jeffrey D.; Dimitrov, Dimiter; Nussenzweig, Michel C.; Martin, Malcolm A.

    2013-11-01

    Neutralizing antibodies can confer immunity to primate lentiviruses by blocking infection in macaque models of AIDS. However, earlier studies of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies administered to infected individuals or humanized mice reported poor control of virus replication and the rapid emergence of resistant variants. A new generation of anti-HIV-1 monoclonal antibodies, possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals. These neutralizing antibodies target different regions of the HIV-1 envelope glycoprotein including the CD4-binding site, glycans located in the V1/V2, V3 and V4 regions, and the membrane proximal external region of gp41 (refs 9, 10, 11, 12, 13, 14). Here we have examined two of the new antibodies, directed to the CD4-binding site and the V3 region (3BNC117 and 10-1074, respectively), for their ability to block infection and suppress viraemia in macaques infected with the R5 tropic simian-human immunodeficiency virus (SHIV)-AD8, which emulates many of the pathogenic and immunogenic properties of HIV-1 during infections of rhesus macaques. Either antibody alone can potently block virus acquisition. When administered individually to recently infected macaques, the 10-1074 antibody caused a rapid decline in virus load to undetectable levels for 4-7days, followed by virus rebound during which neutralization-resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viraemia for 3-5weeks in some long-term chronically SHIV-infected animals with low CD4+ T-cell levels. A second cycle of anti-HIV-1 monoclonal antibody therapy, administered to two previously treated animals, successfully controlled virus rebound. These results indicate that immunotherapy or a combination of immunotherapy plus conventional antiretroviral drugs might be useful as a treatment for chronically HIV-1-infected

  7. Monoclonal antibody "gold rush".

    Science.gov (United States)

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  8. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide......-antibody interface and the antibody intraface.the microenvironment and ecology of Acaryochloris and Prochloron, and in this thesis we attempted to further describe the distribution, growth characteristics and adaptive/regulatory mechanisms of these two cyanobacteria, both in their natural habitat and under defined...

  9. Serum herpes simplex antibodies

    Science.gov (United States)

    ... causes cold sores (oral herpes). HSV-2 causes genital herpes. How the Test is Performed A blood sample ... person has ever been infected with oral or genital herpes . It looks for antibodies to herpes simplex virus ...

  10. Anti-sulfotyrosine antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM

    2009-09-15

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  11. Bifunctional antibodies for radioimmunotherapy.

    Science.gov (United States)

    Chatal, J F; Faivre-Chauvet, A; Bardies, M; Peltier, P; Gautherot, E; Barbet, J

    1995-04-01

    In two-step targeting technique using bifunctional antibodies, a nonradiolabeled immunoconjugate with slow uptake kinetics (several days) is initially injected, followed by a small radiolabeled hapten with fast kinetics (several hours) that binds to the bispecific immunoconjugate already taken up by the tumor target. In patients with colorectal or medullary thyroid cancer, clinical studies performed with an anti-CEA/anti-DTPA-indium bifunctional antibody and an indium-111-labeled di-DTPA-TL bivalent hapten showed that tumor uptake was not modified compared to results for F(ab')2 fragments of the same anti-CEA antibody directly labeled with indium-111, whereas the radioactivity of normal tissues was significantly reduced (3- to 6-fold). The fast tumor uptake kinetics (several hours) and high or very high tumor-to-normal tissue ratios obtained with the bifunctional antibody technique are favorable parameters for efficient radioimmunotherapy.

  12. Antibody Blood Tests

    Science.gov (United States)

    Antibody Blood Tests Researchers have discovered that people with celiac disease who eat gluten have higher than normal levels of ... do I do if I have a negative blood test (or panel) but I’m still having symptoms? ...

  13. Therapeutic Treatment of Arthritic Mice with 15-Deoxy Δ12,14-Prostaglandin J2 (15d-PGJ2 Ameliorates Disease through the Suppression of Th17 Cells and the Induction of CD4+CD25−FOXP3+ Cells

    Directory of Open Access Journals (Sweden)

    Vanessa Carregaro

    2016-01-01

    Full Text Available The prostaglandin, 15-deoxy Δ12,14-prostaglandin J2 (15d-PGJ2, is a lipid mediator that plays an important role in the control of chronic inflammatory disease. However, the role of prostanoid in rheumatoid arthritis (RA is not well determined. We demonstrated the therapeutic effect of 15d-PGJ2 in an experimental model of arthritis. Daily administration of 15d-PGJ2 attenuated the severity of CIA, reducing the clinical score, pain, and edema. 15d-PGJ2 treatment was associated with a marked reduction in joint levels of proinflammatory cytokines. Although the mRNA expression of ROR-γt was profoundly reduced, FOXP3 was enhanced in draining lymph node cells from 15d-PGJ2-treated arthritic mice. The specific and polyclonal CD4+ Th17 cell responses were limited during the addition of prostaglandin to cell culture. Moreover, in vitro 15d-PGJ2 increased the expression of FOXP3, GITR, and CTLA-4 in the CD4+CD25− population, suggesting the induction of Tregs on conventional T cells. Prostanoid addition to CD4+CD25− cells selectively suppressed Th17 differentiation and promoted the enhancement of FOXP3 under polarization conditions. Thus, 15d-PGJ2 ameliorated symptoms of collagen-induced arthritis by regulating Th17 differentiation, concomitant with the induction of Tregs, and, consequently, protected mice from diseases aggravation. Altogether, these results indicate that 15d-PGJ2 may represent a potential therapeutic strategy in RA.

  14. Unraveling the role of maternal anti-HLA class I antibodies in fetal and neonatal thrombocytopenia-Antibody specificity analysis using epitope data.

    Science.gov (United States)

    Dahl, Jesper; Refsum, Erle; Ahlen, Maria Therese; Egeland, Torstein; Jensen, Tore; Viken, Marte K; Stuge, Tor Brynjar; Acharya, Ganesh; Husebekk, Anne; Skogen, Bjørn; Tiller, Heidi

    2017-08-01

    Anti-HLA class I antibodies have been suggested as a possible cause of fetal and neonatal alloimmune thrombocytopenia (FNAIT). The aim of this study was to characterize maternal anti-HLA class I alloantibodies in suspected cases of FNAIT. The study population consisted of all nationwide referrals of neonates with suspected FNAIT to the National Unit for Platelet Immunology in Tromsø, Norway, during 1998-2009 (cases), and 250 unselected pregnancies originally included in a prospective study (controls). Inclusion criterion was a positive screening for maternal anti-HLA class I antibodies. Neonates with other identifiable causes of thrombocytopenia, including maternal anti-human platelet antigens (HPA) antibodies, were excluded. Ultimately, 50 cases with suspected FNAIT were compared with 60 controls. The median neonatal platelet count nadir among cases was 24×10 9 /L (range 4-98×10 9 /L). Five children (10%) were reported to have intracranial hemorrhage. Maternal and neonatal HLA class I genotype was available for 33 mother/child pairs (66%). Immunization was not tied to any particular HLA class I antigen. Using epitope mapping, we could demonstrate that the maternal anti-HLA class I antibodies were specific towards mismatched paternally-inherited fetal epitopes, with little reactivity towards any third-party epitopes. Antibody reactivity patterns were similar to those found among controls, although the mean fluorescence intensities (MFI) among cases were significantly higher. This study demonstrates the value of using data on HLA epitope expression, instead of HLA antigens, to examine alloimmune responses in connection with neonatal thrombocytopenia. Our findings support the idea that maternal anti-HLA class I antibodies are involved in FNAIT. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Anti-CD8 antibodies can trigger CD8+ T-cell effector function in the absence of TCR engagement and improve pMHCI tetramer staining

    Science.gov (United States)

    Clement, Mathew; Ladell, Kristin; Ekeruche-Makinde, Julia; Miles, John J.; Edwards, Emily S. J.; Dolton, Garry; Williams, Tamsin; Schauenburg, Andrea J. A.; Cole, David K.; Lauder, Sarah N.; Gallimore, Awen M.; Godkin, Andrew J.; Burrows, Scott R.; Price, David A.; Sewell, Andrew K.; Wooldridge, Linda

    2011-01-01

    CD8+ T-cells recognize immunogenic peptides presented at the cell surface bound to major histocompatibility complex class I (MHCI) molecules. Antigen recognition involves the binding of both T-cell receptor (TCR) and CD8 co-receptor to the same peptide-MHCI (pMHCI) ligand. Specificity is determined by the TCR, whereas CD8 mediates effects on antigen sensitivity. Anti-CD8 antibodies have been used extensively to examine the role of CD8 in CD8+ T-cell activation. However, as previous studies have yielded conflicting results, it is unclear from the literature whether anti-CD8 antibodies per se are capable of inducing effector function. Here, we report on the ability of seven monoclonal anti-human CD8 antibodies to activate six human CD8+ T-cell clones with a total of five different specificities. Six out of seven anti-human CD8 antibodies tested did not activate CD8+ T-cells. In contrast, one anti-human CD8 antibody, OKT8, induced effector function in all CD8+ T-cells examined. Moreover, OKT8 was found to enhance TCR/pMHCI on-rates and, as a consequence, could be used to improve pMHCI tetramer staining and the visualization of antigen-specific CD8+ T-cells. The anti-mouse CD8 antibodies, CT-CD8a and CT-CD8b, also activated CD8+ T-cells despite opposing effects on pMHCI tetramer staining. The observed heterogeneity in the ability of anti-CD8 antibodies to trigger T-cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of antibody-mediated CD8-engagement to deliver an activation signal underscores the importance of CD8 in CD8+ T-cell signalling. PMID:21677135

  16. Delivering HIV Gagp24 to DCIR Induces Strong Antibody Responses In Vivo.

    Directory of Open Access Journals (Sweden)

    Anne-Laure Flamar

    Full Text Available Targeting dendritic cell-specific endocytic receptors using monoclonal antibodies fused to desired antigens is an approach widely used in vaccine development to enhance the poor immunogenicity of protein-based vaccines and to induce immune responses. Here, we engineered an anti-human DCIR recombinant antibody, which cross-reacts with the homologous cynomolgous macaque receptor and was fused via the heavy chain C-terminus to HIV Gagp24 protein (αDCIR.Gagp24. In vitro, αDCIR.Gagp24 expanded multifunctional antigen-specific memory CD4+ T cells recognizing multiple Gagp24 peptides from HIV-infected patient peripheral blood mononuclear cells. In non human primates, priming with αDCIR.Gagp24 without adjuvant elicited a strong anti-Gagp24 antibody response after the second immunization, while in the non-targeted HIV Gagp24 protein control groups the titers were weak. The presence of the double-stranded RNA poly(I:C adjuvant significantly enhanced the anti-Gagp24 antibody response in all the groups and reduced the discrimination between the different vaccine groups. The avidity of the anti-Gagp24 antibody responses was similar with either αDCIR.Gagp24 or Gagp24 immunization, but increased from medium to high avidity in both groups when poly(I:C was co-administered. This data provides a comparative analysis of DC-targeted and non-targeted proteins for their capacity to induce antigen-specific antibody responses in vivo. This study supports the further development of DCIR-based DC-targeting vaccines for protective durable antibody induction, especially in the absence of adjuvant.

  17. Polyclonal antibodies directed against human placental Fcgamma receptor. Characterization of the antibodies and their interaction with the receptor.

    Science.gov (United States)

    Mikulska, J; Lisowski, J

    1987-01-01

    Antibodies to the putative Fc gamma receptor (Fc gamma R) of human placenta were raised by immunization of rabbits with the receptor purified form syncytiotrophoblast plasma membranes of human placenta. The rabbit antibodies were of IgG class and their F(ab')2 fragment interacted with Fc receptors in solubilized form and membrane-bound, as well. Immunological reactivity of the antibodies with Fc gamma R was demonstrated using immunodiffusion, solid-phase immunoassay, and ELISA. Studies on interaction of the antibodies with the isolated placental Fc gamma R showed that antigenic determinants of the receptor were different from the IgG-binding site. Rabbit anti-human placental Fc gamma R crossreacted, to various extent, with Fc gamma R-positive human cell lines showing antigenic relatedness of the placental receptor with Fc gamma R on other cell types. The antibodies showed only a weak crossreactivity with guinea pig peritoneal macrophage Fc gamma R. SDS-PAGE analysis of immunoprecipitates obtained by treatment of detergent lysates of 3H-labeled human placental trophoblasts membranes with the rabbit antibodies or with human IgG showed the presence of the some components which were observed in the case of the isolated, purified placental Fc gamma R: Mr of 123,000 and 52,000-56,000 under nonreducing conditions, and Mr of 64,000-67,000, 52,000-56,000, and 26,000-29,000, under reducing conditions. The polypeptide chains of the purified human placental receptor resolved in SDS-PAGE and transferred on nitrocellulose strips were able to interact both with the rabbit anti-placental receptor IgG F(ab')2 fragments and with human IgG. This gives an evidence that human placental Fc gamma R polypeptide chains Mr of approx. 64,000, 54,000, and 28,000 contain antigenic determinants of the receptor and binding sites for the Fc region of IgG, as well.

  18. Clinical relevance of HLA donor-specific antibodies detected by single antigen assay in kidney transplantation.

    Science.gov (United States)

    Caro-Oleas, José Luis; González-Escribano, María Francisca; González-Roncero, Francisco Manuel; Acevedo-Calado, María José; Cabello-Chaves, Virginia; Gentil-Govantes, Miguel Ángel; Núñez-Roldán, Antonio

    2012-03-01

    Clinical relevance of donor-specific antibodies (DSAs) detected by a single antigen Luminex virtual crossmatch in pre-transplant serum samples from patients with a negative cytotoxicity-dependent complement crossmatch is controversial. The aim of this study was to analyse the influence of a pre-transplant positive virtual crossmatch in the outcome of kidney transplantation. A total of 892 patients who received a graft from deceased donors after a negative cytotoxicity crossmatch were included. Presence of anti-human leucocyte antigen (HLA) antibodies was investigated using a Luminex screening assay and anti-HLA specificities were assigned performing a Luminex single antigen assay. Graft survival was significantly worse among patients with anti-HLA DSA compared to both patients with anti-HLA with no DSA (P = 0.001) and patients without HLA antibodies (P HLA with no DSA and no HLA antibodies patient groups were observed (P = 0.595). Influence of both anti-Class I and anti-Class II DSA was detected (P 1500 (global P > 0.05). The presence of preformed HLA DSA in transplanted patients with a negative cytotoxicity crossmatch is associated with a lower allograft survival. The detection of anti-HLA with no DSA has no influence in the graft outcome. Finally, there were no demonstrable effects of mean fluorescence intensity (MFI) values >1500 on graft survival.

  19. Quantitation of anti-tetanus and anti-diphtheria antibodies by enzymoimmunoassay: methodology and applications.

    Science.gov (United States)

    Virella, G; Hyman, B

    1991-01-01

    We have developed enzymoimmunoassays (EIA) for the quantitation of antibodies (Ab) to tetanus and diphtheria toxoids (TT, DT) using Immulon I plates coated with the appropriate toxoid. A preparation of human tetanus immunoglobulin with a known concentration of anti-TT Ab was used as calibrator of the anti-TT antibody assay. The assay of anti-DT Ab is calibrated with a pool of human sera whose anti-DT Ab concentration was determined by quantitative immunoelectrophoresis, using a horse anti-DT with known Ab concentration as calibrator. A peroxidase-conjugated anti-human IgG was used in both assays. ABTS was used as substrate, and the reaction was stopped after 1 min incubation with citric acid and the OD measured at 414 nm on a Vmax reader. The assays have been applied to a variety of clinical situations. In patients suspected of having tetanus, the quantitation of antibodies has been helpful in establishing a diagnosis. In patients with a history of hypersensitivity to tetanus toxoid, verification of the levels of anti-TT antibody may prevent unnecessary and potentially harmful immunizations. The assays have also been used for the diagnostic evaluation of the humoral immune response to TT and DT, both in pediatric patients and in immunosuppressed patients. Several non-responders have been detected, and we have recently used the assay to monitor the effects of fish oil administration on the humoral immune response.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568- and Fluorescein Isothiocyanate- conjugated Antibody.

    Science.gov (United States)

    Mahmoudian, Jafar; Hadavi, Reza; Jeddi-Tehrani, Mahmood; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Shaban, Elham; Vafakhah, Mohtaram; Darzi, Maryam; Tarahomi, Majid; Ghods, Roya

    2011-01-01

    Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. Based on dye chemical structures, their photobleaching and photostability indices are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate (FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleahing profiles than FITC. In this experimental study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleaching profiles and photostability indices. Then, the fluorophore/antibody ratios were calculated using a spectrophotometer. The photobleaching profiles and photostability indices of conjugated antibodies were subsequently studied by immunocytochemistry (ICC). Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software. Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC. Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC.

  1. Detection of IgG anti-beta 2 glycoprotein-I antibodies in Saudi patients with systemic lupus erythematosis

    International Nuclear Information System (INIS)

    Alenzi, Faris Q.

    2008-01-01

    Objective was to develop an assay for the measurement of this anti-human beta2-glycoprotein I. This study was conducted from September 2004 to December 2006. The patients attending the Rheumatology Clinic were chosen from several centers in the Eastern region of Saudi Arabia because they had complications. An enzyme-linked immunosorbent (ELISA) assay was optimized and developed to measure antibody levels in humans. Fifty normal blood donors and 50 systemic lupus erythrematosis (SLE) patients were selected for this experiment. Raised IGg a beta 2 GPI antibody levels were associated with the presence of venous thrombosis and thrombocytopenia. The real value of IgG a beta 2 GPI as a predictor for the future clinical complications needs to be confirmed in prospective controlled studies investigating clinical complications in relationship to IgG a beta 2 GPI and to other risk factors for thrombosis. (author)

  2. Natural and Man-made Antibody Repertories for Antibody Discovery

    Directory of Open Access Journals (Sweden)

    Juan C eAlmagro

    2012-11-01

    Full Text Available Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of human, mice and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity and composition of a repertoire impact the antibody discovery process.

  3. Anti-Human Trafficking Interventions

    Science.gov (United States)

    Davy, Deanna

    2016-01-01

    Since the early 2000s, a significant number of programs and policies have been developed and implemented to prevent and combat human trafficking. At the international, regional and national levels, government, and international, and nongovernment organizations have established plans of action, conducted training, developed policy tools, and…

  4. Quantitative in vitro and in vivo models to assess human IgE B cell receptor crosslinking by IgE and EMPD IgE targeting antibodies.

    Science.gov (United States)

    Vigl, Benjamin; Salhat, Nina; Parth, Michela; Pankevych, Halyna; Mairhofer, Andreas; Bartl, Stefan; Smrzka, Oskar W

    2017-10-01

    Targeting plasma IgE by therapeutic mABs like Omalizumab (Xolair ® ) is current clinical practice for severe allergic conditions or other IgE related diseases like chronic urticaria. As an alternative to soluble IgE targeting, IgE supply can be lowered by targeting the Extracellular Membrane Proximal Domain (EMPD) of the IgE B cell receptor (BCR) present on IgE switched B cells. This ultimately leads to apoptosis of these cells upon IgE BCR crosslinking. Since tools to selectively assess the efficacy of IgE BCR crosslinking by IgE targeting antibodies are limited, a readily quantifiable cell model was developed that allows to specifically address IgE BCR crosslinking activity in vitro. The new cell model allowed for a direct quantitative comparison of anti-EMPD IgE therapeutic prototype antibody 47H4 with anti-IgE(Ce3) directed therapeutic antibody Omalizumab and with a newly selected anti-human EMPD IgE monoclonal antibody, designated mAB 15cl12. Furthermore, a complementing mouse model was developed that allows for in vivo validation of antibodies addressing human EMPD IgE. It carries a targetable humanized EMPD IgE sequence that has been introduced by seamless genomic replacement of the endogenous EMPD encoding sequence. The model allowed to directly compare IgE lowering activity of two anti-human EMPD IgE therapeutic antibodies in vivo. Our tools provide the means for quantitative assessment of IgE BCR crosslinking activity which is increasingly gaining attention with respect to forthcoming second generation anti-IgE clinical candidates such as Ligelizumab or other clinical candidates featuring additional effector functions such as IgE BCR crosslinking activity. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

    Shirai, Hiroki; Prades, Catherine; Vita, Randi

    2014-01-01

    to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic...... infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies...... for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii...

  6. Comparative binding of bovine, human and rat insulin-like growth factors to membrane receptors and to antibodies against human insulin-like growth factor-1.

    OpenAIRE

    Read, L C; Ballard, F J; Francis, G L; Baxter, R C; Bagley, C J; Wallace, J C

    1986-01-01

    The immunological properties of human, bovine and rat insulin-like growth factors (IGF) and insulin were compared in competitive binding studies with Tr10 and NPA polyclonal antisera raised in rabbits against human IGF-1. Bovine IGF-1 was 11-19% as effective as human IGF-1 in competing for binding with 125I-labelled human IGF-1, whereas IGF-2 reacted poorly and insulin did not compete. Similar competitive binding curves were obtained with the mouse monoclonal anti-(human IGF-1) antibody 3D1, ...

  7. Radioimmunodetection (RAID) using radiolabeled monoclonal antibodies (MoAbs) has potential clinical useful application in diagnosis and management of breast carcinoma

    International Nuclear Information System (INIS)

    Rosner, D.; Nabi, H.A.; Wild, L.; Nabi, J.O.; Hreshchyshyn, M.

    1994-01-01

    The current study attempted to assess the potential proficiency of radioimmunodetection (RAID) of primary, residual, multicentric and recurrent breast carcinoma using two-radiolabeled murine monoclonal antibodies (MoAbs), anti-Human Milk Fat Globuli n (HMFG1) labeled with Iodine (I 123 ) and anti-Carcinoembryonic Antigen (CEA) labeled with Technetium (Tch 99 ). The MoAbs used bound specifically to breast cancer lesions, with accurate RAID in 10 of 12 studied patients. Accuracy 83.3%, sensitivity 87.5% and specificity 75.0%, suggesting that radiolabeled MoAbs used in this study are potential diagnostic agents for breast carcinoma

  8. Interleukin-9 receptor α chain mRNA formation in CD8+ T cells producing anti-human immunodeficiency virus type 1 substance(s)

    International Nuclear Information System (INIS)

    Hossain, M.M.; Tsuchie, H.; Detorio, M.A.; Shirono, H.; Hara, C.; Nishimoto, A.; Saji, A.; Koga, J.; Takata, N.; Maniar, J.K.; Saple, D.G.; Taniguchi, K.; Kageyama, S.; Ichimura, H.; Kurimura, T.

    1998-01-01

    A search for gene(s) associated with anti-human immunodeficiency virus type 1 (HIV-l) activity of CD8 + T cells was attempted using molecular cloning and the relation between the anti-HIV activity of CD8 + T cells and the interleukin-9 receptor a chain (IL-9R-α) mRNA expression from the cDNA clones obtained was examined. The anti-HIV-l activity of CD8 + T cell culture supernatants was assessed by measuring the level of HIV-l replication in a CD4 + T cell line transfected with an infectious HIV-l DNA clone. IL-9R-a mRNA was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR). Of 5 cases showing high level of anti-HIV-l activity (more than 80% suppression of HIV-l replication), the mRNA was detected in 4 cases. Of 10 cases showing low level of anti-HIV-l activity (less than 80% suppression of HIV-l replication), the mRNA was detected in one case. Soluble recombinant human IL-9 receptor (rhIL-9sR) did not suppress HIV-l replication at a concentration of 1 μg/ml. These data suggest that the IL-9R-a mRNA formation in CD8 + T cells may correlate with and play some role in the anti-HIV-l activity of CD8+ T cells from HIV-l-infected individuals. Key words: CD8+ T cells; anti-HIV-l activity; cytokines; interleukin-9 receptor (authors)

  9. Elevated myeloid: plasmacytoid dendritic cell ratio associates with late, but not early liver rejection in children induced with, anti-human thymocyte globulin1

    Science.gov (United States)

    Gupta, Ankit; AshokKumar, Chethan; Ningappa, Mylarappa; Sun, Qing; Higgs, Brandon W; Snyder, Sara; Zeevi, Adriana; Thomson, Angus W; Mazariegos, George V; Sindhi, Rakesh

    2009-01-01

    Background Dendritic cells (DC) play an important role in the induction and regulation of immune responses. Methods Myeloid CD11c+DC (MDC), which may have inflammatory functions, and plasmacytoid CD123+ DC (PDC), which may have tolerogenic potential, were measured by flow cytometric analysis, cross-sectionally, once, in 48 children, and longitudinally (pre-transplant, and at days 1–60, 61–200, 201–400 post transplant) in 30 children following liver transplantation (LTx). All children received 53/25 cadaveric/live donor liver allografts with rabbit anti-human thymocyte globulin (rATG) induction, and steroid-free Tacrolimus therapy. Rejectors in both groups were those children (n=35), who experienced biopsy-proven acute cellular rejection (ACR) within 60 days of DC monitoring. Results Among rejectors in the longitudinal and cross-sectional cohorts, the MDC: PDC ratio was higher, and was associated with decreased PDC frequencies. Logistic regression analysis, leave-one out cross-validation, and receiver operating characteristic analysis applied to 30 cross-sectional subjects revealed that an MDC:PDC ratio 1.78 was associated with rejector status with sensitivity/specificity of 76.9/88.2%. Sensitivity and specificity were replicated in the 18 remaining cross-sectional subjects (88.8 and 78.8%, respectively), but not in longitudinally-monitored subjects, during the early, 60-day period after LTx (30.76 and 62.50%, respectively). A significant negative correlation was observed between Tacrolimus whole blood concentrations and PDC frequencies (Spearman r = −0.370, p=0.005) in 48 cross-sectional subjects in whom DC subsets were monitored 1–3 years after LTx, but not during the early post-LTx period. Conclusion We conclude that an elevated MDC: PDC ratio associates with liver graft rejection, which occurs after first year in children induced with rATG. PMID:19696644

  10. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    International Nuclear Information System (INIS)

    Lee, Gwon Jun; Hong, Key Sak; Choi, Kang Won; Lee, Kyu; Koh, Chang Soon; Lee, Mun Ho; Park, Sung Hoe; Chi, Je Geun; Lee, Sang Kook

    1979-01-01

    The authors investigated the incidence of antithyroglobulin antibodies and antibodies and antimicrosomal antibodies measured by tanned red cell hemagglutination method in subjects suffering from various thyroid disorders. 1) In 15 normal patients, neither suffering from any thyroid diseases nor from any other autoimmune disorders, the antithyroglobulin antibodies were all negative, but the antimicrosomal antibody was positive only in one patient (6.7%). 2) The antithyroglobulin antibodies were positive in 31.5% (34 patients) of 108 patients with various thyroid diseases, and the antimicrosomal antibodies were positive in 37.0% (40 patients). 3) of the 25 patients with Graves' diseases, 7 patients (28.0%) showed positive for the antithyroglobulin antibodies, and 9 (36.0%) for the antimicrosomal antibodies. There was no definite differences in clinical and thyroid functions between the groups with positive and negative results. 4) Both antibodies were positive in 16 (88.9%) and 17 (94.4%) patients respectively among 18 patients with Hashimoto's thyroiditis, all of them were diagnosed histologically. 5) Three out of 33 patients with thyroid adenoma showed positive antibodies, and 3 of 16 patients with thyroid carcinoma revealed positive antibodies. 6) TRCH antibodies demonstrated negative results in 2 patients with subacute thyroiditis, but positive in one patient with idiopathic primary myxedema. 7) The number of patients with high titers(>l:802) was 16 for antithyroglobulin antibody, and 62.5% (10 patients) of which was Hashimoto's thyroiditis. Thirteen (65.0) of 20 patients with high titers (>l:802) for antimicrosomal antibody was Hashimoto's thyroiditis. TRCH test is a simple, sensitive method, and has high reliability and reproducibility. The incidences and titers of antithyroglobulin antibody and antimicrosomal antibody are especially high in Hashimoto's thyroiditis.

  11. Maraviroc (UK-427,857), a Potent, Orally Bioavailable, and Selective Small-Molecule Inhibitor of Chemokine Receptor CCR5 with Broad-Spectrum Anti-Human Immunodeficiency Virus Type 1 Activity

    OpenAIRE

    Dorr, Patrick; Westby, Mike; Dobbs, Susan; Griffin, Paul; Irvine, Becky; Macartney, Malcolm; Mori, Julie; Rickett, Graham; Smith-Burchnell, Caroline; Napier, Carolyn; Webster, Rob; Armour, Duncan; Price, David; Stammen, Blanda; Wood, Anthony

    2005-01-01

    Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades a...

  12. Compositions, antibodies, asthma diagnosis methods, and methods for preparing antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hongjun; Zangar, Richard C.

    2017-01-17

    Methods for preparing an antibody are provided with the method including incorporating 3-bromo-4-hydroxy-benzoic acid into a protein to form an antigen, immunizing a mammalian host with the antigen, and recovering an antibody having an affinity for the antigen from the host. Antibodies having a binding affinity for a monohalotyrosine are provided as well as composition comprising an antibody bound with monohalotyrosine. Compositions comprising a protein having a 3-bromo-4-hydroxy-benzoic acid moiety are also provided. Methods for evaluating the severity of asthma are provide with the methods including analyzing sputum of a patient using an antibody having a binding affinity for monohalotyrosine, and measuring the amount of antibody bound to protein. Methods for determining eosinophil activity in bodily fluid are also provided with the methods including exposing bodily fluid to an antibody having a binding affinity for monohalotyrosine, and measuring the amount of bound antibody to determine the eosinophil activity.

  13. A four-step sandwich radioimmunoassay for direct selection of monoclonal antibodies to allergen molecules

    International Nuclear Information System (INIS)

    Ley, V.; Corbi, A.L.; Sanchez-Madrid, F.; Carreira, J.C.

    1985-01-01

    A 4-step radioimmunoassay has been devised for direct identification of monoclonal antibodies (MAb) directed to IgE-binding molecules. Polyvinyl chloride wells coated with purified anti-mouse kappa chain MAb (187-1) were successively incubated with: (1) MAb-containing hybridoma supernatants, (2) allergen extract, (3) allergic patients' serum pool, and (4) 125 I-labeled anti-human IgE antiserum, to detect MAb-allergen-IgE complexes. MAb to allergens from Parietaria judaica pollen and Dermatophagoides mites have been selected with this screening procedure. The affinity-purified allergen molecules competed the binding of IgE to allergen extracts coated to paper discs in a RAST inhibition assay, confirming the anti-allergen specificity of the selected MAb. This screening method is sensitive enough to allow detection of MAb directed to poorly represented allergens. (Auth.)

  14. Electrochemical detection of celiac disease-related anti-tissue transglutaminase antibodies using thiol based surface chemistry.

    Science.gov (United States)

    Dulay, Samuel; Lozano-Sánchez, Pablo; Iwuoha, Emmanuel; Katakis, Ioanis; O'Sullivan, Ciara K

    2011-05-15

    Celiac disease is an autoimmune disorder that affects the gastrointestinal tract upon ingestion of gluten, which triggers the production of antibodies against gliadin and tissue transglutaminase, activating an inflammatory response and inducing tissue damage in the small intestine resulting in malabsorption. The measurement of these antibodies in an individual's blood can be used to screen for celiac disease and the criteria for definitive diagnosis is currently being revised to be based on serological analysis rather than biopsy. In the work reported here, an electrochemical immunosensor for the detection of human anti-tissue transglutaminase antibodies was developed, consisting of gold-based self-assembled monolayers of a carboxylic group terminated bipodal alkanethiol that is covalently linked to tissue transglutaminase, the antigen for the immunorecognition of circulating autoantibodies. The presence of the autoantibodies was recorded using horseradish peroxidase labeled anti-human antibodies, which provided an enzyme based electrochemical signal. Optimization and characterization of the surface of the sensor was carried out by electrochemical impedance spectroscopy and surface plasmon resonance. The immunosensor gave a stable quantitative response to different antibody concentrations after 30 min with a limit of detection of 390 ng/mL and an RSD of 9%, n=3. The developed immunosensor was tested with calibrator solutions as well as with real patients' samples, and the results compared to those obtained from Eurospital's Eu-tTG IgA and IgG ELISA kits, showing an excellent degree of correlation. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Development of electrochemical immunosensors based on different serum antibody immobilization methods for detection of Japanese encephalitis virus

    International Nuclear Information System (INIS)

    Tran, Quang Huy; Hanh Nguyen, Thi Hong; Phan, Thi Nga; Mai, Anh Tuan; Nguyen, Thi Thuy; Vu, Quang Khue

    2012-01-01

    This paper describes the development of electrochemical immunosensors based on human serum antibodies with different immobilization methods for detection of Japanese encephalitis virus (JEV). Human serum containing anti-JEV antibodies was used to immobilize onto the surface of silanized interdigitated electrodes by four methods: direct adsorption (APTES-serum), covalent binding with a cross linker of glutaraldehyde (APTES-GA-serum), covalent binding with a cross linker of glutaraldehyde combined with anti-human IgG (APTES-GA-anti-HIgG-serum) and covalent binding with a cross linker of glutaraldehyde combined with a bioaffinity of protein A (APTES-GA-PrA-serum). Atomic force microscopy was used to verify surface characteristics of the interdigitated electrodes before and after treatment with serum antibodies. The output signal of the immunosensors was measured by the change of conductivity resulting from the specific binding of JEV antigens and serum antibodies immobilized on the electrodes, with the help of horseradish peroxidase (HRP)-labeled secondary antibody against JEV. The results showed that the APTES-GA-PrA-serum method provided the highest signal of the electrochemical immunosensor for detection of JEV antigens, with the linear range from 25 ng ml −1 to 1 μg ml −1 , and the limit of detection was about 10 ng ml −1 . This study shows a potential development of novel electrochemical immunosensors applied for virus detection in clinical samples in case of possible outbreaks

  16. Antithyroid microsomal antibody

    Science.gov (United States)

    ... that you have a higher chance of developing thyroid disease in the future. Antithyroid microsomal antibodies may be ... PA: Elsevier; 2016:chap 11. Weiss RE, Refetoff S. Thyroid function testing. In: Jameson JL, De Groot LJ, eds. Endocrinology: Adult and ... Lupus Read more ...

  17. Antibodies Targeting EMT

    Science.gov (United States)

    2017-10-01

    determine their targets on the cell. The newly discovered antibodies will then be engineered for utility as new highly specific drugs and diagnostics in...are from the aldo-keto reductase family (AKRs). Remarkably, 3 of the top 10 genes with induction in the mesenchymal TES2b cells Figure 1. Amino

  18. Monoclonal antibodies in haematopathology

    Energy Technology Data Exchange (ETDEWEB)

    Grignani, F.; Martelli, M.F.; Mason, D.Y.

    1985-01-01

    This book contains over 40 selections. Some of the titles are: Oncogene (c-myc, c-myb) amplification in acute myelogenous leukaemia; Ultrastructural characterization of leukaemic cells with monoloclonal antibodies; Origin of B-cell malignancies; Immunohistology of gut lymphomas; and Spurious evidence of lineage infidelity in monocytic leukaemia.

  19. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.

    2015-01-01

    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  20. The Amaranthus leucocarpus lectin enhances the anti-CD3 antibody-mediated activation of human peripheral blood CD4+ T cells.

    Science.gov (United States)

    Urrea, Francisco; Ortiz-Quintero, Blanca; Sanchez-Garcia, Francisco Javier; Blanco-Favela, Francisco; Garfias, Yonathan; Lascurain, Ricardo; Zenteno, Edgar

    2010-08-01

    Activation of CD4(+) T cells plays a main role in adaptive immune response by regulating cellular and humoral immunity via processes associated with changes in cell surface oligosaccharide receptors. Lectins are glycoproteins that specifically recognize oligosaccharides and have been used to characterize changes in oligosaccharides present on T cell surface and their effects on activation. A lectin from Amaranthus leucocarpus seeds (ALL) is specific for glycoprotein structures containing galactose-N-acetylgalactosamine and is able to bind to human and murine CD4(+) T cells, however, its effect on activation remains unclear. We examined the effect of ALL on the activation of peripheral blood human CD4(+) T cells and analyzed cell proliferation, expression of the activation-associated molecule CD25, secretion of the activation-dependent cytokine interleukin (IL)-2 and intracellular calcium influx changes using flow cytometry. CD4(+) T cells were stimulated with anti-CD3 antibodies that provided the first activation signal in the presence or absence of ALL. ALL alone did not induce CD4(+) T cell activation but when also stimulated with anti-CD3 antibodies, ALL up-regulated CD25 expression, cell proliferation, IL-2 secretion and an intracellular calcium influx in a dose-dependent manner. In addition, ALL recognized CD4(+) T cells expressing the CD69 and Ki67 molecules expressed only by activated T cells and induced production of the TH1-type cytokine interferon-gamma. Our findings indicate that ALL binds to human activated CD4(+) T cells and enhances the degree of activation of CD4(+) T cells that are stimulated with anti-CD3 antibodies. ALL provides a new tool for analyzing T cell activation mechanisms.

  1. Flow cytometric immunophenotyping of regulatory T cells in chronic lymphocytic leukemia: comparative assessment of various markers and use of novel antibody panel with CD127 as alternative to transcription factor FoxP3.

    Science.gov (United States)

    Dasgupta, Alakananda; Mahapatra, Manoranjan; Saxena, Renu

    2013-04-01

    This study analyzed the frequency of regulatory T cells (Tregs) in chronic lymphocytic leukemia (CLL) by multiparameter flow cytometric immunophenotyping. Patients showed significantly increased frequencies of Tregs as compared to controls, a significantly higher percentage than that identified by previous studies, possibly indicating a different prognosis of CLL in different parts of the world and, more precisely, a worse prognosis of CLL in the Indian population. A higher frequency of Tregs was also seen in advanced stage of disease with significantly reduced frequencies of Tregs in patients with CLL after chemotherapy. A significant proportion of CD127low/-FoxP3+ Tregs expressed only low levels of CD25. Thus, CD127 appears to be a better marker than CD25 for the identification of CD4+FoxP3+ T cells as potential Tregs. Our results suggest that the specificity and sensitivity of CD4+CD127low/- cells are comparable to those of CD4+FoxP3+, which is the gold standard, and can be used as an alternative. This novel flow cytometric antibody panel with fewer number of antibodies is cost-effective and can be used to enumerate Tregs in resource-limited settings.

  2. The development and validation of a sensitive, dual-flow cell, SPR-based biosensor immunoassay for the detection, semi-quantitation, and characterization of antibodies to darbepoetin alfa and epoetin alfa in human serum.

    Science.gov (United States)

    Mytych, Daniel T; La, Stephon; Barger, Troy; Ferbas, John; Swanson, Steven J

    2009-02-20

    A surface plasmon resonance (SPR)-based biosensor immunoassay was developed and validated using the Biacore 3000 instrument to detect, semi-quantitate, and characterize serum antibodies against darbepoetin alfa (Aranesp) and epoetin alfa (EPOGEN). In this sensitive, dual-flow cell assay, epoetin alfa and darbepoetin alfa are covalently immobilized onto consecutive flow cells of a carboxymethyl dextran-coated sensor chip. Diluted human serum samples are injected sequentially over both surfaces. The binding of serum antibodies to the immobilized proteins are detected and recorded in real time based on the principles of SPR. Furthermore, antibody binding is confirmed with a secondary anti-human immunoglobulin antibody. Positive samples are further characterized to determine the relative concentration of the antibodies using an affinity-purified, rabbit anti-epoetin alfa antibody as a reference control. The assay can detect 80ng/ml and 100ng/ml of antibody to epoetin alfa and darbepoetin alfa, respectively. The dynamic range of the assay is from 0.078microg/ml to 10microg/ml using a rabbit antibody with demonstrated accuracy and intra- and inter-assay precision. Approximately 80 serum samples can be analyzed on each sensor chip while maintaining a stable baseline and consistent immunological reactivity. The analysis of serum samples from subjects administered with epoetin alfa or darbepoetin alfa provided evidence that the assay can detect varying concentrations of antibodies of different off rates, isotypes, and IgG subclasses.

  3. Humanized Antibodies for Antiviral Therapy

    Science.gov (United States)

    Co, Man Sung; Deschamps, Marguerite; Whitley, Richard J.; Queen, Cary

    1991-04-01

    Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.

  4. Cross-reactive antibodies induced by xenogeneic IgA can cause selective IgA deficiency.

    Science.gov (United States)

    Klartag, Ayelet; Chen, Chiann-Chyi; Dougherty, Joseph P; Ron, Yacov

    2010-03-01

    Selective immunoglobulin A deficiency (sIgAD) is the most common immunodeficiency in humans. Auto-reactive antibodies to human immunoglobulin A (IgA) are found in the serum of 20-40% of individuals with sIgAD. It is unknown whether these antibodies play a role in the pathogenesis of this immunodeficiency and although the prevailing thought is that they are secondary to the onset of sIgAD, there is very little, if any, support for this notion. Here, we propose that anti-IgA antibodies are in fact responsible for the removal of IgA from serum, and that the inducing antigen is most probably a xenogeneic IgA. This hypothesis is based on data obtained from an sIgAD patient in whom changes in dietary consumption of beef and/or bovine dairy products resulted in changes in anti-IgA levels in the serum. To test the hypothesis, the presence of anti-bovine IgA antibodies was tested by a highly specific enzyme-linked immunosorbent assay in serum samples from IgA-deficient and control individuals. All 13 sIgAD individuals with anti-IgA antibodies had a higher titer against bovine IgA than against human IgA. Of 23 control individuals, a surprisingly high proportion (65%) was also found to have IgG anti-bovine IgA antibodies. These results support the hypothesis that the anti-human IgA antibodies found in IgA-deficient individuals are originally produced against bovine IgA. These antibodies are found in many normal individuals, but only in cases where they cross react with endogenous human IgA, sIgAD may develop.

  5. Anti-human CD73 monoclonal antibody inhibits metastasis formation in human breast cancer by inducing clustering and internalization of CD73 expressed on the surface of cancer cells

    DEFF Research Database (Denmark)

    Terp, Mikkel G; Olesen, Kristina A; Christensen, Eva Arnspang

    2013-01-01

    of circulating tumor cells to extravasate and colonize, leading to inhibition of metastasis. Ab-based CD73 cancer therapy should include a combination of Abs that target the catalytic activity of CD73, as well as those with the characteristics described in this article.......-linking of CD73, because both whole IgG anti-CD73 AD2 mAb and Fab' fragments thereof exhibited this effect. Ex vivo treatment of different breast cancer cell lines with anti-CD73 AD2 mAb before i.v. injection into mice inhibited extravasation/colonization of circulating tumor cells and significantly reduced...... metastasis development. This effect was also observed when the cancer cell-surface expression of CD73 was significantly reduced by small interfering RNA knockdown. The antimetastatic activity is epitope specific, as another Ab that efficiently binds CD73-expressing live cancer cells did not lead to CD73...

  6. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery...

  7. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    N.D. Zegers (Netty)

    1995-01-01

    textabstractSynthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps

  8. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.

    1995-01-01

    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the

  9. Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA-transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy.

    Science.gov (United States)

    Hung, Chien-Fu; Xu, Xuequn; Li, Linhong; Ma, Ying; Jin, Qiu; Viley, Angelia; Allen, Cornell; Natarajan, Pachai; Shivakumar, Rama; Peshwa, Madhusudan V; Emens, Leisha A

    2018-04-02

    CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to "on-target off-tumor" toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of "on-target off-tumor" toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 10 9 peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival

  10. Magnetic Purification of Antibodies

    Science.gov (United States)

    Dhadge, Vijaykumar Laxman

    This work aimed at the development of magnetic nanoparticles for antibody purification and at the evaluation of their performance in Magnetic fishing and in a newly developed hybrid technology Magnetic Aqueous Two Phase Systems. Magnetic materials were produced by coprecipitation and solvothermal approaches. Natural polymers such as dextran, extracellular polysaccharide and gum Arabic were employed for coating of iron oxide magnetic supports. Polymer coated magnetic supports were then modified with synthetic antibody specific ligands,namely boronic acid, a triazine ligand (named 22/8) and an Ugi ligand (named A2C7I1). To optimize the efficacy of magnetic nanoparticles for antibody magnetic fishing, various solutions of pure and crude antibody solutions along with BSA as a non-specific binding protein were tested. The selectivity of magnetic nanoparticle for antibody, IgG, was found effective with boronic acid and ligand 22/8. Magnetic supports were then studied for their performance in high gradient magnetic separator for effective separation capability as well as higher volume handling capability. The magnetic materials were also supplemented to aqueous two phase systems, devising a new purification technology. For this purpose, magnetic particles modified with boronic acid were more effective. This alternative strategy reduced the time of operation,maximized separation capability (yield and purity), while reducing the amount of salt required. Boronic acid coated magnetic particles bound 170 +/- 10 mg hIgG/g MP and eluted 160 +/- 5 mg hIgG/g MP, while binding only 15 +/- 5 mg BSA/g MP. The affinity constant for the interaction between hIgG and APBA_MP was estimated as 4.9 x 105 M-1 (Ka) with a theoretical maximum capacity of 492 mg hIgG adsorbed/g MP (Qmax). APBA_MPs were also tested for antibody purification directly from CHO cell supernatants. The particles were able to bind 98% of IgG loaded and to recover 95% of pure IgG (purity greater than 98%) at extremely

  11. The detection of rubella-specific IgM antibodies by radioimmunoassay

    International Nuclear Information System (INIS)

    Kangro, H.O.; Pattison, J.R.; Heath, R.B.

    1978-01-01

    An indirect solid-phase radioimmunoassay (RIA) has been developed for the detection of immunoglobulin (Ig) class-specific rubella antibodies. A commercial rubella haemagglutinin is dried and fixed on to the wells of flexible microtitre plates and allowed to react with serial dilutions of whole or fractionated human sera. Class-specific rubella antibodies are then detected by determining the specific binding of 125 I-labelled anti-human IgG or IgM. The RIA was first evaluated by comparison with the heamagglutination-inhibition (HI) test for the detection of rubella-specific IgM in gel-filtration fractions. RIA was found to be as specific as HI but 50-150 times more sensitive. Rubella-specific IgG antibodies did not interfere in specific IgM determinations by RIA and therefore the latter technique applied to unfractionated sera. The results obtained indicate that RIA on unfractionated sera is a practical, sensitive and specific technique which could provide a reliable method for the diagnosis of rubella. The rubella-specific IgM titres obtained by RIA were not increased by the removal of IgG by pretreatment of sera with Staphylococcal Protein A. (author)

  12. Vaccination of B-CLL patients with autologous dendritic cells can change the frequency of leukemia antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T cells toward an antileukemia response.

    Science.gov (United States)

    Hus, I; Schmitt, M; Tabarkiewicz, J; Radej, S; Wojas, K; Bojarska-Junak, A; Schmitt, A; Giannopoulos, K; Dmoszyńska, A; Roliński, J

    2008-05-01

    Recently, we described that vaccination with allogeneic dendritic cells (DCs) pulsed with tumor cell lysate generated specific CD8+ T cell response in patients with B-cell chronic lymphocytic leukemia (B-CLL). In the present study, the potential of autologous DCs pulsed ex vivo with tumor cell lysates to stimulate antitumor immunity in patients with B-CLL in early stages was evaluated. Twelve patients at clinical stage 0-2 as per Rai were vaccinated intradermally up to eight times with a mean number of 7.4 x 10(6) DCs pulsed with B-CLL cell lysate. We observed a decrease of peripheral blood leukocytes and CD19+/CD5+ leukemic cells in five patients, three patients showed a stable disease and four patients progressed despite DC vaccination. A significant increase of specific cytotoxic CD8+ T lymphocytes against the leukemia-associated antigens RHAMM or fibromodulin was detected in four patients after DC vaccination. In patients with a clinical response, an increase of interleukin 12 (IL-12) serum levels and a decrease of the frequency of CD4+CD25(+)FOXP3+ T regulatory cells were observed. Taken together, the study demonstrated that vaccination with autologous DC in CLL patients is feasible and safe. Immunological and to some extend hematological responses could be noted, justifying further investigation on this immunotherapeutical approach.

  13. Clinical use of antibodies

    International Nuclear Information System (INIS)

    Baum, R.P.; Hoer, Gustav; Cox, P.H.; Buraggi, G.L.

    1991-01-01

    Use of monoclonal antibodies as tumour specific carrier molecules for therapeutic agents or as in vivo diagnostic reagents when labelled with radionuclides or NMR signal enhancers is attracting more and more attention. The potential is enormous but the technical problems are also considerable requiring the concerted action of many different scientific disciplines. This volume is based upon a symposium organised in Frankfurt in 1990 under the auspices of the European Association of Nuclear Medicines' Specialist Task Groups on Cardiology and the Utility of Labelled Antibodies. It gives a multidisciplinary review of the state of the art and of problems to be solved as well as recording the not inconsiderable successes which have been booked to date. The book will be of value as a reference to both clinicians and research scientists. refs.; figs.; tabs

  14. Antibody Production with Synthetic Peptides.

    Science.gov (United States)

    Lee, Bao-Shiang; Huang, Jin-Sheng; Jayathilaka, Lasanthi P; Lee, Jenny; Gupta, Shalini

    2016-01-01

    Peptides (usually 10-20 amino acid residues in length) can be used as effectively as proteins in raising antibodies producing both polyclonal and monoclonal antibodies routinely with titers higher than 20,000. Peptide antigens do not function as immunogens unless they are conjugated to proteins. Production of high quality antipeptide antibodies is dependent upon peptide sequence selection, the success of peptide synthesis, peptide-carrier protein conjugation, the humoral immune response in the host animal, the adjuvant used, the peptide dose administered, the injection method, and the purification of the antibody. Peptide sequence selection is probably the most critical step in the production of antipeptide antibodies. Although the process for designing peptide antigens is not exact, several guidelines and computational B-cell epitope prediction methods can help maximize the likelihood of producing antipeptide antibodies that recognize the protein. Antibodies raised by peptides have become essential tools in life science research. Virtually all phospho-specific antibodies are now produced using phosphopeptides as antigens. Typically, 5-20 mg of peptide is enough for antipeptide antibody production. It takes 3 months to produce a polyclonal antipeptide antibody in rabbits that yields ~100 mL of serum which corresponds to ~8-10 mg of the specific antibody after affinity purification using a peptide column.

  15. Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte

    International Nuclear Information System (INIS)

    Jain, Swati; Chattopadhyay, Sruti; Jackeray, Richa; Singh, Harpal

    2009-01-01

    Pendent nitrile groups of multifilamentous polyacrylonitrile (PAN) fibers were reduced to amino groups using lithium aluminum hydride for different time of reduction and amine content was estimated by performing acid-base titrations. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC) were used for the characterization of the generated amino groups and thermal properties of the reduced fibers, respectively. The surface morphology of the fibers after reduction and immobilization was characterized using Scanning Electron Microscope (SEM). The newly formed amino groups of the fibers were activated by using glutaraldehyde for the covalent linking of Goat anti-Rabbit IgG-HRP (GAR-HRP) antibody enzyme conjugate. Modified PAN fibers were evaluated as a matrix for sandwich ELISA by using Goat anti-Rabbit antibody (GAR-IgG), Rabbit anti-Goat (RAG-IgG) as analyte and enzyme conjugate GAR-HRP. The fibers reduced for 24 h were able to detect the analyte RAG-IgG at a concentration as low as 3.75 ng mL -1 with 12% skimmed milk as blocking reagent for the optimized concentration of primary antibody GAR-IgG 3 μg mL -1 and peroxidase conjugate GAR-HRP dilution of 8000 fold. The sensitivity, specificity and reproducibility of the developed immunoassay was further established with antibodies present in human blood using Rabbit anti-Human (RAH-IgG) antibody and the corresponding HRP enzyme conjugate. As low as 0.1 μL of human blood was sufficient to perform the assay with the modified fibers.

  16. Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte.

    Science.gov (United States)

    Jain, Swati; Chattopadhyay, Sruti; Jackeray, Richa; Singh, Harpal

    2009-11-10

    Pendent nitrile groups of multifilamentous polyacrylonitrile (PAN) fibers were reduced to amino groups using lithium aluminum hydride for different time of reduction and amine content was estimated by performing acid-base titrations. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC) were used for the characterization of the generated amino groups and thermal properties of the reduced fibers, respectively. The surface morphology of the fibers after reduction and immobilization was characterized using Scanning Electron Microscope (SEM). The newly formed amino groups of the fibers were activated by using glutaraldehyde for the covalent linking of Goat anti-Rabbit IgG-HRP (GAR-HRP) antibody enzyme conjugate. Modified PAN fibers were evaluated as a matrix for sandwich ELISA by using Goat anti-Rabbit antibody (GAR-IgG), Rabbit anti-Goat (RAG-IgG) as analyte and enzyme conjugate GAR-HRP. The fibers reduced for 24h were able to detect the analyte RAG-IgG at a concentration as low as 3.75ng mL(-1) with 12% skimmed milk as blocking reagent for the optimized concentration of primary antibody GAR-IgG 3microg mL(-1) and peroxidase conjugate GAR-HRP dilution of 8000 fold. The sensitivity, specificity and reproducibility of the developed immunoassay was further established with antibodies present in human blood using Rabbit anti-Human (RAH-IgG) antibody and the corresponding HRP enzyme conjugate. As low as 0.1microL of human blood was sufficient to perform the assay with the modified fibers.

  17. Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte

    Energy Technology Data Exchange (ETDEWEB)

    Jain, Swati, E-mail: swatijain.iitd@gmail.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India); Chattopadhyay, Sruti, E-mail: srutic@hotmail.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India); Jackeray, Richa, E-mail: richajackeray.iitd@gmail.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India); Singh, Harpal, E-mail: harpal2000@yahoo.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India)

    2009-11-10

    Pendent nitrile groups of multifilamentous polyacrylonitrile (PAN) fibers were reduced to amino groups using lithium aluminum hydride for different time of reduction and amine content was estimated by performing acid-base titrations. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC) were used for the characterization of the generated amino groups and thermal properties of the reduced fibers, respectively. The surface morphology of the fibers after reduction and immobilization was characterized using Scanning Electron Microscope (SEM). The newly formed amino groups of the fibers were activated by using glutaraldehyde for the covalent linking of Goat anti-Rabbit IgG-HRP (GAR-HRP) antibody enzyme conjugate. Modified PAN fibers were evaluated as a matrix for sandwich ELISA by using Goat anti-Rabbit antibody (GAR-IgG), Rabbit anti-Goat (RAG-IgG) as analyte and enzyme conjugate GAR-HRP. The fibers reduced for 24 h were able to detect the analyte RAG-IgG at a concentration as low as 3.75 ng mL{sup -1} with 12% skimmed milk as blocking reagent for the optimized concentration of primary antibody GAR-IgG 3 {mu}g mL{sup -1} and peroxidase conjugate GAR-HRP dilution of 8000 fold. The sensitivity, specificity and reproducibility of the developed immunoassay was further established with antibodies present in human blood using Rabbit anti-Human (RAH-IgG) antibody and the corresponding HRP enzyme conjugate. As low as 0.1 {mu}L of human blood was sufficient to perform the assay with the modified fibers.

  18. [Study of anti-idiotype antibodies to human monoclonal antibody].

    Science.gov (United States)

    Harada, R; Takahashi, N; Owaki, I; Kannagi, R; Endo, N; Morita, N; Inoue, M

    1992-02-01

    A human monoclonal antibody, ll-50 (IgM, lambda), was generated, which reacted specifically with a major of glycolipid present in LS174T colon cancer cells. The glycolipid antigen which reacted with the ll-50 antibody was expected to four sugar residues from its TLC mobility, and it was ascertained that the glycolipid antigen which reacted with ll-50 antibody might be Lc4 antigen [Gal beta 1----3 GLcNAc beta 1----3 Gal beta 1----4 Glc beta 1----1 Cer] judging from TLC immunostaining and ELISA when the reactivity of ll-50 antibody was tested using various pure glycolipids in 3-5 sugar residues as an antigen. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated ll-50 antibody. The serum of the Lc4 antigen recognized by ll-50 antibody was significantly higher in patients with malignant disorders than that in healthy individuals (p less than 0.05). Three mouse monoclonal anti-idiotype antibodies, G3, B3 and C5 (all IgG1), were generated by the immunization of BALB/c mice with ll-50 antibody. These anti-idiotype antibodies specifically bound to to human monoclonal antibody, ll-50 and had a significant inhibitory activity towards the binding of ll-50 antibody to the Lc4 antigen. This indicated that these anti-idiotype antibodies, G3, B3, and C5, were paratope-related anti-idiotype antibodies. G3, B3, and C5 were expected to define the nearest idiotope because they could mutually inhibit ll-50 antibody. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated anti-idiotype antibodies, G3, B3, and C5. As to the ll-50 like antibodies defined by C5 (Id-C5+), the mean serum level in patients with malignant disorders was significantly higher than that in healthy individuals (p less than 0.05). As to the ll-50 like antibodies defined by B3 (Id-B3+), the mean serum level in patients with malignant disorders was significantly higher

  19. Comparisons of anti-human immunodeficiency virus activities, cellular transport, and plasma and intracellular pharmacokinetics of 3'-fluoro-3'-deoxythymidine and 3'-azido-3'-deoxythymidine.

    Science.gov (United States)

    Kong, X B; Zhu, Q Y; Vidal, P M; Watanabe, K A; Polsky, B; Armstrong, D; Ostrander, M; Lang, S A; Muchmore, E; Chou, T C

    1992-01-01

    3'-Fluoro-3'-deoxythymidine (FLT), a candidate anti-AIDS compound in clinical trials, showed anti-human immunodeficiency virus type 1 (HIV-1) potency (50% effective concentration, 0.0052 microM) slightly better than or equal to that of 3'-azido-3'-deoxythymidine (AZT) in MT4 cells and was threefold more potent in H9 cells. There was no FLT resistance demonstrable in the AZT-resistant HIV-1 strains. Both FLT and AZT showed low cytotoxicity for MT4 cells, with selectivity indices (efficacy/toxicity ratio) of greater than 47,000 and greater than 33,000, respectively. Cellular permeation of FLT and thymidine (dThd) was greater than that of AZT, and FLT and dThd permeated the cell membranes by a carrier-mediated mechanism as well as by simple diffusion, as indicated by the existence of nitrobenzylthioinosine-5'-monophosphate-sensitive and -insensitive components. By contrast, transport of AZT into cells was by simple diffusion. The intracellular level of the triphosphate of FLT (FLTTP) in MT4 cells was two- to threefold higher than that of AZT (AZTTP) after exposure to 1.8 microM each compound for 12 h. The elimination kinetics of FLTTP and AZTTP in HIV-1-infected MT4 cells in fresh medium showed biphasic patterns, with initial half-lives of 1.03 and 1.09 h, respectively. In phytohemagglutinin-stimulated human peripheral blood lymphocytes, the FLTTP level was increased 59-fold compared with that in unstimulated cells at 12 h, was four- to sixfold higher than the level of AZTTP in stimulated cells at 12 h, and remained four- to fivefold higher during a 4-h elimination period in fresh medium and twofold higher at the end of a 12-h elimination period. Two- to eightfold more [3H]AZT than [3H]FLT was incorporated into the host cell DNA, and both [3H]AZT and [3H]FLT remained persistently incorporated for over 24 h. The incorporated [3H]AZT and [3H]FLT were alkali labile, whereas incorporated [3H]dThd was alkali stable. Pharmacokinetics of FLT in plasma of monkeys after

  20. The antibody Hijikata Tatsumi

    Directory of Open Access Journals (Sweden)

    Éden Peretta

    2012-11-01

    Full Text Available Considered one of the most influential modern dance representatives in Japan, Tatsumi Hijikata’s work was a milestone in the Japanese post-war experimental artistic scene. Heretic son of his time, he staged a fertile mix of artistic and cultural influences, overlapping subversive elements of European arts and philosophy with radical references from pre-modern Japanese culture. In this way he built the foundations of its unstable antibody, its political-artistic project of dissolution of a organism, both physical and social.

  1. Lower incidence of acute GVHD is associated with the rapid recovery of CD4+CD25+CD45RA+ regulatory T cells in patients who received haploidentical allografts from NIMA-mismatched donors: A retrospective (development) and prospective (validation) cohort-based study.

    Science.gov (United States)

    Wang, Yu; Zhao, Xiang-Yu; Xu, Lan-Ping; Zhang, Xiao-Hui; Han, Wei; Chen, Huan; Wang, Feng-Rong; Mo, Xiao-Dong; Zhang, Yuan-Yuan; Zhao, Xiao-Su; Y, Kong; Liua, Kai-Yan; Huang, Xiao-Jun; Yu, Xue-Zhong; Chang, Ying-Jun

    2016-01-01

    To investigate the effects of non-inherited maternal antigen (NIMA) on clinical outcomes and immune recovery, especially of regulatory T cells (Tregs), in patients who underwent unmanipulated haploidentical transplantation. A retrospective cohort (n = 57) and a prospective cohort (n = 88) were included. All patients received haploidentical allografts from sibling donors. Reconstitution of immune subsets, including Tregs, was determined using multicolor flow cytometry. In the retrospective cohort, the cumulative incidence of grades II-IV acute GVHD in patients with NIMA-mismatched donors was significantly lower than that of cases with NIPA-mismatched donors (14.8% vs. 43.30%, p = 0.018). Patients with higher percentages of CD4 + CD25 + CD45RA + T cells (naive Tregs) within CD4 + T cells recovered on day 30 (≥1.55%) experienced a significantly lower incidence of grades II-IV acute GVHD than that of cases with lower percentages of naive Tregs (<1.55%) (13.8% vs. 46.4%, p = 0.010). Multivariate analysis showed that NIMA mismatch and the percentages of naive Tregs were associated with the incidence of grades II-IV acute GVHD [ p = 0.050, and 0.031, respectively]. In the prospective cohort, the association of NIMA mismatch [HR = 0.365, 95% CI, 0.169-0.786, p = 0.010] or higher percentages of naive Tregs recovered on day 30 (≥1.55%) [HR = 0.114, 95% CI, 0.027-0.479, p = 0.003] with a lower cumulative incidence of grades II-IV acute GVHD was further demonstrated. No effects of NIMA mismatch on chronic GVHD, transplant-related mortality, relapse, disease-free survival, or overall survival were found. Our results confirmed the role of NIMA mismatch in acute GVHD and provided the first demonstration, based on clinical data, that recovered Tregs may be involved in the effects of NIMA on acute GVHD in a haploidentical transplant setting.

  2. The future of monoclonal antibody technology

    OpenAIRE

    Zider, Alexander; Drakeman, Donald L

    2010-01-01

    With the rapid growth of monoclonal antibody-based products, new technologies have emerged for creating modified forms of antibodies, including fragments, conjugates and multi-specific antibodies. We created a database of 450 therapeutic antibodies in development to determine which technologies and indications will constitute the “next generation” of antibody products. We conclude that the antibodies of the future will closely resemble the antibodies that have already been approved for commer...

  3. Monoclonal antibodies for treating cancer

    International Nuclear Information System (INIS)

    Dillman, R.O.

    1989-01-01

    The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references

  4. Incidence of clinically significant antibodies in patients and healthy blood donors: a prospective cross-sectional study from a tertiary healthcare center in India.

    Science.gov (United States)

    Tiwari, Aseem K; Pandey, Prashant; Sharma, Jyoti; Shailja, Kumari; Dixit, Surbhi; Raina, Vimarsh

    2014-04-01

    Since there is scarce data available on incidence and type of irregular antibodies in patients and donors in India, a study was undertaken to find the incidence of irregular antibodies in patients and irregular antibodies and a positive Direct Antiglobulin Test (DAT) in blood donors. Antibody screening was performed using commercial pooled "O" cells and three-cell panel for donors and patients respectively and an 11-cell panel for identification using Column Agglutination Technology (CAT) with Low-Ionic-Strength Saline-Indirect Antiglobulin Test (LISS-IAT) technique (Ortho Clinical Diagnostics, Johnson & Johnson, USA). The cassettes used were Anti-human Globulin (AHG) type. DAT on donors was also performed using the AHG cassettes. Cumulative incidence of irregular antibodies amongst patients (n=32,560) and donors (n=31,367) were 0.12-0.009%, respectively. In patients, the commonest antibodies were from Rh system with anti-D being the most common antibody type (14/40; 35%) while in donors it was the MNS system with anti M (2/3) being the most common. Incidence of a positive DAT amongst the healthy blood donors was 0.04% (n=13/32,560). Incidence of irregular antibodies in patients at 0.12% was slightly lower than published reports and could be because of the fact that the patient population studied comprised largely of surgical patients many of whom have never received blood transfusion. This data could also be more representative since the number of patients studied was much higher as compared to the previous Indian studies (32,560 vs. 531-2026). The report on incidence of irregular antibodies and DAT (0.009-0.04%) in blood donors was first such attempt in India. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Validation of anti-FXR1 antibodies in the canine species and application to an immunohistochemical study of canine oral melanomas

    Directory of Open Access Journals (Sweden)

    Laura Nordio

    2017-05-01

    Full Text Available FXR1 (Fragile X mental retardation-related protein 1 is a cytoplasmic RNA binding protein, which genetic expression has been related to metastatic potential in human melanoma. The aims of the present study were: the validation of two commercially available clones of polyclonal anti-human FXR1 antibody in dogs; their application to investigate FXR1 expression in a group of canine oral melanomas. Anti-FXR1 antibody was not previously validated in the canine species. Two different commercially available polyclonal anti-FXR1 antibodies (respectively made in goat and in rabbit were used. FXR1 protein in canine serum was identified by western blot after SDS-PAGE, using human serum as control. FXR1 immunohistochemical expression was tested in a series of normal tissues, that are expected to express FXR1, and in 31 cases of oral melanomas. The final immunohistochemical protocol used heat-induced unmasking and overnight incubation. FXR1 protein bands in canine serum were detected by tested antibodies, in a more specific way by the rabbit antibody. FXR1 immunohistochemical staining was positive in all tested organs, with different levels of expression. FXR1 was also expressed in 31/31 tested melanomas, with variable intensity and percentage of positive cells (Figure 1. Equal results were achieved with the two antibodies in 8 cases of melanoma, whereas there were variable differences in 22, and one case stained only with goat antibody. The rabbit antibody gave less background staining. This study validated anti-FXR1 antibodies for use in the canine species. This protein was expressed in various normal tissues, as well as in the tested neoplasms. Significance of different level of expression is undergoing evaluation with further studies.

  6. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna

    2015-01-01

    and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps...... elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity...... of the humanization experiment protocol....

  7. Development of ELISAs for quantification of HMFG1-specific human anti-mouse IgG and IgM antibodies.

    Science.gov (United States)

    Oei, A L M; Boerman, O C; Geurts-Moespot, A; van Eerd, J E; van Tienoven, D; Courtenay-Luck, N; Thomas, C M G; Massuger, L F A G; Sweep, F C G J

    2007-01-01

    The aim of this study was to develop and validate ELISAs for quantification of HAMA-IgM and HAMA-IgG in serum of patients with ovarian cancer who enrolled in a large international randomized phase III trial of intraperitoneal Yttrium-90-labeled HMFG1 murine monoclonal antibody therapy. The capture antibody of these 2 assays was the murine antibody HMFG1, while mouse anti-human IgM-HRP or mouse anti-human IgG(Fc)-HRP served as tracer antibodies. A pool of HAMA-positive serum samples was used to prepare a series of assay standards and another pool served as reference preparation. The analytical sensitivity of the HAMA-IgM assay was 2.5 arbitrary units per mL (AU/mL) and 4.7 AU/mL for the HAMA-IgG ELISA. Diluted serum samples showed good parallelism with the HAMA-IgM and HAMA-IgG standard dose-response curves. Within-assay coefficient of variation was 7.5% for HAMA-IgM and 6.5% for HAMA-IgG. Between-assay variation was 14.2% for HAMA-IgM and 15.3% for HAMA-IgG. The developed HAMA-IgM and HAMA-IgG ELISAs show satisfactory reliability criteria (sensitivity, parallelism and precision) and are suitable for monitoring of HAMA-IgM and HAMA-IgG responses in ovarian cancer patients. These ELISAs will be used to monitor the development of HAMAs in patients who received radioimmunotherapy with murine HMFG1.

  8. Oriented immobilized anti-LDL antibody carrying poly(hydroxyethyl methacrylate) cryogel for cholesterol removal from human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Bereli, Nilay [Department of Chemistry, Hacettepe University, Beytepe, Ankara (Turkey); Sener, Guelsu [Nanotechnology and Nanomedicine Division, Hacettepe University, Ankara (Turkey); Yavuz, Handan, E-mail: handany@hacettepe.edu.tr [Department of Chemistry, Hacettepe University, Beytepe, Ankara (Turkey); Denizli, Adil [Department of Chemistry, Hacettepe University, Beytepe, Ankara (Turkey)

    2011-07-20

    Low density lipoprotein (LDL) cholesterol is a major ingredient of the plaque that collects in the coronary arteries and causes coronary heart diseases. Among the methods used for the extracorporeal elimination of LDL from intravasal volume, immunoaffinity technique using anti-LDL antibody as a ligand offers superior selectivity and specificity. Proper orientation of the immobilized antibody is the main issue in immunoaffinity techniques. In this study, anti-human {beta}-lipoprotein antibody (anti-LDL antibody) molecules were immobilized and oriented through protein A onto poly(2-hydroxyethyl methacrylate) (PHEMA) cryogel in order to remove LDL from hypercholesterolemic human plasma. PHEMA cryogel was prepared by free radical polymerization initiated with N,N,N',N'-tetramethylene diamine (TEMED). PHEMA cryogel with a swelling degree of 8.89 g H{sub 2}O/g and 67% macro-porosity was characterized by swelling studies, scanning electron microscope (SEM) and blood compatibility tests. All the clotting times were increased when compared with control plasma. The maximum immobilized anti-LDL antibody amount was 63.2 mg/g in the case of random antibody immobilization and 19.6 mg/g in the case of oriented antibody immobilization (protein A loading was 57.0 mg/g). Random and oriented anti-LDL antibody immobilized PHEMA cryogels adsorbed 111 and 129 mg LDL/g cryogel from hypercholesterolemic human plasma, respectively. Up to 80% of the adsorbed LDL was desorbed. The adsorption-desorption cycle was repeated 6 times using the same cryogel. There was no significant loss of LDL adsorption capacity. - Research highlights: {yields} LDL cholesterol is a risk factor in the development of coronary heart diseases. {yields} Antibodies against LDL are used for the selective extracorporeal removal of LDL. {yields} Protein A is used for the oriented immobilization of anti LDL onto PHEMA cryogel. {yields} PHEMA cryogels are biocompatible, exhibit a low pressure drop, lack diffusion

  9. Oriented immobilized anti-LDL antibody carrying poly(hydroxyethyl methacrylate) cryogel for cholesterol removal from human plasma

    International Nuclear Information System (INIS)

    Bereli, Nilay; Sener, Guelsu; Yavuz, Handan; Denizli, Adil

    2011-01-01

    Low density lipoprotein (LDL) cholesterol is a major ingredient of the plaque that collects in the coronary arteries and causes coronary heart diseases. Among the methods used for the extracorporeal elimination of LDL from intravasal volume, immunoaffinity technique using anti-LDL antibody as a ligand offers superior selectivity and specificity. Proper orientation of the immobilized antibody is the main issue in immunoaffinity techniques. In this study, anti-human β-lipoprotein antibody (anti-LDL antibody) molecules were immobilized and oriented through protein A onto poly(2-hydroxyethyl methacrylate) (PHEMA) cryogel in order to remove LDL from hypercholesterolemic human plasma. PHEMA cryogel was prepared by free radical polymerization initiated with N,N,N',N'-tetramethylene diamine (TEMED). PHEMA cryogel with a swelling degree of 8.89 g H 2 O/g and 67% macro-porosity was characterized by swelling studies, scanning electron microscope (SEM) and blood compatibility tests. All the clotting times were increased when compared with control plasma. The maximum immobilized anti-LDL antibody amount was 63.2 mg/g in the case of random antibody immobilization and 19.6 mg/g in the case of oriented antibody immobilization (protein A loading was 57.0 mg/g). Random and oriented anti-LDL antibody immobilized PHEMA cryogels adsorbed 111 and 129 mg LDL/g cryogel from hypercholesterolemic human plasma, respectively. Up to 80% of the adsorbed LDL was desorbed. The adsorption-desorption cycle was repeated 6 times using the same cryogel. There was no significant loss of LDL adsorption capacity. - Research highlights: → LDL cholesterol is a risk factor in the development of coronary heart diseases. → Antibodies against LDL are used for the selective extracorporeal removal of LDL. → Protein A is used for the oriented immobilization of anti LDL onto PHEMA cryogel. → PHEMA cryogels are biocompatible, exhibit a low pressure drop, lack diffusion resistance and viscous samples can be

  10. Theranostics Using Antibodies and Antibody-Related Therapeutics

    NARCIS (Netherlands)

    Moek, Kirsten L; Giesen, Danique; Kok, Iris C; de Groot, Derk Jan A; Jalving, Mathilde; Fehrmann, Rudolf S N; Lub-de Hooge, Marjolijn N; Brouwers, Adrienne H; de Vries, Elisabeth G E

    In theranostics, radiolabeled compounds are used to determine a treatment strategy by combining therapeutics and diagnostics in the same agent. Monoclonal antibodies (mAbs) and antibody-related therapeutics represent a rapidly expanding group of cancer medicines. Theranostic approaches using these

  11. Antibodies and Plasmodium falciparum merozoites

    NARCIS (Netherlands)

    Ramasamy, R; Ramasamy, M; Yasawardena, S

    There is considerable interest in using merozoite proteins in a vaccine against falciparum malaria. Observations that antibodies to merozoite surface proteins block invasion are a basis for optimism. This article draws attention to important and varied aspects of how antibodies to Plasmodium

  12. Catalytic Antibodies: Concept and Promise

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 11. Catalytic Antibodies: Concept and Promise. Desirazu N Rao Bharath Wootla. General Article Volume 12 Issue ... Keywords. Catalytic antibodies; abzymes; hybridome technology; Diels– Alder reaction; Michaelis– Menten kinetics; Factor VIII.

  13. Antiphospholipid antibodies: standardization and testing.

    Science.gov (United States)

    Riley, R S; Friedline, J; Rogers, J S

    1997-09-01

    A phenomenon originally scorned as a laboratory nuisance has turned out to be an important cause of thromboembolism, fetal death, and other forms of human disease. Investigations of this inaptly named "lupus anticoagulant" has led to the discovery of at least two distinct types of autoimmune antibodies. In spite of recent discoveries regarding the pathophysiology of these antibodies, their clinical significance is still controversial.

  14. Educational paper: Primary antibody deficiencies

    NARCIS (Netherlands)

    G.J.A. Driessen (Gertjan); M. van der Burg (Mirjam)

    2011-01-01

    textabstractPrimary antibody deficiencies (PADs) are the most common primary immunodeficiencies and are characterized by a defect in the production of normal amounts of antigen-specific antibodies. PADs represent a heterogeneous spectrum of conditions, ranging from often asymptomatic selective IgA

  15. [Antibody induction after intrauterine interventions].

    Science.gov (United States)

    Hoch, J; Giers, G; Bald, R; Hansmann, M; Hanfland, P

    1993-06-01

    Immunohematologic and clinical data, i.e., antibody profile, location of the placenta, mode of cordocentesis, obtained from 48 pregnant patients with irregular erythrocyte antibodies during the last 2 years have been retrospectively evaluated. All fetuses of the patients received intrauterine transfusions for the treatment of fetal erythroblastosis. In 16 (33%) patients (group I) a secondarily induced antibody was detected after the onset of intrauterine transfusion therapy. 32 (67%) patients (group II) did not further develop new antibody specificities. Group I exhibited a significantly different distribution in the location of the placenta (p pregnant women. In group I a 5-fold higher rate of anterior than posterior placenta location was found. The mode of cordocentesis differed significantly (p antibodies by invasive intrauterine interventions in our patients depended indirectly on the location of the placenta and directly on the mode of the puncture (trans- vs. paraplacental access).

  16. Thermodynamic and kinetic approaches for evaluation of monoclonal antibody - Lipoprotein interactions.

    Science.gov (United States)

    Multia, Evgen; Sirén, Heli; Andersson, Karl; Samuelsson, Jörgen; Forssén, Patrik; Fornstedt, Torgny; Öörni, Katariina; Jauhiainen, Matti; Riekkola, Marja-Liisa

    2017-02-01

    Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low-Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Correlation between levels of human papillomavirus (HPV)-16 and 18 antibodies in serum and cervicovaginal secretions in girls and women vaccinated with the HPV-16/18 AS04-adjuvanted vaccine.

    Science.gov (United States)

    Schwarz, Tino F; Kocken, Mariëlle; Petäjä, Tiina; Einstein, Mark H; Spaczynski, Marek; Louwers, Jacqueline A; Pedersen, Court; Levin, Myron; Zahaf, Toufik; Poncelet, Sylviane; Hardt, Karin; Descamps, Dominique; Dubin, Gary

    2010-12-01

    This pooled analysis of data from four Phase III clinical trials was undertaken to assess the correlation between levels of anti-human papillomavirus (HPV)-16/18 antibodies in serum and cervicovaginal secretions (CVS) in girls and women vaccinated with the HPV-16/18 AS04-adjuvanted vaccine. Serum and CVS samples were collected from a subset of women aged 10-65 years (N=350) at pre-specified time-points from 7 to 36 months post-vaccination. Anti-HPV-16/18 antibody levels in serum and CVS were measured by enzyme-linked immunosorbent assay. Pearson correlation coefficients between serum and CVS antibody levels, standardized for total immunoglobulin G, were calculated at each time-point in women with detectable antibodies in both serum and CVS. All subjects had seroconverted at Month 7 and remained seropositive through Month 36 for both antigens. Geometric mean titers of anti-HPV-16/18 antibodies in serum were substantially higher at all time-points than those in a control group of women who had cleared a natural HPV infection in another trial. In women with detectable antibodies in both serum and CVS, good correlation was seen between HPV-16/18 antibody levels at all time-points (Pearson correlation coefficient: 0.84-0.92 for HPV-16 and 0.90-0.91 for HPV-18). The strong correlation between levels of HPV-16/18 antibodies in serum and CVS up to 36 months post-vaccination in girls and women vaccinated with the HPV-16/18 AS04-adjuvanted vaccine supports transudation of serum antibodies as the mechanism by which antibodies are introduced into CVS. These CVS antibodies may play a role in the protective efficacy of this vaccine.

  18. 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.

  19. The humanized anti-human AMHRII mAb 3C23K exerts an anti-tumor activity against human ovarian cancer through tumor-associated macrophages.

    Science.gov (United States)

    Bougherara, Houcine; Némati, Fariba; Nicolas, André; Massonnet, Gérald; Pugnière, Martine; Ngô, Charlotte; Le Frère-Belda, Marie-Aude; Leary, Alexandra; Alexandre, Jérôme; Meseure, Didier; Barret, Jean-Marc; Navarro-Teulon, Isabelle; Pèlegrin, André; Roman-Roman, Sergio; Prost, Jean-François; Donnadieu, Emmanuel; Decaudin, Didier

    2017-11-21

    Müllerian inhibiting substance, also called anti-Müllerian hormone (AMH), inhibits proliferation and induces apoptosis of AMH type II receptor-positive tumor cells, such as human ovarian cancers (OCs). On this basis, a humanized glyco-engineered monoclonal antibody (3C23K) has been developed. The aim of this study was therefore to experimentally confirm the therapeutic potential of 3C23K in human OCs. We first determined by immunofluorescence, immunohistochemistry and cytofluorometry analyses the expression of AMHRII in patient's tumors and found that a majority (60 to 80% depending on the detection technique) of OCs were positive for this marker. We then provided evidence that the tumor stroma of OC is enriched in tumor-associated macrophages and that these cells are responsible for 3C23K-induced killing of tumor cells through ADCP and ADCC mechanisms. In addition, we showed that 3C23K reduced macrophages induced-T cells immunosuppression. Finally, we evaluated the therapeutic efficacy of 3C23K alone and in combination with a carboplatin-paclitaxel chemotherapy in a panel of OC Patient-Derived Xenografts. In those experiments, we showed that 3C23K significantly increased the proportion and the quality of chemotherapy-based in vivo responses. Altogether, our data support the potential interest of AMHRII targeting in human ovarian cancers and the evaluation of 3C23K in further clinical trials.

  20. 125I anti-immunoglobulin binding assay for the detection and characterization of anti-platelet antibodies

    International Nuclear Information System (INIS)

    Kirkley, J.; Fabre, J.W.

    1980-01-01

    The binding assay as described in this paper is a very versatile system, and in this study it has been evaluated specifically for the detection of allo- or autoantibodies to platelets in man. The basic assay involves the incubation of a standard number of platelets with dilutions of test sera and the detection of platelet bound immunoglobulin by a second incubation with 125I labeled, immunoadsorbent purified rabbit F(ab') anti-human F(ab')2 (RAH). Of most importance, by varying the number of target platelets in the titrations and looking for binding plateaus, one can readily define conditions of optimum sensitivity for particular serum/platelet combinations. In addition, the assay can be used in conjunction with quantitative absorptions to subdivide complex sera into subspecificities and to give an estimate of the relative amounts of particular antigens on different platelets or other tissue or cell suspensions. One can also use saturating concentrations of RAH in the second incubation, in which case the amount of platelet bound radioactivity is directly related to the amount of first antibody bound to the platelets, and this can be manipulated to give information about serum antibody concentrations and amounts of antigen on the target tissue. The problem of ABO antibodies in this system, optimal conditions for platelet storage for the assay, and techniques for reducing assay backgrounds resulting from immunoglobulin adsorbed to the platelet surface are all evaluated

  1. Dissecting Immunogenicity of Monoclonal Antibodies

    National Research Council Canada - National Science Library

    Snyder, Christopher

    2002-01-01

    The potential of monoclonal antibodies, (mAbs), for use in therapeutic and diagnostic applications has not been fully realized in part due to counter-immune responses that often arise in patient recipients of mAb...

  2. Dissecting Immunogenicity of Monoclonal Antibodies

    National Research Council Canada - National Science Library

    Snyder, Christopher

    2003-01-01

    The potential of monoclonal antibodies, (mAbs), for use in therapeutic and diagnostic applications has not been fully realized in part due to counter-immune responses that often arise in patient recipients of mAb...

  3. Antisperm antibodies and fertility association.

    Science.gov (United States)

    Restrepo, B; Cardona-Maya, W

    2013-10-01

    To evaluate the relation between antisperm antibodies (ASA) and human fertility by reviewing the scientific literature of the last 45 years. We carried out a review of scientific literature about antisperm antibodies and infertility published in spanish or english in databases as Pubmed, Medline, Scielo, some books and another gray literature include information related to this review and that is published in the last 45 years. Infertile couples suffer infertility by immunological mechanisms mainly by the presence of antisperm antibodies ASA in blood, semen or cervicovaginal secretions; the formation of ASA in men and women may be associated with disturbance in immunomodulatory mechanisms that result in functional impairment of sperm and thus its inability to fertilize the oocyte. Immunological infertility caused by ASA is the result of interference of these antibodies in various stages of fertilization process, inhibiting the ability of interaction between sperm and oocyte. Copyright © 2012 AEU. Published by Elsevier Espana. All rights reserved.

  4. Antibody Drug Conjugates: Preclinical Considerations.

    Science.gov (United States)

    Bornstein, Gadi G

    2015-05-01

    The development path for antibody drug conjugates (ADCs) is more complex and challenging than for unmodified antibodies. While many of the preclinical considerations for both unmodified and antibody drug conjugates are shared, special considerations must be taken into account when developing an ADC. Unlike unmodified antibodies, an ADC must preferentially bind to tumor cells, internalize, and traffic to the appropriate intracellular compartment to release the payload. Parameters that can impact the pharmacological properties of this class of therapeutics include the selection of the payload, the type of linker, and the methodology for payload drug conjugation. Despite a plethora of in vitro assays and in vivo models to screen and evaluate ADCs, the challenge remains to develop improved preclinical tools that will be more predictive of clinical outcome. This review will focus on preclinical considerations for clinically validated small molecule ADCs. In addition, the lessons learned from Mylotarg®, the first in class FDA-approved ADC, are highlighted.

  5. Monoclonal antibodies technology. Protocols

    International Nuclear Information System (INIS)

    Acevado Castro, B.E.

    1997-01-01

    Full text: Immunization. The first step in preparing useful monoclonal antibodies (MAbs) is to immunize an animal (Balb/c for example) with an appropriate antigen. Methods (only for soluble antigen): Solubilize selected antigen in Phosphate buffer solution (PBS) at pH 7.2-7.4, ideally at a final concentration per animal between 10 to 50 μg/ml. It is recommended that the antigen under consideration be incorporated into the emulsion adjuvants in 1:1 volumetric relation. We commonly use Frend's adjuvant (FA) to prepared immunized solution. The first immunization should be prepared with complete FA, and the another could be prepared with incomplete FA. It is recommended to inject mice with 0.2 ml intraperitoneal (ip) or subcutaneous (sc). Our experience suggests the sc route is the preferred route. A minimum protocol for immunizing mice to generate cells for preparing hybridomas is s follows: immunize sc on day 0, boost sc on day 21, take a trial bleeding on day 26; if antibody titters are satisfactory, boost ip on day 35 with antigen only, and remove the spleen to obtain cells for fusion on day 38. Fusion protocol. The myeloma cell line we are using is X63 Ag8.653. At the moment of fusion myeloma cells need a good viability (at least a 95%). 1. Remove the spleen cells from immunized mice using sterile conditions. An immune spleen should yield between 7 a 10x10 7 nucleated cells. 2. Place the spleen in 20 ml of serum-free RPMI 1640 in a Petri dish. Using a needle and syringe, inject the spleen with medium to distend and disrupt the spleen stroma and free the nucleated cells. 3. Flush the cell suspension with a Pasteur pipet to disperse clumps of cells. 4. Centrifuge the spleen cell suspension at 250g for 10 min. Resuspend the pellet in serum-free RPMI 1640. Determine cell concentration using Neuhabuer chamber. 5. Mix the myeloma cells and spleen cells in a conical 50-ml tube in serum-free RPMI 1640, 1 x10 7 spleen cells to 1x10 6 myeloma cells (ratio 10:1). Centrifuge

  6. Radiolabeled monoclonal antibodies: a review

    International Nuclear Information System (INIS)

    Toledo e Souza, I.T. de; Okada, H.

    1990-05-01

    Since the description by Kohler and Milstein 1975 of their technique for producing monoclonal antibodies of predefined specificity, it has become a mainstay in most laboratories that utilize immunochemical techniques to study problems in basic, applied or clinical research. Paradoxically, the very success of monoclonal antibodies has generated a literature which is now so vast and scattered that it has become difficult to obtain a perspective. This brief review represents the distillation of many publications relating to the production and use of monoclonaal antibodies as radiopharmaceuticals. Significant advances were made possible in the last few years by combined developments in the fields of tumor-associated antigens and of monoclonal antibodies. In fact monoclonal antibodies against some well defined tumor-associated antigens, has led to significantly greater practical possibilities for producing highly specific radiolabeled antibodies as radiopharmaceuticals for diagnosis and therapy of human tumors. One of the main requirements of this methodology is the availability of stable radiopharmaceutical reagents which after labeling in vivo injection retain the capacity of specific interaction with the defined antigen and their molecular integrity. Since injection into human is the objetive of this kind of study all the specifications of radiopharmaceutical have to be fulfilled e.g. sterility, apirogenicity and absence of toxicity. (author) [pt

  7. Avian Diagnostic and Therapeutic Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, David Sherman [UND SMHS

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  8. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo

    2014-10-09

    SUMMARY: Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: http://www.biocomputing.it/tabhu CONTACT: anna.tramontano@uniroma1.it, pierpaolo.olimpieri@uniroma1.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  10. Prevalence of human anti-mouse antibodies (HAMAs) in routine examinations.

    Science.gov (United States)

    Koshida, Saori; Asanuma, Koichi; Kuribayashi, Kageaki; Goto, Maki; Tsuji, Naoki; Kobayashi, Daisuke; Tanaka, Maki; Watanabe, Naoki

    2010-03-01

    Circulating heterophilic antibodies interfere with immunological assays in laboratory examinations; however, their rate of incidence is currently questionable. We developed an enzyme-linked immunosorbent assay (ELISA) to detect human anti-mouse antibodies (HAMAs) in routine examinations. The study samples were comprised of serum samples obtained from 290 inpatients and outpatients at our hospital. Mouse immunoglobulin G1 (mIgG1), mIgG2a, and mIgG2b were used as the antigens and horseradish peroxidase (HRP)-conjugated anti-human IgG and IgM were used to identify the HAMA isotype. HAMAs were detected in 11.7% (34/290) of the samples. We observed 18 and 20 samples positive for IgG- and IgM-type HAMAs, respectively. Four samples contained both IgG- and IgM-type HAMAs. HAMAs against mIgG1, mIgG2a, and mIgG2b were found in 21, 14, and 13 samples, respectively. Existence of HAMAs was confirmed by western blotting using mIgG's as the antigens and HAMAs as the primary antibodies. Heterophilic blocking reagent (HBR) was also used to block the heterophilic interactions. Unexpectedly, a low HBR concentration rather enhanced the interactions instead of blocking them. A considerable number of HAMA-positive samples, reacting with the heavy chain of mIg, were found in routine examinations. A sufficient amount of HBR should be used for blocking the heterophilic interactions. Copyright 2009 Elsevier B.V. All rights reserved.

  11. Antibodies to human serum amyloid P component eliminate visceral amyloid deposits.

    Science.gov (United States)

    Bodin, Karl; Ellmerich, Stephan; Kahan, Melvyn C; Tennent, Glenys A; Loesch, Andrzej; Gilbertson, Janet A; Hutchinson, Winston L; Mangione, Palma P; Gallimore, J Ruth; Millar, David J; Minogue, Shane; Dhillon, Amar P; Taylor, Graham W; Bradwell, Arthur R; Petrie, Aviva; Gillmore, Julian D; Bellotti, Vittorio; Botto, Marina; Hawkins, Philip N; Pepys, Mark B

    2010-11-04

    Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one in a thousand deaths in developed countries. Localized amyloid can also have serious consequences; for example, cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present. There is therefore a major unmet need for therapy that safely promotes the clearance of established amyloid deposits. Over 20 different amyloid fibril proteins are responsible for different forms of clinically significant amyloidosis and treatments that substantially reduce the abundance of the respective amyloid fibril precursor proteins can arrest amyloid accumulation. Unfortunately, control of fibril-protein production is not possible in some forms of amyloidosis and in others it is often slow and hazardous. There is no therapy that directly targets amyloid deposits for enhanced clearance. However, all amyloid deposits contain the normal, non-fibrillar plasma glycoprotein, serum amyloid P component (SAP). Here we show that administration of anti-human-SAP antibodies to mice with amyloid deposits containing human SAP triggers a potent, complement-dependent, macrophage-derived giant cell reaction that swiftly removes massive visceral amyloid deposits without adverse effects. Anti-SAP-antibody treatment is clinically feasible because circulating human SAP can be depleted in patients by the bis-d-proline compound CPHPC, thereby enabling injected anti-SAP antibodies to reach residual SAP in the amyloid deposits. The unprecedented capacity of this novel combined therapy to eliminate amyloid deposits should be applicable to all forms of systemic and local amyloidosis.

  12. The preparation, characterization, and application of environment-friendly monoclonal antibodies for human blood cell.

    Science.gov (United States)

    Zhou, Chenjie; Gao, Xuechao; He, Shixiang; Gao, Xiaoling; Zhuang, Jialin; Huang, Lirong; Guo, Hengchang

    2017-03-01

    Monoclonal anti-human blood group A (51A8) and B (63B6) antibody reagents were prepared using the serum-free technique. The aims of this research were to characterize the serum-free reagents and prove their reliabilities in routine use. Experiments including antigen-antibody agglutination testing, stability testing, SDS-PAGE, protein and IgM quantification, flow cytometry, and variable domain sequencing were performed to characterize the anti-A (51A8) and anti-B (63B6) reagents. Over 12 000 samples were tested using these reagents as routine blood grouping reagents. Serum-free anti-A (51A8) and anti-B (63B6) reagents were stable in longitudinal and accelerated testing, and their high purity was shown in SDS-PAGE and IgM quantification. These reagents have high specificity to red blood cells in serologic agglutination testing and flow cytometric analysis. A1 and A2 subgroup antigens can be distinguished clearly by patterns of flow cytometric histograms. No discrepancy was found in clinical trials of 12 000 samples. To reduce the risk of being affected by any animal additives, a serum-free culture system was applied to get mass-production of monoclonal anti-A/B antibodies. The high specificity and the high purity of the reagents were verified by the lab experiments. Lab research and clinical trial showed that serum-free monoclonal anti-A (51A8) and anti-B (63B6) reagents meet the requirements of routine blood grouping reagents. Moreover, these reagents featured ultra-high purity that is missing in other commercial counterparts, and therefore are recommended as more environment-friendly reagents.

  13. Antibody tagged gold nanoparticles as scattering probes for the pico molar detection of the proteins in blood serum using nanoparticle tracking analyzer.

    Science.gov (United States)

    Kashid, Sahebrao Balaso; Tak, Rajesh D; Raut, Rajesh Warluji

    2015-09-01

    We report a rapid one-step immunoassay to detect protein using antibody conjugated gold nanoparticles (AbGNPs) where the targeted protein concentration was determined by analyzing the gold nanoparticle aggregation caused by antibody-antigen interactions using nanoparticles tracking analysis (NTA) technique. The sandwich structure constituting the binding of the targeted human IgG to the gold nanoparticle conjugates with goat anti human monoclonal IgG (AbGNPs) was confirmed by transmission electron microscopy. The binding of human IgG (antigen, mentioned hence forth as AT) induce AbGNPs to form dimers or trimers through a typical antibody-antigen-antibody sandwich structure that can be analyzed for the sensitive determination on the basis of change in hydrodynamic diameter of AbGNPs. By this method the minimum detectable concentration of AT is found to be below 2pg/ml. We expect that a significant change in the hydrodynamic diameter of AbGNP could form the basis for the rapid one-step immunoassay development. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Monoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133.

    Directory of Open Access Journals (Sweden)

    Kristina Thamm

    Full Text Available The pentaspan membrane glycoprotein prominin-1 (CD133 is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.

  15. Replacing reprogramming factors with antibodies selected from combinatorial antibody libraries.

    Science.gov (United States)

    Blanchard, Joel W; Xie, Jia; El-Mecharrafie, Nadja; Gross, Simon; Lee, Sohyon; Lerner, Richard A; Baldwin, Kristin K

    2017-10-01

    The reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) is usually achieved by exogenous induction of transcription by factors acting in the nucleus. In contrast, during development, signaling pathways initiated at the membrane induce differentiation. The central idea of this study is to identify antibodies that can catalyze cellular de-differentiation and nuclear reprogramming by acting at the cell surface. We screen a lentiviral library encoding ∼100 million secreted and membrane-bound single-chain antibodies and identify antibodies that can replace either Sox2 and Myc (c-Myc) or Oct4 during reprogramming of mouse embryonic fibroblasts into iPSCs. We show that one Sox2-replacing antibody antagonizes the membrane-associated protein Basp1, thereby de-repressing nuclear factors WT1, Esrrb and Lin28a (Lin28) independent of Sox2. By manipulating this pathway, we identify three methods to generate iPSCs. Our results establish unbiased selection from autocrine combinatorial antibody libraries as a robust method to discover new biologics and uncover membrane-to-nucleus signaling pathways that regulate pluripotency and cell fate.

  16. Electrochemical aptamer/antibody based sandwich immunosensor for the detection of EGFR, a cancer biomarker, using gold nanoparticles as a signaling probe.

    Science.gov (United States)

    Ilkhani, Hoda; Sarparast, Morteza; Noori, Abolhassan; Zahra Bathaie, S; Mousavi, Mir F

    2015-12-15

    Detection of epidermal growth factor receptor (EGFR) in biological fluids is of paramount importance, since it has significant application in cancer diagnosis, drug development, and therapy monitoring. EGFR is a cancer biomarker, and its overexpression is associated with the development of some types of cancer. Herein, we report on the development of a sensitive and selective electrochemical aptamer/antibody (Apt/Ab) sandwich immunosensor for detection of EGFR. In this study, a biotinylated anti-human EGFR Apt was immobilized on streptavidin-coated magnetic beads (MB) and served as a capture probe. A polyclonal anti-human EGFR Ab was conjugated to citrate-coated gold nanoparticles (AuNPs) and used as a signaling probe. In the presence of EGFR, an Apt-EGFR-Ab sandwich was formed on the MB surface. The extent of the complexation was evaluated by differential pulse voltammetry of AuNPs after their dissolution in HCl. Under optimal conditions, the dynamic concentration range of the immunosensor for EGFR spanned from 1 to 40 ng/mL, with a low detection limit of 50 pg/mL, and RSD percent of less than 4.2%. The proposed approach takes advantage of sandwich assay for high specificity, MBs for fast separation, and electrochemical method for cost-effective and sensitive detection. In this proof-of-principle study, we demonstrate the potential clinical efficacy of the immunosensor for monitoring of chemotherapy effectiveness in breast cancer samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Radioiodination of antibodies for tumor imaging

    International Nuclear Information System (INIS)

    Saha, G.B.

    1983-01-01

    In view of the great potential of radioiodinated antibody for the detection and treatment of cancer, the present article deals with the various techniques of radioiodination of antibody and their uses. Topics include methods of iodination of antibody, advantages and disadvantages of different methods, and effects of radioiodination on the antibody molecules with respect to their physiochemical and immunologic reactivity. In addition, the clinical usefulness of radioiodinated antibodies is discussed. (Auth.)

  18. Antibodies from plants for bionanomaterials.

    Science.gov (United States)

    Edgue, Gueven; Twyman, Richard M; Beiss, Veronique; Fischer, Rainer; Sack, Markus

    2017-11-01

    Antibodies are produced as part of the vertebrate adaptive immune response and are not naturally made by plants. However, antibody DNA sequences can be introduced into plants, and together with laboratory technologies that allow the design of antibodies recognizing any conceivable molecular structure, plants can be used as 'green factories' to produce any antibody at all. The advent of plant-based transient expression systems in particular allows the rapid, convenient, and safe production of antibodies, ranging from laboratory-scale expression to industrial-scale manufacturing. The key features of plant-based production include safety, speed, low cost, and convenience, allowing newcomers to rapidly master the technology and use it to its full advantage. Manufacturing in plants has recently achieved significant milestones and offers more than just an alternative to established microbial and mammalian cell platforms. The use of plants for product development in particular offers the power and flexibility to easily coexpress many different genes, allowing the plug-and-play construction of novel bionanomaterials, perfectly complementing existing approaches based on plant virus-like particles. As well as producing single antibodies for applications in medicine, agriculture, and industry, plants can be used to produce antibody-based supramolecular structures and scaffolds as a new generation of green bionanomaterials that promise a bright future based on clean and renewable nanotechnology applications. WIREs Nanomed Nanobiotechnol 2017, 9:e1462. doi: 10.1002/wnan.1462 For further resources related to this article, please visit the WIREs website. © 2017 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals, Inc.

  19. Antibody-Directed Phototherapy (ADP

    Directory of Open Access Journals (Sweden)

    M. Adil Butt

    2013-04-01

    Full Text Available Photodynamic therapy (PDT is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs, which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs.

  20. Antibody Validation by Western Blotting.

    Science.gov (United States)

    Signore, Michele; Manganelli, Valeria; Hodge, Alex

    2017-01-01

    Validation of antibodies is an integral part of translational research, particularly for biomarker discovery. Assaying the specificity of the reagent (antibody) and confirming the identity of the protein biomarker is of critical importance prior to implementing any biomarker in clinical studies, and the lack of such quality control tests may result in unexpected and/or misleading results.Antibody validation is the procedure in which a single antibody is thoroughly assayed for sensitivity and specificity. Although a plethora of commercial antibodies exist, antibody specificity must be extensively demonstrated using diverse complex biological samples, rather than purified recombinant proteins, prior to use in clinical translational research. In the simplest iteration, antibody specificity is determined by the presence of a single band in a complex biological sample, at the expected molecular weight, on a Western blot.To date, numerous Western blotting procedures are available, based on either manual or automated systems and spanning the spectrum of single blots to multiplex blots. X-ray film is still employed in many research laboratories, but digital imaging has become a gold standard in immunoblotting. The basic principles of Western blotting are (a) separation of protein mixtures by gel electrophoresis, (b) transfer of the proteins to a blot, (c) probing the blot for a protein or proteins of interest, and (d) subsequent detection of the protein by chemiluminescent, fluorescent, or colorimetric methods. This chapter focuses on the chemiluminescent detection of proteins using a manual Western blotting system and a vacuum-enhanced detection system (SNAP i.d.™, Millipore).

  1. Antibodies: an alternative for antibiotics?

    Science.gov (United States)

    Berghman, L R; Abi-Ghanem, D; Waghela, S D; Ricke, S C

    2005-04-01

    In 1967, the success of vaccination programs, combined with the seemingly unstoppable triumph of antibiotics, prompted the US Surgeon General to declare that "it was time to close the books on infectious diseases." We now know that the prediction was overly optimistic and that the fight against infectious diseases is here to stay. During the last 20 yr, infectious diseases have indeed made a staggering comeback for a variety of reasons, including resistance against existing antibiotics. As a consequence, several alternatives to antibiotics are currently being considered or reconsidered. Passive immunization (i.e., the administration of more or less pathogen-specific antibodies to the patient) prior to or after exposure to the disease-causing agent is one of those alternative strategies that was almost entirely abandoned with the introduction of chemical antibiotics but that is now gaining interest again. This review will discuss the early successes and limitations of passive immunization, formerly referred to as "serum therapy," the current use of antibody administration for prophylaxis or treatment of infectious diseases in agriculture, and, finally, recent developments in the field of antibody engineering and "molecular farming" of antibodies in various expression systems. Especially the potential of producing therapeutic antibodies in crops that are routine dietary components of farm animals, such as corn and soy beans, seems to hold promise for future application in the fight against infectious diseases.

  2. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A; Thompson, Vicki S

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  3. Antibody profiling sensitivity through increased reporter antibody layering

    International Nuclear Information System (INIS)

    Apel, William A.; Thompson, Vickie S.

    2013-01-01

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  4. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  5. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S

    2010-04-13

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  6. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.

    2017-03-28

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  7. Hu and Yo antibodies have heterogeneous avidity.

    Science.gov (United States)

    Totland, Cecilie; Aarseth, Jan; Vedeler, Christian

    2007-04-01

    Onconeural antibodies such as anti-Hu and anti-Yo may be important in the pathogenesis of paraneoplastic neurological syndromes. The avidity of these antibodies is not known. In this study, we compared the avidity of Hu and Yo antibodies both at single time points and over a time range of 2 months to 6 years. The avidity of Yo and Hu antibodies differed among the patients, but anti-Yo generally had higher avidity than anti-Hu. Whether Yo antibodies are more pathogenic than Hu antibodies are presently unknown.

  8. High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

    Science.gov (United States)

    Batsuli, Glaivy; Deng, Wei; Healey, John F.; Parker, Ernest T.; Baldwin, W. Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete

    2016-01-01

    Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. PMID:27381905

  9. Differential ability of tissue factor antibody clones on detection of tissue factor in blood cells and microparticles.

    Science.gov (United States)

    Basavaraj, Manjunath Goolyam; Olsen, Jan Ole; Østerud, Bjarne; Hansen, John-Bjarne

    2012-09-01

    Tissue factor (TF), the primary initiator of coagulation in vivo, plays a major role in both thrombosis and hemostasis. The expression of TF in monocytes is well documented, but its presence in other blood cells has been disputed, possibly due to methodological variations among different studies. We studied TF expression on platelets, monocytes, lymphocytes and microparticles (MPs) by flow cytometry (FCM) with five commercially available mouse anti-human TF antibodies (HTF-1, TF9-10H10, CLB/TF-5, VIC7 and VD8). The ability of different TF antibodies to inhibit cell surface TF activity was explored by incubating LPS-stimulated monocytes and MPs derived from LPS-stimulated monocytes (MMPs) with TF antibodies followed by measuring TF activity. HTF-1 detected TF only on LPS-stimulated monocytes, whereas, TF9-10H10 and VD8 detected TF associated with MPs and MMPs in addition to LPS stimulated monocytes. Surprisingly, CLB/TF-5 and VIC7 detected TF on platelets, monocytes even under unstimulated conditions, in addition to MPs and MMPs. CLB/TF-5 also detected TF on unstimulated lymphocytes. Inhibitory studies showed that at a final concentration of 10 μg/mL, HTF-1, CLB/TF-5 and VD8 inhibited monocyte TF activity by 81-84% and MMP TF activity by 92-96%; whereas TF9-10H10 had no inhibitory effect on TF activity in monocytes and MMPs. Our results suggest non-specific binding by the CLB/TF-5 and VIC7 antibodies in a FCM test system and explain at least some of the reports on TF presence in blood cells, particularly TF associated with platelets and MPs. TF9-10H10 and VD8 are more suitable to detect TF on MPs by FCM. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors.

    Science.gov (United States)

    Batsuli, Glaivy; Deng, Wei; Healey, John F; Parker, Ernest T; Baldwin, W Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete; Meeks, Shannon L

    2016-10-20

    Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. © 2016 by The American Society of Hematology.

  11. Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

    Science.gov (United States)

    Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

    2015-01-01

    Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

  12. 9 CFR 113.452 - Erysipelothrix Rhusiopathiae Antibody.

    Science.gov (United States)

    2010-01-01

    ..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD... Antibody is a specific antibody product containing antibodies directed against one or more somatic antigens...

  13. Monoclonal antibodies to Treponema Pallidum.

    NARCIS (Netherlands)

    H.J.M. van de Donk; J.D.A. van Embden; M.F. van Olderen; A.D.M.E. Osterhaus (Albert); J.C. de Jong (Jan)

    1984-01-01

    textabstractThree successive fusions of mouse myeloma cells and spleen lymphocytes of a mouse immunized with Treponema Pallidum resulted in one hybridoma producing anti T. pallidum antibodies for each fusion. The mice were immunized with live pallidum cells respectively 1, 3 and 5 months before

  14. Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment

    International Nuclear Information System (INIS)

    Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

    2014-01-01

    Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies

  15. Selenoprotein P-neutralizing antibodies improve insulin secretion and glucose sensitivity in type 2 diabetes mouse models.

    Science.gov (United States)

    Mita, Yuichiro; Nakayama, Kaho; Inari, Shogo; Nishito, Yukina; Yoshioka, Yuya; Sakai, Naoko; Sotani, Kanade; Nagamura, Takahiro; Kuzuhara, Yuki; Inagaki, Kumi; Iwasaki, Miki; Misu, Hirofumi; Ikegawa, Masaya; Takamura, Toshinari; Noguchi, Noriko; Saito, Yoshiro

    2017-11-21

    Selenoprotein P (SeP) functions as a selenium (Se)-supply protein. SeP is identified as a hepatokine, promoting insulin resistance in type 2 diabetes. Thus, the suppression of Se-supply activity of SeP might improve glucose metabolism. Here, we develop an anti-human SeP monoclonal antibody AE2 as with neutralizing activity against SeP. Administration of AE2 to mice significantly improves glucose intolerance and insulin resistance that are induced by human SeP administration. Furthermore, excess SeP administration significantly decreases pancreas insulin levels and high glucose-induced insulin secretion, which are improved by AE2 administration. Epitope mapping reveals that AE2 recognizes a region of human SeP adjacent to the first histidine-rich region (FHR). A polyclonal antibody against the mouse SeP FHR improves glucose intolerance and insulin secretion in a mouse model of diabetes. This report describes a novel molecular strategy for the development of type 2 diabetes therapeutics targeting SeP.

  16. Radioimmunological determination of growth hormone antibodies

    International Nuclear Information System (INIS)

    Kracmar, P.; Hnikova, O.

    1979-01-01

    The method is based on the assumption of the presence of antibodies in the serum of the patient and the formation of the complex antibody-tracer ( 125 I-STH). For separation the principle is used of two antibodies and subsequent ultrafiltration with membrane ultrafilters. Clinical experience, reproducibility and the procedure recommended for simple monitoring and the determination of the amount of antibodies in the serum of patients are presented. (author)

  17. Antibody therapeutics - the evolving patent landscape.

    Science.gov (United States)

    Petering, Jenny; McManamny, Patrick; Honeyman, Jane

    2011-09-01

    The antibody patent landscape has evolved dramatically over the past 30 years, particularly in areas of technology relating to antibody modification to reduce immunogenicity in humans or improve antibody function. In some cases antibody techniques that were developed in the 1980s are still the subject of patent protection in the United States or Canada. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Antibodies against chromosomal beta-lactamase

    DEFF Research Database (Denmark)

    Giwercman, B; Rasmussen, J W; Ciofu, Oana

    1994-01-01

    A murine monoclonal anti-chromosomal beta-lactamase antibody was developed and an immunoblotting technique was used to study the presence of serum and sputum antibodies against Pseudomonas aeruginosa chromosomal group 1 beta-lactamase in patients with cystic fibrosis (CF). The serum antibody resp...... against the infection. On the other hand, immune complexes between the beta-lactamase and corresponding antibodies could play a role in the pathogenesis of bronchopulmonary injury in CF by mediating hyperimmune reactions....

  19. Radioimmunoassay method for detection of gonorrhea antibodies

    International Nuclear Information System (INIS)

    1975-01-01

    A novel radioimmunoassay for the detection of gonorrhea antibodies in serum is described. A radionuclide is bound to gonorrhea antigens produced by a growth culture. In the presence of gonorrhea antibodies in the serum, an antigen-antibody conjugate is formed, the concentration of which can be measured with conventional radiometric methods. The radioimmunoassay is highly specific

  20. Antibodies Against Melanin | Wassermann | South African Medical ...

    African Journals Online (AJOL)

    This study reports on unsuccessful attempts to produce antibodies against melanoprotein in rabbits. Available evidence suggests antibodies against melanocytes in the aetiology of vitiligo, but there is no convincing evidence for antibodies against melanin per se. It is suggested that the demonstration of antibodif's against ...

  1. Comparative analysis of bispecific antibody and streptavidin-targeted radioimmunotherapy for B cell cancers

    Science.gov (United States)

    Green, Damian J.; Frayo, Shani L.; Lin, Yukang; Hamlin, Donald K.; Fisher, Darrell R.; Frost, Sofia H.L.; Kenoyer, Aimee L.; Hylarides, Mark D.; Gopal, Ajay K.; Gooley, Theodore A.; Orozco, Johnnie J.; Till, Brian G.; O’Steen, Shyril; Orcutt, Kelly D.; Wilbur, D. Scott; Wittrup, K. Dane; Press, Oliver W.

    2016-01-01

    Streptavidin (SA)-biotin pretargeted radioimmunotherapy (PRIT) that targets CD20 in non-Hodgkin lymphoma (NHL) exhibits remarkable efficacy in model systems, but SA immunogenicity and interference by endogenous biotin may complicate clinical translation of this approach. In this study, we engineered a bispecific fusion protein (FP) that evades the limitations imposed by this system. Briefly, one arm of the FP was an anti-human CD20 antibody (2H7) with the other arm of the FP an anti-chelated radiometal trap for a radiolabeled ligand (yttrium[Y]-DOTA) captured by a very high-affinity anti-Y-DOTA scFv antibody (C825). Head-to-head biodistribution experiments comparing SA-biotin and bispecific FP (2H7-Fc-C825) PRIT in murine subjects bearing human lymphoma xenografts demonstrated nearly identical tumor targeting by each modality at 24 hrs. However, residual radioactivity in the blood and normal organs was consistently higher following administration of 1F5-SA compared to 2H7-Fc-C825. Consequently, tumor-to-normal tissue ratios of distribution were superior for 2H7-Fc-C825 (p<0.0001). Therapy studies in subjects bearing either Ramos or Granta subcutaneous lymphomas demonstrated that 2H7-Fc-C825 PRIT is highly effective and significantly less myelosuppressive than 1F5-SA (p<0.0001). All animals receiving optimal doses of 2H7-Fc-C825 followed by 90Y-DOTA were cured by 150 days, whereas the growth of tumors in control animals progressed rapidly with complete morbidity by 25 days. In addition to demonstrating reduced risk of immunogenicity and an absence of endogenous biotin interference, our findings offer a preclinical proof of concept for the preferred use of bispecific PRIT in future clinical trials, due to a slightly superior biodistribution profile, less myelosuppression and superior efficacy. PMID:27590740

  2. Detection of antibodies to co-trimoxazole (preservative drug interfering with routine red cell antibody screening

    Directory of Open Access Journals (Sweden)

    Deepti Sachan

    2018-01-01

    Full Text Available Drug-dependent antibodies can rarely cause interference in pretransfusion antibody screening. The diluents for commercial reagent red blood cells contain different antibiotics, such as chloramphenicol, neomycin sulfate, and gentamycin as a preservative. The presence of antibodies to a given drug in patient may lead to positive results when performing antibody identification. We present a rare case of detection of anti-co-trimoxazole antibody during routine antibody screening in a female patient undergoing neurosurgery. These antibodies mimicked as antibody against high-frequency red cell antigens reacting in both saline phase as well as antiglobulin phase. Anti-co-trimoxazole antibody was confirmed by repeating antibody screen using reagent red cells of different manufacturers with and without co-trimoxazole drug as preservative as well as using washed red cell panels. There were no associated clinical or laboratory evidence of hemolysis.

  3. T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab.

    Directory of Open Access Journals (Sweden)

    Usha Bughani

    Full Text Available CD6 is associated with T-cell modulation and is implicated in several autoimmune diseases. We previously demonstrated that Itolizumab, a CD6 domain 1 (CD6D1 specific humanized monoclonal antibody, inhibited the proliferation and cytokine production by T lymphocytes stimulated with anti-CD3 antibody or when co-stimulated with ALCAM. Aberrant IL-17 producing CD4+ helper T-cells (Th17 have been identified as pivotal for the pathogenesis of certain inflammatory autoimmune disorders, including psoriasis. Itolizumab has demonstrated efficacy in human diseases known to have an IL-17 driven pathogenesis. Here, in in vitro experiments we show that by day 3 of human PBMC activation using anti-CD3 and anti-CD28 co-stimulation in a Th17 polarizing milieu, 15-35% of CD4+ T-cells overexpress CD6 and there is an establishment of differentiated Th17 cells. Addition of Itolizumab reduces the activation and differentiation of T cells to Th17 cells and decreases production of IL-17. These effects are associated with the reduction of key transcription factors pSTAT3 and RORγT. Further, transcription analysis studies in these conditions indicate that Itolizumab suppressed T cell activation by primarily reducing cell cycle, DNA transcription and translation associated genes. To understand the mechanism of this inhibition, we evaluated the effect of this anti-human CD6D1 mAb on ALCAM-CD6 as well as TCR-mediated T cell activation. We show that Itolizumab but not its F(ab'2 fragment directly inhibits CD6 receptor hyper-phosphorylation and leads to subsequent decrease in associated ZAP70 kinase and docking protein SLP76. Since Itolizumab binds to CD6 expressed only on human and chimpanzee, we developed an antibody binding specifically to mouse CD6D1. This antibody successfully ameliorated the incidence of experimental autoimmune encephalitis in the mice model. These results position CD6 as a key molecule in sustaining the activation and differentiation of T cells and an

  4. Solid phase double-antibody radioimmunoassay procedure

    International Nuclear Information System (INIS)

    Niswender, G.D.

    1977-01-01

    The present invention is concerned with the radioimmunoassay (RIA) procedure for assaying body fluid content of an antigenic substance which may either be an antigen itself or a hapten capable of being converted, such as by means of reaction with a protein, to an antigenic material. The present invention is concerned with a novel and improved modification of a double-antibody RIA technique in which there is a first antibody that is specific to the antigenic substance suspected to be present in a body fluid from which the assay is intended. The second antibody, however, is not specific to the antigenic substance or analyte, but is an antibody against the first antibody

  5. Production of Monoclonal Antibody against Human Nestin

    OpenAIRE

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-01-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140?250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such a...

  6. Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy.

    Science.gov (United States)

    Goldenberg, David M; Sharkey, Robert M; Paganelli, Giovanni; Barbet, Jacques; Chatal, Jean-François

    2006-02-10

    This article reviews the methods of pretargeting, which involve separating the targeting antibody from the subsequent delivery of an imaging or therapeutic agent that binds to the tumor-localized antibody. This provides enhanced tumor:background ratios and the delivery of a higher therapeutic dose than when antibodies are directly conjugated with radionuclides, as currently practiced in cancer radioimmunotherapy. We describe initial promising clinical results using streptavidin-antibody constructs with biotin-radionuclide conjugates in the treatment of patients with malignant gliomas, and of bispecific antibodies with hapten-radionuclides in the therapy of tumors expressing carcinoembryonic antigen, such as medullary thyroid and small-cell lung cancers.

  7. Serum Antibody Biomarkers for ASD

    Science.gov (United States)

    2015-10-01

    45-56. Singh VK. (2009) Phenotypic expression of autoimmune autistic disorder (AAD): A major subset of autism. Ann Clin Psychiat. 21:148-160. 5...spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in communication (verbal and nonverbal), social interactions, and... autoimmunity ; in particular, the generation of antibodies reactive against brain and CNS proteins. The goal of this grant is to identify serum

  8. Antibody Repertoire Development in Swine

    Czech Academy of Sciences Publication Activity Database

    Butler, J. E.; Wertz, N.; Šinkora, Marek

    2017-01-01

    Roč. 5, FEB 17 (2017), s. 255-279 ISSN 2165-8102 R&D Projects: GA ČR GA15-02274S; GA ČR(CZ) GA16-09296S Institutional support: RVO:61388971 Keywords : swine * pre-immune antibody repertoire * ileal Peyer's patches Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.708, year: 2016

  9. Pharmacokinetics and immunogenicity investigation of a human anti-interleukin-17 monoclonal antibody in non-naïve cynomolgus monkeys.

    Science.gov (United States)

    Han, Chao; Gunn, George R; Marini, Joseph C; Shankar, Gopi; Han Hsu, Helen; Davis, Hugh M

    2015-05-01

    The pharmacokinetics (PK) of biologic therapeutics, especially monoclonal antibodies (mAbs), in monkeys generally presents the most relevant predictive PK information for humans. However, human mAbs, xenogeneic proteins to monkeys, are likely to be immunogenic. Monkeys previously treated with a human mAb (non-naïve) may have developed antidrug antibodies (ADAs) that cross-react with another test mAb in subsequent studies. Unlike PK studies for small-molecule therapeutics, in which animals may be reused, naïve monkeys have been used almost exclusively for preclinical PK studies of biologic therapeutics to avoid potential pre-existing immunologic cross-reactivity issues. The propensity and extent of pre-existing ADAs have not been systematically investigated to date. In this study, the PK and immunogenicity of mAb A, a human anti-human interkeukin-17 mAb, were investigated in a colony of 31 cynomolgus monkeys previously exposed to other human mAbs against different targets. We screened the monkeys for pre-existing antibodies to mAb A prior to the PK study and showed that 44% of the monkeys had pre-existing cross-reactive antibodies to mAb A, which could affect the PK characterization of the antibody. In the subcolony of monkeys without measurable pre-existing ADAs, PK and immunogenicity of mAb A were successfully characterized. The impact of ADAs on mAb A PK was also demonstrated in the monkeys with pre-existing ADAs. Here we report the results and propose a pragmatic approach for the use of non-naïve monkeys when conducting PK studies of biologic therapeutics. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  10. Phase I dose escalation pharmacokinetic assessment of intravenous humanized anti-MUC1 antibody AS1402 in patients with advanced breast cancer.

    Science.gov (United States)

    Pegram, Mark D; Borges, Virginia F; Ibrahim, Nuhad; Fuloria, Jyotsna; Shapiro, Charles; Perez, Susan; Wang, Karen; Schaedli Stark, Franziska; Courtenay Luck, Nigel

    2009-01-01

    MUC1 is a cell-surface glycoprotein that establishes a molecular barrier at the epithelial surface and engages in morphogenetic signal transduction. Alterations in MUC1 glycosylation accompany the development of cancer and influence cellular growth, differentiation, transformation, adhesion, invasion, and immune surveillance. A 20-amino-acid tandem repeat that forms the core protein of MUC1 is overexpressed and aberrantly glycosylated in the majority of epithelial tumors. AS1402 (formerly R1550) is a humanized IgG1k monoclonal antibody that binds to PDTR sequences within this tandem repeat that are not exposed in normal cells. AS1402 is a potent inducer of antibody-dependent cellular cytotoxicity (ADCC), specifically against MUC1-expressing tumor cells. The objective of this study was to determine the safety, tolerability, and pharmacokinetic (PK) characteristics of AS1402 monotherapy in patients with locally advanced or metastatic MUC1-positive breast cancer that had progressed after anthracyclines- and taxane-based therapy. Patients received AS1402 over a 1- to 3-hour intravenous (i.v.) infusion at doses between 1 and 16 mg/kg, with repeated dosing every 1 to 3 weeks (based on patient-individualized PK assessment) until disease progression. Serum AS1402 levels were measured at multiple times after i.v. administration. Human anti-human antibody (HAHA) responses were measured to determine the immunogenicity of AS1402. Noncompartmental pharmacokinetic parameters were determined and were used to assess dose dependency across the dose range studied. Twenty-six patients were treated. AS1402 was generally well tolerated. Two grade 3/4 drug-related adverse events were reported, both at the 3-mg/kg dose. Neither was observed in expanded or subsequent dosing cohorts. No anti-human antibodies were detected. Plasma concentrations of AS1402 appeared to be proportional to dose within the 1- to 16-mg/kg dose range assessed, with a mean terminal half-life of 115.4 +/- 37.1 hours

  11. Donor-derived HLA antibody production in patients undergoing SCT from HLA antibody-positive donors.

    Science.gov (United States)

    Taniguchi, K; Yoshihara, S; Maruya, E; Ikegame, K; Kaida, K; Hayashi, K; Kato, R; Inoue, T; Fujioka, T; Tamaki, H; Okada, M; Onuma, T; Fujii, N; Kusunoki, Y; Soma, T; Saji, H; Ogawa, H

    2012-10-01

    Pre-existing donor-specific HLA antibodies in patients undergoing HLA-mismatched SCT have increasingly been recognized as a risk factor for primary graft failure. However, the clinical implications of the presence of HLA antibodies in donors remain unknown. We prospectively examined 123 related donors for the presence of HLA antibodies by using a Luminex-based single antigen assay. Of these, 1/57 (1.8%) male, 6/27 (22%) parous female and 0/39 (0%) nonparous female donors were HLA antibody-positive. Then, we determined the presence of HLA antibodies in seven patients who received SCT from antibody-positive donors. Of these, four became HLA antibody-positive after SCT. The specificities of the antibodies that emerged in the patients closely resembled those of the antibodies found in the donors, indicating their production by donor-derived plasma cells. Moreover, the kinetics of the HLA antibody levels were similar in all four patients: levels started increasing within 1 week after SCT and peaked at days 10-21, followed by a gradual decrease. These results suggest that donor-derived HLA antibody production frequently occurs in patients undergoing SCT from antibody-positive donors. Further studies are warranted for clarifying the clinical significance of donor-derived HLA antibodies, including the role of these antibodies in post transplant platelet transfusion refractoriness.

  12. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity.

    Science.gov (United States)

    Dorr, Patrick; Westby, Mike; Dobbs, Susan; Griffin, Paul; Irvine, Becky; Macartney, Malcolm; Mori, Julie; Rickett, Graham; Smith-Burchnell, Caroline; Napier, Carolyn; Webster, Rob; Armour, Duncan; Price, David; Stammen, Blanda; Wood, Anthony; Perros, Manos

    2005-11-01

    Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin (geometric mean 90% inhibitory concentration of 2.0 nM). Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. There was little difference in the sensitivity of the 200 viruses to maraviroc, as illustrated by the biological cutoff in this assay (= geometric mean plus two standard deviations [SD] of 1.7-fold). The mechanism of action of maraviroc was established using cell-based assays, where it blocked binding of viral envelope, gp120, to CCR5 to prevent the membrane fusion events necessary for viral entry. Maraviroc did not affect CCR5 cell surface levels or associated intracellular signaling, confirming it as a functional antagonist of CCR5. Maraviroc has no detectable in vitro cytotoxicity and is highly selective for CCR5, as confirmed against a wide range of receptors and enzymes, including the hERG ion channel (50% inhibitory concentration, >10 microM), indicating potential for an excellent clinical safety profile. Studies in preclinical in vitro and in vivo models predicted maraviroc to have human pharmacokinetics consistent with once- or twice-daily dosing following oral administration. Clinical trials are ongoing to further investigate the potential of using maraviroc for the treatment of HIV-1 infection and AIDS.

  13. A natural sulfated polysaccharide, calcium spirulan, isolated from Spirulina platensis: in vitro and ex vivo evaluation of anti-herpes simplex virus and anti-human immunodeficiency virus activities.

    Science.gov (United States)

    Hayashi, K; Hayashi, T; Kojima, I

    1996-10-10

    A sulfated polysaccharide named calcium spirulan (Ca-SP) has been isolated from a sea alga, Spirulina platensis, as an antiviral component. The anti-human immunodeficiency virus type 1 (HIV-1) and anti-herpes simplex virus type 1 (HSV-1) activities of Ca-SP were compared with those of dextran sulfate (DS) as a representative sulfated polysaccharide. Anti-HIV-1 activities of these agents were measured by three different assays: viability of acutely infected CD4-positive cells, or a cytopathology assay; determination of HIV-1 p24 antigen released into culture supernatants; and inhibition of HIV-induced syncytium formation. Anti-HSV-1 activity was assessed by plaque yield reduction. In addition, their effects on the blood coagulation processes and stability in the blood were evaluated. These data indicate that Ca-SP is a potent antiviral agent against both HIV-1 and HSV-1. Furthermore, Ca-SP is quite promising as an anti-HIV agent because even at low concentrations of Ca-SP an enhancement of virus-induced syncytium formation was not observed, as was observed in DS-treated cultures, Ca-SP had very low anticoagulant activity, and showed a much longer half-life in the blood of mice when compared with that of DS. Thus, Ca-SP can be a candidate agent for an anti-HIV therapeutic drug that might overcome the disadvantages observed in many sulfated polysaccharides. When the role of chelation of calcium ion with sulfate groups was examined by removing calcium or its replacement by sodium, the presence of calcium ion in the molecule was shown to be essential for the dose-dependent inhibition of cytopathic effect and syncytium formation induced by HIV-1.

  14. Construction of Rabbit Immune Antibody Libraries.

    Science.gov (United States)

    Nguyen, Thi Thu Ha; Lee, Jong Seo; Shim, Hyunbo

    2018-01-01

    Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

  15. Effective therapy for a murine model of adult T-cell leukemia with the humanized anti-CD2 monoclonal antibody, MEDI-507.

    Science.gov (United States)

    Zhang, Zhuo; Zhang, Meili; Ravetch, Jeffrey V; Goldman, Carolyn; Waldmann, Thomas A

    2003-07-01

    Adult T-cell leukemia (ATL) develops in a small proportion of individuals infected with the retrovirus human T-cell leukemia virus (HTLV-1). We evaluated the efficacy of MEDI-507 (a humanized monoclonal antibody directed against CD2) alone and in combination with humanized anti-Tac (HAT) directed toward CD25, the interleukin-2 receptor alpha (IL-2Ralpha) using a human adult T-cell leukemia xenograft model. Weekly treatments (4) with HAT significantly prolonged the survival of the ATL-bearing mice when compared with phosphate-buffered saline (PBS)-treated controls (P MEDI-507 (100 microg/wk for 4 weeks) survived longer than those treated with HAT (P MEDI-507 significantly improved the outcome when compared with a short course (4 weeks) of therapy (P MEDI-507 for 6 months led to a prolonged survival of the ATL-bearing mice that was comparable with the survival observed in the control group of mice that did not receive a tumor or therapeutic agent. We also found that the expression of Fcgamma receptors (FcRgamma) on polymorphonuclear leukocytes and monocytes was required for MEDI-507-mediated tumor killing in vivo. Thus, the tumor-killing mechanism with MEDI-507 in vivo required the expression of the receptor FcRgammaIII on polymorphonuclear leukocytes and monocytes, suggesting that it is mediated by a form of antibody-dependent cellular cytotoxicity. These results demonstrate that MEDI-507 has therapeutic efficacy on ATL in vivo and provides support for a clinical trial involving this monoclonal antibody in the treatment of patients with CD2-expressing leukemias and lymphomas.

  16. Humanization of an anti-CCR4 antibody that kills Cutaneous T-Cell Lymphoma cells and abrogates suppression by T-regulatory cells

    Science.gov (United States)

    Chang, De-Kuan; Sui, Jianhua; Geng, Shusheng; Muvaffak, Asli; Bai, Mei; Fuhlbrigge, Robert C.; Lo, Agnes; Yammanuru, Anuradha; Hubbard, Luke; Sheehan, Jared; Campbell, James J.; Zhu, Quan; Kupper, Thomas S.; Marasco, Wayne A.

    2012-01-01

    Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of neoplastic disorders characterized by clonally derived and skin-homing malignant T-cells that express high level of chemokine receptor CCR4, which is associated with their skin-homing capacity. CCR4 is also highly expressed on T-regulatory cells (Tregs) that can migrate to several different types of chemotactic ligand CCL17 and CCL22 secreting tumors to facilitate tumor cell evasion from immune surveillance. Thus, its high level expression on CTCL cells and Tregs makes CCR4 a potential ideal target for antibody-based immunotherapy for CTCL and other types of solid tumors. Here we performed humanization and affinity optimization of a murine anti-CCR4 monoclonal antibody (mAb), mAb1567, that recognizes both the N-terminal and extracellular domains of CCR4 with high affinity and inhibits chemotaxis of CCR4+ CTCL cells. In a mouse CTCL tumor model, mAb1567 exhibited a potent anti-tumor effect and in vitro mechanistic studies showed that both complement-dependent cytotoxicity (CDC) and neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) likely mediated this effect. MAb1567 also exerts human NK cell-mediated ADCC activity in vitro. Moreover, mAb1567 also effectively inhibits chemotaxis of CD4+CD25high Tregs via CCL22 and abrogates Treg suppression activity in vitro. An affinity optimized variant of humanized mAb1567, mAb2-3, was selected for further preclinical development based on its higher binding affinity and more potent ADCC and CDC activities. Taken together, this high affinity humanized mAb2-3 with potent anti-tumor effect and a broad range of mechanisms of action may provide a novel immunotherapy for CTCL and other solid tumors. PMID:22869555

  17. Update on antiphospholipid antibody syndrome.

    Science.gov (United States)

    Lopes, Michelle Remião Ugolini; Danowski, Adriana; Funke, Andreas; Rêgo, Jozelia; Levy, Roger; Andrade, Danieli Castro Oliveira de

    2017-11-01

    Antiphospholipid syndrome (APS) is an autoimmune disease characterized by antiphospholipid antibodies (aPL) associated with thrombosis and/or pregnancy morbidity. Most APS events are directly related to thrombotic events, which may affect small, medium or large vessels. Other clinical features like thrombocytopenia, nephropathy, cardiac valve disease, cognitive dysfunction and skin ulcers (called non-criteria manifestations) add significant morbidity to this syndrome and represent clinical situations that are challenging. APS was initially described in patients with systemic lupus erythematosus (SLE) but it can occur in patients without any other autoimmune disease. Despite the autoimmune nature of this syndrome, APS treatment is still based on anticoagulation and antiplatelet therapy.

  18. Phase Separation in Solutions of Monoclonal Antibodies

    Science.gov (United States)

    Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil

    2012-02-01

    We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

  19. The future of antibodies as cancer drugs.

    Science.gov (United States)

    Reichert, Janice M; Dhimolea, Eugen

    2012-09-01

    Targeted therapeutics such as monoclonal antibodies (mAbs) have proven successful as cancer drugs. To profile products that could be marketed in the future, we examined the current commercial clinical pipeline of mAb candidates for cancer. Our analysis revealed trends toward development of a variety of noncanonical mAbs, including antibody-drug conjugates (ADCs), bispecific antibodies, engineered antibodies and antibody fragments and/or domains. We found substantial diversity in the antibody sequence source, isotype, carbohydrate residues, targets and mechanisms of action (MOA). Although well-validated targets, such as epidermal growth factor receptor (EGFR) and CD20, continue to provide opportunities for companies, we found notable trends toward targeting less-well-validated antigens and exploration of innovative MOA such as the generation of anticancer immune responses or recruitment of cytotoxic T cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Evaluation of a generic immunoassay with drug tolerance to detect immune complexes in serum samples from cynomolgus monkeys after administration of human antibodies.

    Science.gov (United States)

    Stubenrauch, Kay; Wessels, Uwe; Essig, Ulrich; Vogel, Rudolf; Schleypen, Julia

    2010-06-05

    Current state of the art bridging ELISA technologies for detection of anti-drug antibodies (ADAs) against therapeutic antibodies bear the risk of false-negative results due to interference by circulating drug. Methods to remove the drug in the sample or sample pre-treatment techniques such as acid dissociation of the immune complexes are limited, laborious and may destroy ADAs resulting again in false-negative results. The immune complex ELISA described in this publication provides a simple solution. It is designed to analyze samples from cynomolgus monkeys dosed with human antibodies; it can be used for all human antibodies since it is independent of the specific antibody and its target. The generic applicability of the ADA assay is enabled by the use of (1) a murine anti-human Fc monoclonal antibody (MAb) as capture reagent; (2) a murine anti-cynomolgus monkey IgG MAb as detection reagent; and (3) an ADA positive control conjugate consisting of cynomolgus IgG complexed with human IgG. In its basic version, the generic ADA ELISA specifically detects only immune complexes formed in vivo. Validation of the ADA assay revealed a lower limit of quantitation of 15.6 ng/mL in serum samples. Intra-assay and inter-assay precision was characterized by a coefficient of variation of less than 10% and accuracy was within 8%. Matrix effects were low as evidenced by a mean recovery of 95%. In vitro pre-incubation of the serum samples with drug makes also the free ADA in the sample amenable to measurement by the immune complex ELISA as demonstrated by analysis of ADAs from two cynomolgus monkey studies with two different antibodies. The generic and versatile nature of this ADA assay favors its use in pilot pharmacokinetic and safety studies in cynomolgus monkeys during candidate selection of antibodies. The assay can help to explain unexpected drug clearance profiles, loss of efficacy or safety events caused by immune complexes and guide further development. Copyright (c) 2010

  1. Warm antibody autoimmune hemolytic anemia.

    Science.gov (United States)

    Kalfa, Theodosia A

    2016-12-02

    Autoimmune hemolytic anemia (AIHA) is a rare and heterogeneous disease that affects 1 to 3/100 000 patients per year. AIHA caused by warm autoantibodies (w-AIHA), ie, antibodies that react with their antigens on the red blood cell optimally at 37°C, is the most common type, comprising ∼70% to 80% of all adult cases and ∼50% of pediatric cases. About half of the w-AIHA cases are called primary because no specific etiology can be found, whereas the rest are secondary to other recognizable underlying disorders. This review will focus on the postulated immunopathogenetic mechanisms in idiopathic and secondary w-AIHA and report on the rare cases of direct antiglobulin test-negative AIHA, which are even more likely to be fatal because of inherent characteristics of the causative antibodies, as well as because of delays in diagnosis and initiation of appropriate treatment. Then, the characteristics of w-AIHA associated with genetically defined immune dysregulation disorders and special considerations on its management will be discussed. Finally, the standard treatment options and newer therapeutic approaches for this chronic autoimmune blood disorder will be reviewed. © 2016 by The American Society of Hematology. All rights reserved.

  2. An anti vimentin antibody promotes tube formation

    DEFF Research Database (Denmark)

    Jørgensen, Mathias Lindh; Møller, Carina Kjeldahl; Rasmussen, Lasse

    2017-01-01

    antibody technology, promotes tube formation of endothelial cells in a 2D matrigel assay. By binding vimentin, the antibody increases the tube formation by 21% after 5 hours of incubation. Addition of the antibody directly to cultured endothelial cells does not influence endothelial cell migration...... or proliferation. The enhanced tube formation can be seen for up to 10 hours where after the effect decreases. It is shown that the antibody-binding site is located on the coil 2 domain of vimentin. To our knowledge this is the first study that demonstrates an enhanced tube formation by binding vimentin in a 2D...

  3. Uses of monoclonal antibody 8H9

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, Nai-Kong V.

    2018-04-10

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  4. Exceptional Antibodies Produced by Successive Immunizations.

    Directory of Open Access Journals (Sweden)

    Patricia J Gearhart

    2015-12-01

    Full Text Available Antibodies stand between us and pathogens. Viruses mutate quickly to avoid detection, and antibodies mutate at similar rates to hunt them down. This death spiral is fueled by specialized proteins and error-prone polymerases that change DNA sequences. Here, we explore how B lymphocytes stay in the race by expressing activation-induced deaminase, which unleashes a tsunami of mutations in the immunoglobulin loci. This produces random DNA substitutions, followed by selection for the highest affinity antibodies. We may be able to manipulate the process to produce better antibodies by expanding the repertoire of specific B cells through successive vaccinations.

  5. High throughput discovery of influenza virus neutralizing antibodies from phage-displayed synthetic antibody libraries.

    Science.gov (United States)

    Chen, Ing-Chien; Chiu, Yi-Kai; Yu, Chung-Ming; Lee, Cheng-Chung; Tung, Chao-Ping; Tsou, Yueh-Liang; Huang, Yi-Jen; Lin, Chia-Lung; Chen, Hong-Sen; Wang, Andrew H-J; Yang, An-Suei

    2017-10-31

    Pandemic and epidemic outbreaks of influenza A virus (IAV) infection pose severe challenges to human society. Passive immunotherapy with recombinant neutralizing antibodies can potentially mitigate the threats of IAV infection. With a high throughput neutralizing antibody discovery platform, we produced artificial anti-hemagglutinin (HA) IAV-neutralizing IgGs from phage-displayed synthetic scFv libraries without necessitating prior memory of antibody-antigen interactions or relying on affinity maturation essential for in vivo immune systems to generate highly specific neutralizing antibodies. At least two thirds of the epitope groups of the artificial anti-HA antibodies resemble those of natural protective anti-HA antibodies, providing alternatives to neutralizing antibodies from natural antibody repertoires. With continuing advancement in designing and constructing synthetic scFv libraries, this technological platform is useful in mitigating not only the threats of IAV pandemics but also those from other newly emerging viral infections.

  6. Not All Antibodies Are Created Equal: Factors That Influence Antibody Mediated Rejection

    Directory of Open Access Journals (Sweden)

    Carrie L. Butler

    2017-01-01

    Full Text Available Consistent with Dr. Paul Terasaki’s “humoral theory of rejection” numerous studies have shown that HLA antibodies can cause acute and chronic antibody mediated rejection (AMR and decreased graft survival. New evidence also supports a role for antibodies to non-HLA antigens in AMR and allograft injury. Despite the remarkable efforts by leaders in the field who pioneered single antigen bead technology for detection of donor specific antibodies, a considerable amount of work is still needed to better define the antibody attributes that are associated with AMR pathology. This review highlights what is currently known about the clinical context of pre and posttransplant antibodies, antibody characteristics that influence AMR, and the paths after donor specific antibody production (no rejection, subclinical rejection, and clinical dysfunction with AMR.

  7. Properties of surface functionalized iron oxide nanoparticles (ferrofluid) conjugated antibody for lateral flow immunoassay application

    Energy Technology Data Exchange (ETDEWEB)

    Nor, Noorhashimah Mohamad [School of Materials and Mineral Resources Engineering, Engineering Campus, Universiti Sains Malaysia, 14300 Nibong Tebal, Penang (Malaysia); Abdul Razak, Khairunisak, E-mail: khairunisak@eng.usm.my [School of Materials and Mineral Resources Engineering, Engineering Campus, Universiti Sains Malaysia, 14300 Nibong Tebal, Penang (Malaysia); NanoBiotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), 11800 USM, Penang (Malaysia); Tan, Soo Choon; Noordin, Rahmah [NanoBiotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), 11800 USM, Penang (Malaysia)

    2012-10-15

    Highlights: Black-Right-Pointing-Pointer The effects of acid functionalization and biocompatible polymer on iron oxide nanoparticles (IONPs) ferrofluid were studied. Black-Right-Pointing-Pointer The IONPs functionalized using citric acid (IONPs-CA) is the most stable ferrofluid with zeta potential value of -49 mV. Black-Right-Pointing-Pointer IONPs-CA can be directly conjugated with antibody without biocompatible polymer coating. Black-Right-Pointing-Pointer IONPs-CA had optimum detection efficiency of 15 min assay time. Black-Right-Pointing-Pointer IONPs-CA showed the highest colour intensity in labelling lateral flow immunoassay. - Abstract: In this study, colloidal stability of iron oxide nanoparticles (IONPs) with several acid functionalizations and biocompatible polymer coating were compared for use as labelling agent in lateral flow immunoassay (LFIA). IONPs were synthesized using the precipitation method and peptized using perchloric acid (PA), nitric acid (NA) and citric acid (CA) to form a stable IONPs ferrofluid. Steric stabilization of IONPs using silane polyethelene glycol (SiPEG) was developed to improve biocompatibility and provide spaces for subsequent conjugation process. From the transmission electron microscopy (TEM) images, the sizes of IONPs obtained with different acids peptization were in range of 11-17 nm. The IONPs peptized using citric acid showed the most stable ferrofluid condition at physiological condition with zeta potential value of -49 mV. The LFIA was also developed to examine the conjugation properties of IONPs to mouse anti-human IgG{sub 4} antibody (M{alpha}HIgG{sub 4}). IONPs functionalized with citric acid can be directly conjugated with the M{alpha}HIgG{sub 4} without the need of SiPEG addition. This is due to the presence of the carboxylic group that acted as a ligand to the extended bond formation with the antibody. Moreover, the conjugation of IONPs with M{alpha}HIgG{sub 4} was also tested in a LFIA to detect brugian

  8. Human anti-Dectin-1 antibody, hybridoma producing said antibody and applications thereof

    OpenAIRE

    Kremer, Leonor; Llorente Gómez, María de las Mercedes; Casasnovas, José María; Fernández Ruíz, Elena; Galán Díez, Marta

    2008-01-01

    [EN] The invention relates to hybridoma MGD3 and the monoclonal antibody produced thereby (also called MGD3), which specifically recognises the human Dectin-1 membrane receptor. Antibody MGD3 is capable of inhibiting the binding of Dectin-1 to the natural ligand thereof, the ss-glucans that are components of the fungal wall. In addition, the aforementioned antibody specifically blocks binding to Candida albicans and the secretion of cytokines induced thereby. The MGD3 antibody obtained enable...

  9. Stratification of Antibody-Positive Subjects by Antibody Level Reveals an Impact of Immunogenicity on Pharmacokinetics

    OpenAIRE

    Zhou, Lei; Hoofring, Sarah A.; Wu, Yu; Vu, Thuy; Ma, Peiming; Swanson, Steven J.; Chirmule, Narendra; Starcevic, Marta

    2012-01-01

    The availability of highly sensitive immunoassays enables the detection of antidrug antibody (ADA) responses of various concentrations and affinities. The analysis of the impact of antibody status on drug pharmacokinetics (PK) is confounded by the presence of low-affinity or low-concentration antibody responses within the dataset. In a phase 2 clinical trial, a large proportion of subjects (45%) developed ADA following weekly dosing with AMG 317, a fully human monoclonal antibody therapeutic....

  10. Anti-idiotypic antibodies to poliovirus antibodies in commercial immunoglubulin preparations, human serum and milk.

    NARCIS (Netherlands)

    M. Hahn-Zoric; B. Carlsson; S. Jeansson; H.P. Ekre; A.D.M.E. Osterhaus (Albert); D. Roberton; L.A. Hanson

    1993-01-01

    textabstractOur previous studies have suggested that fetal antibody production can be induced by maternal antiidiotypic antibodies transferred to the fetus via the placenta. We tested commercial Ig, sera, and milk for the presence of anti-idiotypic antibodies to poliovirus type 1, using affinity

  11. Open-label, dose escalation phase I study in healthy volunteers to evaluate the safety and pharmacokinetics of a human monoclonal antibody to Clostridium difficile toxin A.

    Science.gov (United States)

    Taylor, Claribel P; Tummala, Sanjeev; Molrine, Deborah; Davidson, Lisa; Farrell, Richard J; Lembo, Anthony; Hibberd, Patricia L; Lowy, Israel; Kelly, Ciaran P

    2008-06-25

    Recent data suggest that Clostridium difficile-associated diarrhea is becoming more severe and difficult to treat. Antibody responses to C. difficile toxin A are protective against symptomatic disease and recurrence. We examined the safety and pharmacokinetics (pk) of a novel neutralizing human monoclonal antibody against C. difficile toxin A (CDA1) in healthy adults. Five cohorts with 6 subjects each received a single intravenous infusion of CDA1 at escalating doses of 0.3, 1, 5, 10, and 20 mg/kg. Safety evaluations took place on days 1, 2, 3, 7, 14, 28, and 56 post-infusion. Samples for pk analysis were obtained before and after infusion, and at each safety evaluation. Serum CDA1 antibody concentrations and human anti-human antibody (HAHA) titers were measured with enzyme-linked immunosorbent assays. A noncompartmental model was used for pk analysis. Thirty subjects were enrolled. The median age was 27.5 yrs. There were no serious adverse events (AE) related to CDA1. Twenty-one of the 48 reported non-serious adverse events were possibly related to CDA1, and included transient blood pressure changes requiring no treatment, nasal congestion, headache, abdominal cramps, nausea, and self-limited diarrhea. Serum CDA1 concentrations increased with escalating doses: mean C(max) ranged from 6.82 microg/ml for the 0.3 mg/kg cohort to 511 microg/ml for the 20 mg/kg cohort. The geometric mean values of the half-life of CDA1 ranged between 25.3 and 31.8 days, and the volume of distribution approximated serum. No subject formed detectable HAHA titers. Administration of CDA1 as a single intravenous infusion was safe and well tolerated. C(max) increased proportionally with increasing doses. A randomized study of CDA1 in patients with C. difficile associated diarrhea is underway.

  12. BF*F allotype of the alternative pathway of complement: A marker of protection against the development of antiphospholipid antibodies in patients with systemic lupus erythematosus.

    Science.gov (United States)

    Picceli, V F; Skare, T L; Nisihara, R M; Nass, F R; Messias-Reason, I T; Utiyama, S R R

    2016-04-01

    B factor (BF) from the alternative complement pathway seems to participate in the pathophysiology of systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). To study the allotypic variability of BF in SLE and their associations with clinical and autoantibodies profile. BF allotypes were determined by high-voltage agarose gel electrophoresis, under constant cooling, followed by immunofixation with anti-human BF antibody, in 188 SLE patients and 103 controls. Clinical and serological data were obtained from medical examination and records. No significant differences of BF variants between patients and controls were found, neither in relation to epidemiologic or clinical manifestations. Associations of phenotype BF SS07 and allotype BF*S07 were found with anticardiolipin IgM (aCl-IgM) antibodies (p = 0.014 and p = 0.009 respectively), but not with aCl-IgG, lupus anticoagulant (LA), anti β2GPI or clinical APS. A significant decrease in BF*F allotype (p = 0.043) and BF SF phenotype (p = 0.018) was detected in patients with anti-phospholipid antibodies as a whole (aCl-IgG, aCl-IgM, LA and anti β2GPI). There is a link between phenotype BF SS07 and allotype BF*S07 with aCl-IgM in SLE patients; BF*F allotype could be considered a marker of protection against the development of antiphospholipid antibodies in these patients. © The Author(s) 2015.

  13. Monoclonal antibodies in pediatric allergy

    Directory of Open Access Journals (Sweden)

    Amelia Licari

    2015-10-01

    Full Text Available Production of monoclonal antibodies (mAbs involving human-mouse hybrid cells was first described in 1970s, but these biologics are now used for a variety of diseases including cancers, autoimmune disorders and allergic diseases. The aim of this article is to review current and future applications of mAbs, in particular focusing on anti-IgE therapy, in the field of pediatric allergy. Proceedings of the 11th International Workshop on Neonatology and Satellite Meetings · Cagliari (Italy · October 26th-31st, 2015 · From the womb to the adultGuest Editors: Vassilios Fanos (Cagliari, Italy, Michele Mussap (Genoa, Italy, Antonio Del Vecchio (Bari, Italy, Bo Sun (Shanghai, China, Dorret I. Boomsma (Amsterdam, the Netherlands, Gavino Faa (Cagliari, Italy, Antonio Giordano (Philadelphia, USA

  14. Update on antiphospholipid antibody syndrome

    Directory of Open Access Journals (Sweden)

    Michelle Remião Ugolini Lopes

    Full Text Available Summary Antiphospholipid syndrome (APS is an autoimmune disease characterized by antiphospholipid antibodies (aPL associated with thrombosis and/or pregnancy morbidity. Most APS events are directly related to thrombotic events, which may affect small, medium or large vessels. Other clinical features like thrombocytopenia, nephropathy, cardiac valve disease, cognitive dysfunction and skin ulcers (called non-criteria manifestations add significant morbidity to this syndrome and represent clinical situations that are challenging. APS was initially described in patients with systemic lupus erythematosus (SLE but it can occur in patients without any other autoimmune disease. Despite the autoimmune nature of this syndrome, APS treatment is still based on anticoagulation and antiplatelet therapy.

  15. Generation and characterization of rabbit polyclonal antibodies against Vasohibin-2 for determination of its intracellular localization.

    Science.gov (United States)

    Sun, Jie; Tu, Min; Han, Bei; Xue, Xiaofeng; Zhang, Ye; Wei, Jishu; Chen, Jianmin; Lu, Zipeng; An, Yong; Cai, Baobao; Lv, Nan; Jiang, Kuirong; Miao, Yi; Gao, Wentao

    2013-07-01

    IF, IHC and WB (post cytoplasmic/nuclear protein isolation) showed a quite different intracellular localization of VASH2 protein. The VASH2 (with 355 amino acid residues) was located in the cytoplasm while VASH2 (with 311 amino acid residues) was located in the nucleus. The former was found to be a relatively low abundance protein. We successfully generated three rabbit anti-human Vasohibin-2 polyclonal antibodies which can be used for western blotting, IF, IP and IHC. These antibodies will provide a convenient tool for further studies on Vasohibin-2. This is the first study to report differences in the intracellular localization of the VASH2 protein and, hence, a new research direction on the study of VASH2.

  16. Nano antibody therapy for cancer

    International Nuclear Information System (INIS)

    Venkatachallam, M.; Sivakumar, T.; Nazeema; Venkateswari, P.

    2011-01-01

    Nanomedicine, an offshoot of nanotechnology, refers to highly specific medical intervention at the molecular scale for curing disease or repairing damaged tissues, such as bone, muscle, or nerve. Nanotechnology can have an early, paradigm-changing impact on how clinicians will detect cancer in its earliest stages. Exquisitely sensitive devices constructed of nanoscale components-such as nanocantilevers, nanowires and nanochannels-offer the potential for detecting even the rarest molecular signals associated with malignancy. One of the most pressing needs in clinical oncology is for imaging agents that can identify tumors that are far smaller than is possible with today's technology, at a scale of 100,000 cells rather than 1,000,000,000 cells. A new approach in nanotechnology for treating cancer incorporates nano iron particles and attaches them to an antibody that has targets only cancer cells and not healthy cells. The treatment works in two steps. This treatment is an ingenious way to make localized tumor ablation a systemic treatment. The advantages are incredible. There are absolutely no side effects from this treatment. It is not painful or even uncomfortable. The iron particles get flushed harmlessly from the body. It is not a drug and so the cancer cannot build up a resistance to the treatment. It is a systematic treatment; even cancer cells and tumors that are not known about get heated up and ablated. This treatment can even be used to enhance imaging of the cancer because once the cancer cells are coated with the iron particles, they are easy to identify. Everything depends on how reliably the antibodies target cancer cells and not healthy cells. When used in conjunction with other systemic treatments, such as vaccine treatments, we could be looking at a time when even advanced cancers can be brought under control. (author)

  17. [Radiolabeled antibodies for cancer treatment].

    Science.gov (United States)

    Barbet, Jacques; Chatal, Jean-François; Kraeber-Bodéré, Françoise

    2009-12-01

    The first treatment ever by radio-immunotherapy (RIT) was performed by William H. Beierwaltes in 1951 and was a success. Fifty years later, the main question is to find ways of extending the success of radiolabelled anti-CD20 antibodies in indolent non-Hodgkin's lymphoma to other forms of cancer. Solid tumours are much more radioresistant than lymphomas, but they respond to RIT if the lesions are small. Clinical situations of residual or minimal disease are thus the most likely to benefit from RIT in the adjuvant or consolidation settings. For disseminated disease, like leukemias or myelomas, the problem is different: beta- particles emitted by the radioactive atoms classically used for cancer treatment (iodine-131 or yttrium-90) disperse their energy in large volumes (ranges 1 mm to 1 cm) and are not very effective against isolated cells. Advances in RIT progress in two directions. One is the development of pretargeting strategies in which the antibody is not labelled but used to provide binding sites to small molecular weight radioactivity vectors (biotin, haptens). These techniques have been shown to increase tumour to non-target uptake ratios and anti-tumour efficacy has been demonstrated in the clinic. The other approach is the use of radionuclides adapted to the various clinical situations. Lutetium-177 or copper-67, because of the lower energy of their emission, their relatively long half-life and good gamma emission, may significantly improve RIT efficacy and acceptability. Beyond that, radionuclides emitting particles such as alpha particles or Auger electrons, much more efficient to kill isolated tumour cells, are being tested for RIT in the clinic. Finally, RIT should be integrated with other cancer treatment approaches in multimodality protocols. Thus RIT, now a mature technology, should enter a phase of well designed and focused clinical developments that may be expected to afford significant therapeutic advances.

  18. Applications of recombinant antibodies in plant pathology.

    Science.gov (United States)

    Ziegler, Angelika; Torrance, Lesley

    2002-09-01

    Summary Advances in molecular biology have made it possible to produce antibody fragments comprising the binding domains of antibody molecules in diverse heterologous systems, such as Escherichia coli, insect cells, or plants. Antibody fragments specific for a wide range of antigens, including plant pathogens, have been obtained by cloning V-genes from lymphoid tissue, or by selection from large naive phage display libraries, thus avoiding the need for immunization. The antibody fragments have been expressed as fusion proteins to create different functional molecules, and fully recombinant assays have been devised to detect plant viruses. The defined binding properties and unlimited cheap supply of antibody fusion proteins make them useful components of standardized immunoassays. The expression of antibody fragments in plants was shown to confer resistance to several plant pathogens. However, the antibodies usually only slowed the progress of infection and durable 'plantibody' resistance has yet to be demonstrated. In future, it is anticipated that antibody fragments from large libraries will be essential tools in high-throughput approaches to post-genomics research, such as the assignment of gene function, characterization of spatio-temporal patterns of protein expression, and elucidation of protein-protein interactions.

  19. Monoclonal antibodies against rat leukocyte surface antigens

    NARCIS (Netherlands)

    van den Berg, T. K.; Puklavec, M. J.; Barclay, A. N.; Dijkstra, C. D.

    2001-01-01

    Monoclonal antibodies have proven to be powerful tools for studying the properties of leukocyte surface antigens and the cells that express them. In the past decades many monoclonal antibodies (mAb) for identifying the different rat leukocyte surface antigens have been described. A list of mAb is

  20. Quantitative Changes In Antibodies Against Onchocercal Native ...

    African Journals Online (AJOL)

    Quantitative Changes In Antibodies Against Onchocercal Native Antigens Two Months Postivermectin Treatment Of Onchocerciasis Patients. ... Those without onchocercal skin disease, OSD (n=18) had a significant increase of 20.5±29.6%, with pre- and posttreatment values of 0.59±0.15 versus 0.68±0.13 for IgG antibody ...

  1. Anti-influenza M2e antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M.

    2013-04-16

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  2. Anti-influenza M2e antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M [Santa Fe, NM

    2011-12-20

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  3. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Science.gov (United States)

    NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

  4. Serum Antiphospholipid Antibodies Among Healthy Adults In ...

    African Journals Online (AJOL)

    Background: Antiphospholipid antibodies have been associated with variety of conditions. There is no standard health associated reference values required for the interpretation of antiphospholipid antibodies result available among adults in North- eastern Nigeria and Nigeria in general. The aim of this study is to determine ...

  5. Radiolabeled antibodies for cancer imaging and therapy.

    Science.gov (United States)

    Barbet, Jacques; Bardiès, Manuel; Bourgeois, Mickael; Chatal, Jean-François; Chérel, Michel; Davodeau, François; Faivre-Chauvet, Alain; Gestin, Jean-François; Kraeber-Bodéré, Françoise

    2012-01-01

    Radiolabeled antibodies were studied first for tumor detection by single-photon imaging, but FDG PET stopped these developments. In the meantime, radiolabeled antibodies were shown to be effective in the treatment of lymphoma. Radiolabeling techniques are well established and radiolabeled antibodies are a clinical and commercial reality that deserves further studies to advance their application in earlier phase of the diseases and to test combination and adjuvant therapies including radiolabeled antibodies in hematological diseases. In solid tumors, more resistant to radiations and less accessible to large molecules such as antibodies, clinical efficacy remains limited. However, radiolabeled antibodies used in minimal or small-size metastatic disease have shown promising clinical efficacy. In the adjuvant setting, ongoing clinical trials show impressive increase in survival in otherwise unmanageable tumors. New technologies are being developed over the years: recombinant antibodies and pretargeting approaches have shown potential in increasing the therapeutic index of radiolabeled antibodies. In several cases, clinical trials have confirmed preclinical studies. Finally, new radionuclides, such as lutetium-177, with better physical properties will further improve the safety of radioimmunotherapy. Alpha particle and Auger electron emitters offer the theoretical possibility to kill isolated tumor cells and microscopic clusters of tumor cells, opening the perspective of killing the last tumor cell, which is the ultimate challenge in cancer therapy. Preliminary preclinical and preliminary clinical results confirm the feasibility of this approach.

  6. Determination of antiphospholipid antibodies and Thrombophilia in ...

    African Journals Online (AJOL)

    Background: Recurrent miscarriage is a critical problem in which many factors play a crucial role such as antiphospholipid antibodies (APA) and anticardiolipin antibodies (ACA). Recent studies pointed to a potential role of thrombophilias as a possible cause of recurrent miscarriage (RM). Objectives: This study was ...

  7. A novel polyclonal antibody against human cytomegalovirus ...

    African Journals Online (AJOL)

    Future research should be directed to epitope screening of synthetic HMCV peptides, which could help to understand HCMV infection and virus-neutralising antibodies more fully and to prepare HCMV vaccines and antiviral drugs. Key words: Human cytomegalovirus, AD169 strain, Towne strains, polyclonal antibody.

  8. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia.

  9. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies...... are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors...... such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist...

  10. Antiphospholipid antibody: laboratory, pathogenesis and clinical manifestations

    Directory of Open Access Journals (Sweden)

    T. Ziglioli

    2011-06-01

    Full Text Available Antiphospholipid antibodies (aPL represent a heterogeneous group of antibodies that recognize various antigenic targets including beta2 glycoprotein I (β2GPI, prothrombin (PT, activated protein C, tissue plasminogen activator, plasmin and annexin A2. The most commonly used tests to detect aPL are: lupus anticoagulant (LAC, a functional coagulation assay, anticardiolipin antibody (aCL and anti-β2GPI antibody (anti-β2GPI, which are enzyme-linked immunoassay (ELISA. Clinically aPL are associated with thrombosis and/or with pregnancy morbidity. Apparently aPL alone are unable to induce thrombotic manifestations, but they increase the risk of vascular events that can occur in the presence of another thrombophilic condition; on the other hand obstetrical manifestations were shown to be associated not only to thrombosis but mainly to a direct antibody effect on the trophoblast.

  11. Antibodies against chromosomal beta-lactamase

    DEFF Research Database (Denmark)

    Giwercman, B; Rasmussen, J W; Ciofu, Oana

    1994-01-01

    A murine monoclonal anti-chromosomal beta-lactamase antibody was developed and an immunoblotting technique was used to study the presence of serum and sputum antibodies against Pseudomonas aeruginosa chromosomal group 1 beta-lactamase in patients with cystic fibrosis (CF). The serum antibody...... response was studied with serum samples collected in 1992 from 56 CF patients in a cross-sectional study and with serum samples from 18 CF patients in a longitudinal study. Anti-beta-lactamase immunoglobulin G antibodies were present in all of the serum samples from the patients with chronic...... bronchopulmonary P. aeruginosa infection (CF + P) but in none of the CF patients with no or intermittent P. aeruginosa infection. Anti-beta-lactamase antibodies were present in serum from CF + P patients after six antipseudomonal courses (median) and correlated with infection with a beta-lactam-resistant strain...

  12. Onconeural antibodies: improved detection and clinical correlations.

    Science.gov (United States)

    Storstein, Anette; Monstad, Sissel Evy; Haugen, Mette; Mazengia, Kibret; Veltman, Dana; Lohndal, Emilia; Aarseth, Jan; Vedeler, Christian

    2011-03-01

    Onconeural antibodies are found in many patients with paraneoplastic neurological syndromes (PNS) and define the disease as paraneoplastic. The study describes the presence of onconeural antibodies and PNS in 555 patients with neurological symptoms and confirmed cancer within five years, and compares the diagnostic accuracy of different antibody assays (immunoprecipitation, immunofluorescence and immunoblot). Onconeural antibodies were found in 11.9% of the patients by immunoprecipitation, in 7.0% by immunofluorescence and in 6.3% by immunoblot. PNS were present in 81.8% of the cancer patients that were seropositive by immunoprecipitation. Immunofluorescence and immunoblot failed to detect onconeural antibodies in almost one third of the PNS cases. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Electrochemical immunosensor for anti-tissue transglutaminase antibodies based on the in situ detection of quantum dots.

    Science.gov (United States)

    Martín-Yerga, Daniel; González-García, María Begoña; Costa-García, Agustín

    2014-12-01

    A miniaturized electrochemical biosensor array with in situ detection of quantum dots (QDs) was developed for the detection of anti-transglutaminase IgG antibodies (a celiac disease biomarker) in human sera. For the fabrication of the sensor, a 8-channel screen-printed carbon electrochemical arrays were used as transducers and modified with tissue-transglutaminase by adsorption. The immunologic reaction was carried out in a few simple steps: reaction with human serum, which contains the analyte of interest, followed by the immunoreaction with anti-human IgG labeled with CdSe/ZnS QDs and electrochemical detection of Cd(2+) released from QDs. All steps were performed on the screen-printed arrays as the solid support, and the detection of Cd(2+) was performed in situ after acid attack of the QDs without a transfer step by voltammetric stripping. The electrochemical response was correlated with the anti-transglutaminase IgG concentration. The developed electrochemical immunosensor is a trustful screening tool for celiac disease diagnosis discriminating between positive and negative sera samples with high sensitivity. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Prevalence of serum celiac antibodies in a multiracial Asian population--a first study in the young Asian adult population of Malaysia.

    Science.gov (United States)

    Yap, Theresa Wan-Chen; Chan, Weng-Kai; Leow, Alex Hwong-Ruey; Azmi, Ahmad Najib; Loke, Mun-Fai; Vadivelu, Jamuna; Goh, Khean-Lee

    2015-01-01

    Celiac disease (CD) is an immune-mediated disorder induced by the ingestion of gluten in genetically susceptible persons. The prevalence of CD in Malaysia is unknown. We aim to determine the seroprevalence of CD antibodies and also investigate the correlation between H. pylori infection and CD in the young and healthy multiracial Malaysian population. Healthy young adult volunteers between the ages of 18-30 years were consecutively recruited from June 2012 to May 2014 at the University of Malaya Medical Centre (UMMC), Kuala Lumpur. Serum samples from all the participants were tested for anti-gliadin antibody immunoglobulin A/immunoglobulin G (IgA/IgG) and anti-tissue transglutaminase antibody (tTG) IgA/IgG. Samples positive for both anti-gliadin and anti-tTG were further validated for anti-human endomysial IgA antibodies (EmA). Serological diagnosis of CD was made when anti-gliadin, anti-tTG and anti-EmA were positive. 562 qualified participants with mean age 24 ± 2.4 years old were recruited into our study. CD was found in 7 participants where most of them were asymptomatic and unaware of their CD status. The median of anti-gliadin and anti-tTG IgA/IgG value was 38.2 U/ml (interquartile range, 28.3-60.4 U/ml) and 49.2 U/ml (interquartile range, 41.1-65.9 U/ml), respectively. Seroprevalence of CD antibodies was 1.9% (6 out of 324) in female while only 0.4% (1 out of 238) in male. Seroprevalence among Malay was 0.8% (2 of 236), Chinese was 1.7% (3 of 177) and Indian was 1.3% (2 of 149). Overall, seroprevalence of CD antibodies in healthy asymptomatic adults in the Malaysian population was 1.25% (95% CI, 0.78%-1.72%). No significant relationship was discovered between CD and H. pylori infection. The seroprevalence of CD antibodies in healthy young adults in the Malaysian population was 1.25% (1 in 100). CD is underdiagnosed and it could be a much greater problem in Malaysia than previously thought.

  15. Antibody to human α-fetoprotein inhibits cell growth of human hepatocellular carcinoma cells by resuscitating the PTEN molecule: in vitro experiments.

    Science.gov (United States)

    Ohkawa, Kiyoshi; Asakura, Tadashi; Tsukada, Yutaka; Matsuura, Tomokazu

    2017-06-01

    It has been proposed that α-fetoprotein (AFP) is a new member of the intracellular signaling molecule family of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway via interaction with the phosphatase and tensin homolog (PTEN). In this study, the effects of anti-human AFP antibody on the functions of PTEN were examined using an AFP-producing human hepatoma cell line. The antibody caused significant inhibition of cell growth, compared to a normal IgG control, with the accumulation of intracellular immune complexes followed by significant reduction of cytosolic functional AFP. Decrease in the amount of AKT phosphorylated on serine (S) 473 indicated that PI3K/AKT signaling was suppressed in the cells. S380-phosphorylated PTEN increased markedly by the second day after antibody treatment, with slight but significant increase in the PTEN protein level. Since phosphorylation at S380 is critical for PTEN stability, the increase in S380-phosphorylated PTEN indicated maintenance of the number of PTEN molecules and the related potential to control PI3K/AKT signaling. p53 protein (P53) significantly, but slightly increased during antibody treatment, because PTEN expression increased the stability and function of P53 via both molecular interactions. P53 phosphorylated at S20 or at S392 dramatically increased, suggesting an increase in the stability, accumulation and activation of P53. Glucose transporter 1 (GLUT1) increased immediately after antibody treatment, pointing to a deficiency of glucose in the cells. Immunofluorescence cytology revealed that antibody-treatment re-distributed GLUT1 molecules throughout the cytoplasm with a reduction of their patchy localization on the cell surface. This suggested that translocation of GLUT1 depends on the PI3K/AKT pathway, in particular on PTEN expression. Antibody therapy targeted at AFP-producing tumor cells showed an inhibitory effect on the PI3K/AKT pathway via the liberation, restoration and functional stabilization of

  16. Radiohalogenated half-antibodies and maleimide intermediate therefor

    Science.gov (United States)

    Kassis, Amin I.; Khawli, Leslie A.

    1991-01-01

    N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabelled half-antibody having immunological specific binding characteristics of whole antibody.

  17. Docking of Antibodies into Cavities in DNA Origami

    DEFF Research Database (Denmark)

    Quyang, X; Stefano, Mattia De; Krissanaprasit, Abhichart

    2017-01-01

    microscopy (AFM) and transmission electron microscopy (TEM) validated efficient antibody immobilization in the origami structures. The increased ability to control the orientation of antibodies in nanostructures and at surfaces has potential for directing the interactions of antibodies with targets...

  18. Immunohistochemical Examination of Novel Rat Monoclonal Antibodies against Mouse and Human Podoplanin

    International Nuclear Information System (INIS)

    Kaji, Chiaki; Tsujimoto, Yuta; Kato Kaneko, Mika; Kato, Yukinari; Sawa, Yoshihiko

    2012-01-01

    This study aims to develop new monoclonal antibodies (mAbs) against mouse and human podoplanin. Rats were immunized with synthetic peptides, corresponding to amino acids 38–51 of mouse podoplanin or human podoplanin which is 100% homologous to the same site of monkey podoplanin; anti-mouse podoplanin mAb PMab-1 (IgG 2a ) and anti-human mAb NZ-1.2 (IgG 2a ) were established. In immunocytochemistry, the mouse melanoma B16-F10 and mouse podoplanin (mPDPN)-expressed CHO transfectant were stained by PMab-1; human lymphatic endothelial cells (LEC) and human podoplanin (hPDPN)-expressed squamous cell carcinoma HSC3 transfectant, were stained by NZ-1.2. Western-blot analysis detected an about 40-kDa protein in CHO-mPDPN and B16-F10 by PMab-1, and in HSC3-hPDPN and LEC by NZ-1.2. In frozen sections, PMab-1 reacted with mouse kidney, pulmonary alveoli, pulmonary pleura, and salivary gland myoepithelial cells while NZ-1.2 reacted to the human salivary gland myoepithelial cells. The immunostaining of paraffin-embedded sections also showed the reaction of PMab-1 or NZ-1.2 to the mouse or monkey kidney glomerulus, pulmonary alveoli, and lung lymphatic vessels. These results indicate that the two novel rat mAbs to the mouse and human/monkey podoplanin are useful for Western-blot and immunostaining of somatic tissues on paraffin-embedded sections as well as frozen sections

  19. 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

  20. Post-streptococcal auto-antibodies inhibit protein disulfide isomerase and are associated with insulin resistance.

    Directory of Open Access Journals (Sweden)

    Adi Aran

    2010-09-01

    Full Text Available Post-streptococcal autoimmunity affects millions worldwide, targeting multiple organs including the heart, brain, and kidneys. To explore the post-streptococcal autoimmunity spectrum, we used western blot analyses, to screen 310 sera from healthy subjects with (33% and without (67% markers of recent streptococcal infections [anti-Streptolysin O (ASLO or anti-DNAse B (ADB]. A 58 KDa protein, reacting strongly with post-streptococcal sera, was identified as Protein Disulfide Isomerase (PDI, an abundant protein with pleiotropic metabolic, immunologic, and thrombotic effects. Anti-PDI autoantibodies, purified from human sera, targeted similar epitopes in Streptolysin O (SLO, P51-61 and PDI (P328-338. The correlation between post-streptococcal status and anti-human PDI auto-immunity was further confirmed in a total of 2987 samples (13.6% in 530 ASLO positive versus 5.6% in 2457 ASLO negative samples, p<0.0001. Finally, anti-PDI auto-antibodies inhibited PDI-mediated insulin degradation in vitro (n = 90, p<0.001, and correlated with higher serum insulin (14.1 iu/ml vs. 12.2 iu/ml, n = 1215, p = 0.039 and insulin resistance (Homeostatic Model Assessment (HOMA 4.1 vs. 3.1, n = 1215, p = 0.004, in a population-based cohort. These results identify PDI as a major target of post-streptococcal autoimmunity, and establish a new link between infection, autoimmunity, and metabolic disturbances.

  1. Antiphospholipid Antibodies in Lupus Nephritis.

    Directory of Open Access Journals (Sweden)

    Ioannis Parodis

    Full Text Available Lupus nephritis (LN is a major manifestation of systemic lupus erythematosus (SLE. It remains unclear whether antiphospholipid antibodies (aPL alter the course of LN. We thus investigated the impact of aPL on short-term and long-term renal outcomes in patients with LN. We assessed levels of aPL cross-sectionally in SLE patients diagnosed with (n = 204 or without (n = 294 LN, and prospectively in 64 patients with active biopsy-proven LN (52 proliferative, 12 membranous, before and after induction treatment (short-term outcomes. Long-term renal outcome in the prospective LN cohort was determined by the estimated glomerular filtration rate (eGFR and the Chronic Kidney Disease (CKD stage, after a median follow-up of 11.3 years (range: 3.3-18.8. Cross-sectional analysis revealed no association between LN and IgG/IgM anticardiolipin or anti-β2-glycoprotein I antibodies, or lupus anticoagulant. Both aPL positivity and levels were similar in patients with active LN and non-renal SLE. Following induction treatment for LN, serum IgG/IgM aPL levels decreased in responders (p<0.005 for all, but not in non-responders. Both at active LN and post-treatment, patients with IgG, but not IgM, aPL had higher creatinine levels compared with patients without IgG aPL. Neither aPL positivity nor levels were associated with changes in eGFR from either baseline or post-treatment through long-term follow-up. Moreover, aPL positivity and levels both at baseline and post-treatment were similar in patients with a CKD stage ≥3 versus 1-2 at the last follow-up. In conclusion, neither aPL positivity nor levels were found to be associated with the occurrence of LN in SLE patients. However, IgG aPL positivity in LN patients was associated with a short-term impairment of the renal function while no effect on long-term renal outcome was observed. Furthermore, IgG and IgM aPL levels decreased following induction treatment only in responders, indicating that aPL levels are

  2. Structure Based Antibody-Like Peptidomimetics

    Directory of Open Access Journals (Sweden)

    Mark I. Greene

    2012-02-01

    Full Text Available Biologics such as monoclonal antibodies (mAb and soluble receptors represent new classes of therapeutic agents for treatment of several diseases. High affinity and high specificity biologics can be utilized for variety of clinical purposes. Monoclonal antibodies have been used as diagnostic agents when coupled with radionuclide, immune modulatory agents or in the treatment of cancers. Among other limitations of using large molecules for therapy the actual cost of biologics has become an issue. There is an effort among chemists and biologists to reduce the size of biologics which includes monoclonal antibodies and receptors without a reduction of biological efficacy. Single chain antibody, camel antibodies, Fv fragments are examples of this type of deconstructive process. Small high-affinity peptides have been identified using phage screening. Our laboratory used a structure-based approach to develop small-size peptidomimetics from the three-dimensional structure of proteins with immunoglobulin folds as exemplified by CD4 and antibodies. Peptides derived either from the receptor or their cognate ligand mimics the functions of the parental macromolecule. These constrained peptides not only provide a platform for developing small molecule drugs, but also provide insight into the atomic features of protein-protein interactions. A general overview of the reduction of monoclonal antibodies to small exocyclic peptide and its prospects as a useful diagnostic and as a drug in the treatment of cancer are discussed.

  3. Antibody proteases: induction of catalytic response.

    Science.gov (United States)

    Gabibov, A G; Friboulet, A; Thomas, D; Demin, A V; Ponomarenko, N A; Vorobiev, I I; Pillet, D; Paon, M; Alexandrova, E S; Telegin, G B; Reshetnyak, A V; Grigorieva, O V; Gnuchev, N V; Malishkin, K A; Genkin, D D

    2002-10-01

    Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP(89-104)) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an "internal image" of protease is described. The "second order" monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.

  4. Glycosylation profiles of therapeutic antibody pharmaceuticals.

    Science.gov (United States)

    Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger

    2011-11-01

    Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. HIV antibodies for treatment of HIV infection.

    Science.gov (United States)

    Margolis, David M; Koup, Richard A; Ferrari, Guido

    2017-01-01

    The bar is high to improve on current combination antiretroviral therapy (ART), now highly effective, safe, and simple. However, antibodies that bind the HIV envelope are able to uniquely target the virus as it seeks to enter new target cells, or as it is expressed from previously infected cells. Furthermore, the use of antibodies against HIV as a therapeutic may offer advantages. Antibodies can have long half-lives, and are being considered as partners for long-acting antiretrovirals for use in therapy or prevention of HIV infection. Early studies in animal models and in clinical trials suggest that such antibodies can have