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Target Antigen (target + antigen)
Selected AbstractsWhen monocytes and platelets compete: The effect of platelet count on the flow cytometric measurement of monocyte CD36,CYTOMETRY, Issue 2 2010W.H. Dzik Abstract Background: Flow cytometric measurement of monocyte surface CD36 is relevant to several conditions including diabetes, cardiovascular disease, lipid disorders, platelet isoimmunization, and susceptibility to P falciparum malaria. CD36 is also strongly expressed on platelets where it is also known as platelet glycoprotein IV. Methods: Whole blood samples, containing identical monocyte concentrations, were adjusted to contain platelets ranging from 20,000/uL to 600,000/uL, were stained with fluorescent-labeled anti-CD36, and analyzed by flow cytometry. Results: CD36 median fluorescent intensity (MFI) observed on monocytes decreased as the platelet concentration in the sample increased with more than a 50% decline in monocyte MFI over the normal range of platelet values. The effect was not abolished by using larger volumes of monoclonal antibody and was observed with different clones of reagent anti-CD36. The findings were most consistent with competition by platelets for the CD36 reagent. Similar findings were observed with antibody to class I HLA. Under defined assay conditions, monocyte CD36 MFI declined with rising platelet concentration in a predictable fashion following an inverse linear relationship. Conclusions: Measurement of CD36 expression on monocytes by flow cytometry in whole blood samples is affected by the sample platelet count. When comparing the monocyte CD36 expression among different individuals, our approach can be used to adjust measured monocyte CD36 expression for the effect of the platelet concentration in the sample. Competition by platelets for monoclonal reagents may occur in other settings when whole blood assays are used and when the target antigen is strongly expressed on both platelets and leukocytes. © 2009 Clinical Cytometry Society [source] The clinical pharmacology of therapeutic monoclonal antibodiesDRUG DEVELOPMENT RESEARCH, Issue 3 2004Lorin K. Roskos Abstract Seventeen monoclonal antibodies are currently approved in the United States for therapeutic use in organ transplantation, percutaneous coronary intervention, prophylaxis of respiratory syncytial virus disease, rheumatoid arthritis, Crohn's disease, asthma, chronic lymphocytic leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, breast cancer, and colorectal cancer. All approved antibodies are of the IgG class. Thirteen are unconjugated intact antibodies, three are intact immunoconjugates, and one is a Fab fragment. Three of the antibodies are murine, five are chimeric, eight are humanized, and one is a fully human antibody generated by phage display technology. The antigen target and the structural and binding characteristics of the antibody determine the antibody's mechanism of action, pharmacokinetics, safety, and immunogenicity. Antibodies act through multiple mechanisms that include functional modulation of the antigen, recruitment of ADCC and CDC, and delivery of radionuclide or toxin payloads to target cells. Antibody half-life is usually governed by interaction with the FcRn receptor. In some cases, the antigen may act as a sink for antibody elimination. Safety profiles are determined by the pharmacology and tissue distribution of the target antigen, antibody isotype, the antibody payload, cytokine release, hypersensitivity reactions to xenogeneic protein, and immunogenicity. Fully human antibody technology may allow development of antibodies that have reduced risks of hypersensitivity reactions and immunogenicity, thereby enhancing safety and efficacy. The exquisite target specificity of antibodies, improvements in antibody engineering technology, and the wide availability of novel and validated therapeutic targets provide many current and future opportunities for the clinical development of therapeutic antibodies. Drug Dev. Res. 61:108,120, 2004. © 2004 Wiley-Liss, Inc. [source] Adoptive transfer of an anti-MART-127,35 -specific CD8+ T,cell clone leads to immunoselection of human melanoma antigen-loss variants in SCID miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2003Francesco Lozupone Abstract The identification of appropriate mouse models could be useful in carefully evaluating the actual role of the in vivo development of antigen-loss variants during antigen-specific vaccine therapy of human tumors. In this study we investigated the level of efficacy of a MART-1/Melan-A-specific CD8+ T,cell clone against its autologous melanoma in a severe combined immunodeficiency (SCID) mouse model, in which the tumor cells expressed in vivo heterogeneous and suboptimal levels of MART-1. The subcutaneous co-injection of the MART-1/Melan-A-reactive T,cell clone A42 with MART-1/Melan-A+ autologous human melanoma cells into SCID mice caused a total inhibition of tumor growth. However, the systemic treatment with A42 clone lymphocytes resulted inonly 50,60% inhibition of tumor growth, although the T,cell clone targeted the tumors and the MART-1+ cells virtually disappeared from the tumors. This study suggests that an immunotherapybased on the expansion of an antigen-specific T,cell clone generated in vitro is highly efficient in abolishing tumor growth when the target antigen is fully expressed, but leads to in vivoimmunoselection of antigen-loss variants in the presence of suboptimal levels of antigen expression. Furthermore, this work shows that human tumors/SCID mouse models may be useful in evaluating thein vivo efficacy of adoptive immunotherapies. [source] Antigen-specific oligoclonal bands in cerebrospinal fluid and serum from patients with anti-amphiphysin- and anti-CV2/CRMP5 associated paraneoplastic neurological syndromesEUROPEAN JOURNAL OF NEUROLOGY, Issue 6 2007O. Stich Using isoelectric focusing and affinity blotting employing paraneoplastic recombinant antigens, we investigated cerebrospinal fluid (CSF) and sera from three patients with positive anti-CV2/CRMP5- and one patient with positive anti-amphiphysin serology. CSF and sera were previously adjusted to total IgG concentrations of 20 mg/l. All patients suffered from paraneoplastic neurological syndromes (PNS) with predominant involvement of the central nervous system (CNS). Using affinity blot preloaded with paraneoplastic antigen, we detected in three of four patients more or stronger specific oligoclonal bands (OCB) in the CSF than in the corresponding serum, providing qualitative evidence of antigen specific intrathecal antibody synthesis. These results are in line with previous studies demonstrating specific OCB predominantly in CSF from patients with anti-Hu-, anti-Yo- and anti-Ri-associated PNS, supporting the hypothesis of autoimmunity in the pathogenesis of PNS. One patient harboured extensive anti-amphiphysin specific OCB, although OCB of total IgG could not be detected, indicating a higher sensitivity for detection of intrathecal antibody synthesis of the affinity blot preloaded with the paraneoplastic antigen, compared with investigation of total IgG OCB. These results could have implications concerning pathophysiological autoimmune aspects in other inflammatory diseases of CNS associated with total IgG OCB, provided that the target antigen is known. [source] Mechanisms of blister induction by autoantibodiesEXPERIMENTAL DERMATOLOGY, Issue 12 2005Cassian Sitaru Abstract:, Autoimmune diseases are characterized by defined self-antigens, organ specificity, autoreactive T cells and/or autoantibodies that can transfer disease. Autoimmune blistering diseases are organ-specific autoimmune diseases associated with an immune response directed to structural proteins mediating cell,cell and cell,matrix adhesion in the skin. While both autoreactive T and B cells have been detected and characterized in patients with autoimmune blistering diseases, current evidence generally supports a pathogenic role of autoantibodies for blister formation. The immunopathology associated with blisters induced by autoantibodies relies on several mechanisms of action. Autoantibodies from patients with pemphigus diseases can exert a direct effect just by binding to their target mediated by steric hindrance and/or by triggering the transduction of a signal to the cell. In most subepidermal autoimmune blistering conditions, in addition to the binding to their target antigen, autoantibodies need to interact with factors of the innate immune system, including the complement system and inflammatory cells, in order to induce blisters. Generally, decisive progress has been made in the characterization of the mechanisms of blister formation in autoimmune skin diseases. However, various aspects, including the exact contribution of steric hindrance and signal transduction for pemphigus IgG-induced acantholysis or the fine tuning of the inflammatory cascade triggered by autoantibodies in some subepidermal blistering diseases, still need to be addressed. Understanding the mechanisms by which autoantibodies induce blisters should facilitate the development of more specific therapeutic strategies of autoimmune blistering diseases. [source] Control of T-cell activation by CD4+ CD25+ suppressor T cellsIMMUNOLOGICAL REVIEWS, Issue 1 2001Ethan M. Shevach Summary: Depletion of the minor (,10%) subpopulation of CD4+ T cells that co-expresses CD25 (interleukin (IL)-2 receptor ,-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4+ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4+ CD25+ cells. CD4+ CD25+ -mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-,. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4+ CD25+ T cells are also powerful suppressors of the activation of both CD4+ and CD8+ T cells in vitro. Suppression is mediated by a cell contact-dependent, cytokine-independent T,T interaction. Activation of CD4+ CD25+ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4+ or CD8+ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4+ CD25+ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4+ or CD8+ effector cells to suppress the development of autoimmune disease. [source] Identification of SPARC as a candidate target antigen for immunotherapy of various cancersINTERNATIONAL JOURNAL OF CANCER, Issue 6 2010Mitsuhiro Inoue Abstract To establish efficient anticancer immunotherary, it is important to identify tumor-associated antigens (TAAs) directing the immune system to attack cancer. A genome-wide cDNA microarray analysis identified that secreted protein acidic and rich in cysteine (SPARC) gene is overexpressed in the gastric, pancreatic and colorectal cancer tissues but not in their noncancerous counterparts. This study attempted to identify HLA-A24 (A*2402)-restricted and SPARC-derived CTL epitopes. We previously identified H-2Kd -restricted and SPARC-derived CTL epitope peptides in BALB/c mice, of which H-2Kd -binding peptide motif is comparable with that of HLA-A24 binding peptides. By using these peptides, we tried to induce HLA-A24 (A*2402)-restricted and SPARC-reactive human CTLs and demonstrated an antitumor immune response. The SPARC-A24-1143,151 (DYIGPCKYI) and SPARC-A24-4225,234 (MYIFPVHWQF) peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A24 (A*2402) positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both SPARC and HLA-A24 (A*2402). Furthermore, the adoptive transfer of the SPARC-specific CTLs could inhibit the tumor growth in nonobese diabetic/severe combined immunodeficient mice bearing human cancer cells expressing both HLA-A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy. [source] Melanoma patients respond to a new HLA-A*01-presented antigenic ligand derived from a multi-epitope region of melanoma antigen TRP-2INTERNATIONAL JOURNAL OF CANCER, Issue 6 2005Annette Paschen Abstract Tyrosinase-related protein-2 (TRP-2) is a known target antigen of spontaneous cytotoxic T cell responses in melanoma patients. Its frequent expression in metastatic tumors suggests that it might be an ideal candidate antigen for T cell-based immunotherapy. To provide knowledge about TRP-2-derived T cell epitopes useful for immunotherapy we applied a "reverse immunology strategy" based on repeated in vitro peptide stimulation of peripheral blood lymphocytes (PBL) from normal donors with predicted HLA-A*01 ligands. This led to the identification of TRP-2181,190 as the first HLA-A*01-presented TRP-2-derived epitope. T-cell lines specific for peptide TRP-2181,190 could be established from PBL of 50% of the normal HLA-A*01+ donors tested. Such T cells responded specifically to autologous dendritic cells transduced virally with TRP-2, as well as to HLA-A*01+, TRP-2+ melanoma cells, although tumor cells had to be pretreated with IFN-, to become susceptible to T cell recognition. Interestingly, short-term in vitro peptide stimulation of PBL from HLA-A*01+ melanoma patients showed the presence of TRP-2181,190 -reactive CD8+ T cells in some donors, suggesting their in vivo sensitization. Because TRP-2181,190 overlaps with the known HLA-A*0201-presented epitope TRP-2180,188, an 11mer peptide encompassing both epitopes might be of specific value for vaccination of a broad population of melanoma patients. © 2005 Wiley-Liss, Inc. [source] Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activityJOURNAL OF MOLECULAR RECOGNITION, Issue 5 2008Rui Huo Abstract In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t -butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH,+,VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni2+ -immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0,3.0,mg of proteins from 1,L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288,U/µmol. Copyright © 2008 John Wiley & Sons, Ltd. [source] Paraflagellar rod protein-specific CD8+ cytotoxic T lymphocytes target Trypanosoma cruzi -infected host cellsPARASITE IMMUNOLOGY, Issue 8 2002Ruth A. Wrightsman Summary Our previous studies show that in mice immunized with the paraflagellar rod (PFR) proteins of Trypanosoma cruzi protective immunity against this protozoan parasite requires MHC class I-restricted T cell function. To determine whether PFR-specific CD8+ T cell subsets are generated during T. cruzi infection, potential CTL targets in the PFR proteins were identified by scanning the amino acid sequences of the four PFR proteins for regions of 8,10 amino acids that conform to predicted MHC class I H-2b binding motifs. A subset of the peptide sequences identified were synthesized and tested as target antigen in 51Cr-release assays with effector cells from chronically infected T. cruzi mice. Short-term cytotoxic T lymphocyte (CTL) lines specific for two of the peptides, PFR-1164,171 and PFR-3123,130, showed high levels of lytic activity against peptide-pulsed target cells, secreted interferon (IFN)-, in response to parasite-infected target cells, and were found to be CD8+, CD4,, CD3+, TCR,,+ cells of the Tc1 subset. Challenge of PFR immunized CD8,/, and perforin-deficient (PKO) mice confirmed that while CD8+ cells are required for survival of T. cruzi challenge infection, perforin activity is not required. Furthermore, while lytic activity of PFR-specific CD8+ T cell lines derived from PKO mice was severely impaired, the IFN-, levels secreted by CTLs from PKO mice were equivalent to that of normal mice, suggesting that the critical role played by CD8+ T cells in immunity to the parasite may be secretion of type 1 cytokines rather than lysis of parasite infected host cells. [source] Identification and distribution of thioredoxin-like 2 as the antigen for the monoclonal antibody MC3 specific to colorectal cancerPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2008Yuanyuan Lu Abstract MC3 is a colorectal cancer (CRC)-specific mAb previously prepared in our laboratory that can detect CRC with high sensitivity and specificity. However, the target antigen for MC3 had not been identified due to technological limitations. In the present study, immunocytochemistry and immunohistochemistry revealed the expression patterns of MC3 antigen (MC3-Ag) in colon cancer cell lines and CRC tissues. Western blotting analysis showed that the MC3 antibody reproducibly recognized two ,30,kDa proteins in the total cell lysates of human colon carcinoma cell lines SW480 and HT-29. Using a proteomic approach, we identified two MC3 immunoreactive spots as two isoforms of thioredoxin-like 2 (Txl-2) protein. Further paired immunostaining showed that Txl-2 had the same expression profile as probed by the MC3 antibody. Western blotting also showed that both antibodies could detect the same two bands, further verifying that Txl-2 is the antigen of MC3 antibody. Additionally, tissue arrays revealed the expression patterns of Txl-2 in various normal and cancer tissues. Further analysis showed that Txl-2 mRNA was elevated in 18 cases of CRC tissues compared to paracancerous tissues and adjacent normal tissues. [source] Identification of the heterogeneous nuclear ribonucleoprotein A2/B1 as the antigen for the gastrointestinal cancer specific monoclonal antibody MG7PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2005Chi-ho Lee Abstract MG7 is an early gastrointestinal cancer specific monoclonal antibody. It can detect gastric cancer with high sensitivity and specificity. However, the target antigen for MG7 has not been identified. Western blot analysis revealed that the MG7 antibody reproducibly recognized two ,35 kDa proteins in the total cell lysates of human gastric carcinoma cell lines KATO III and MKN-45. Using a proteomic approach, we identified these MG7 immunoreactive proteins as the human heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). Western blot analysis of nuclear and cytosolic fraction of KATO III cells using either MG7 or hnRNP A2/B1 antibodies confirmed that the target antigen is located exclusively in the nucleus. With the use of archival samples, we also found that the level of hnRNP A2/B1 protein was increased in gastric cancer tissues (4 out of 5 patients), when compared to their corresponding matching normal stomach tissue. [source] Antibody and T-cell responses specific for the androgen receptor in patients with prostate cancerTHE PROSTATE, Issue 16 2007Brian M. Olson Abstract BACKGROUND The androgen receptor (AR) is a steroid hormone receptor that is an essential regulator of prostate development, and the primary molecular target for the treatment of metastatic prostate cancer. In this report, we evaluated whether patients with prostate cancer have pre-existing immune responses specific for the AR as evidence that the AR also might be pursued as an immunological target antigen. METHODS The detection of auto-antibodies specific for the AR in patient sera was evaluated by ELISA and Western blotting. Peripheral blood mononuclear cells were analyzed for the presence of AR-specific T-cells, as measured by T-cell proliferation, interferon gamma (IFN,) and interleukin-10 secretion. RESULTS We found that a significantly higher frequency of prostate cancer patients have AR LBD-specific antibody responses than do healthy male volunteers [18/105 cancer patients (17.1%) vs. 0/41 healthy volunteers, P,=,0.0049], and that these responses were present regardless of the patients' disease stage [8/46 organ-confined prostate cancer patients (17.4%), 3/22 metastatic androgen-dependent patients (13.6%), and 7/37 metastatic, androgen-independent patients (18.9%)]. These antibodies were pre-dominantly of the IgG isotype, and furthermore of the IgG2 sub-isotype. In addition, we found that patients with antibody responses also had concurrent antigen-specific CD4+ and CD8+ T-cell proliferation and IFN, secretion when compared to patients without antibody responses. CONCLUSIONS These data demonstrate that some patients with prostate cancer have pre-existing humoral and cellular immune responses specific for the AR, suggesting that tolerance against the AR is not absolute and that the AR may be a potential immunotherapeutic target antigen. Prostate 67: 1729,1739, 2007. © 2007 Wiley-Liss, Inc. [source] Antibodies to native myelin oligodendrocyte glycoprotein in children with inflammatory demyelinating central nervous system disease,ANNALS OF NEUROLOGY, Issue 6 2009Fabienne Brilot PhD Objective Myelin oligodendrocyte glycoprotein (MOG) is a candidate target antigen in demyelinating diseases of the central nervous system (CNS). Although MOG is encephalitogenic in different animal models, the relevance of this antigen in human autoimmune diseases of the CNS is still controversial. Methods We investigated the occurrence and biological activity of antibodies to native MOG (nMOG) in 47 children during a first episode of CNS demyelination (acute disseminated encephalomyelitis [ADEM], n = 19 and clinical isolated syndrome [CIS], n = 28) by a cell-based bioassay. Results High serum immunoglobulin G (IgG) titers to nMOG were detected in 40% of children with CIS/ADEM but 0% of the control children affected by other neurological diseases, healthy children, or adults with inflammatory demyelinating diseases, respectively. By contrast, IgM antibodies to nMOG occurred in only 3 children affected by ADEM. Children with high anti-nMOG IgG titer were significantly younger than those with low IgG titer. Anti-nMOG IgG titers did not differ between the ADEM and CIS group, and did not predict conversion from CIS to MS during a mean 2-year follow-up. However, intrathecal IgG anti-MOG antibody synthesis was only seen in CIS children. IgG antibodies to nMOG not only bound to the extracellular domain of nMOG, but also induced natural killer cell-mediated killing of nMOG-expressing cells in vitro. Interpretation Overall, these findings suggest nMOG as a major target of the humoral immune response in a subgroup of children affected by inflammatory demyelinating diseases of the CNS. Children may provide valuable insight into the earliest immune mechanisms of CNS demyelination. Ann Neurol 2009;66:833,842 [source] Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid,ANNALS OF NEUROLOGY, Issue 6 2009Gregory P. Owens PhD Objective Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B-cell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic. Methods We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy- and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections. Results Whereas humanized control rAbs derived from anti-myelin hybridomas and anti-myelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immunoreactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells. Interpretation The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein. Ann Neurol 2009;65:639,649 [source] Highly avid magnetic bead capture: An efficient selection method for de novo protein engineering utilizing yeast surface displayBIOTECHNOLOGY PROGRESS, Issue 3 2009Margaret Ackerman Abstract Protein engineering relies on the selective capture of members of a protein library with desired properties. Yeast surface display technology routinely enables as much as million-fold improvements in binding affinity by alternating rounds of diversification and flow cytometry-based selection. However, flow cytometry is not well suited for isolating de novo binding clones from naïve libraries due to limitations in the size of the population that can be analyzed, the minimum binding affinity of clones that can be reliably captured, the amount of target antigen required, and the likelihood of capturing artifactual binders to the reagents. Here, we demonstrate a method for capturing rare clones that maintains the advantages of yeast as the expression host, while avoiding the disadvantages of FACS in isolating de novo binders from naïve libraries. The multivalency of yeast surface display is intentionally coupled with multivalent target presentation on magnetic beads,allowing isolation of extremely weak binders from billions of non-binding clones, and requiring far less target antigen for each selection, while minimizing the likelihood of isolating undesirable alternative solutions to the selective pressure. Multivalent surface selection allows 30,000-fold enrichment and almost quantitative capture of micromolar binders in a single pass using less than one microgram of target antigen. We further validate the robust nature of this selection method by isolation of de novo binders against lysozyme as well as its utility in negative selections by isolating binders to streptavidin-biotin that do not cross-react to streptavidin alone. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Imaging for staging bladder cancer: a clinical study of intravenous 111indium-labelled anti-MUC1 mucin monoclonal antibody C595BJU INTERNATIONAL, Issue 1 2001O.D.M. Hughes Objective To investigate the clinical application of an 111In-labelled anti-MUC1 mucin monoclonal antibody (mAb) imaging for staging invasive bladder cancer. Patients and methods Indirect immunohistochemistry was used to confirm the expression of the MUC1 target antigen by metastatic tumours. Twelve patients with bladder cancer (two with superficial and 10 with locally invasive/metastatic disease) underwent planar ,-scintigraphy 48 h after an intravenous injection with 111In-labelled anti-MUC1 mucin mAb C595. Results No bladder uptake was detected in the two patients with superficial disease, but scintigraphy showed primary and recurrent bladder tumours and metastases in nine of the remaining 10 patients with invasive disease. In three patients additional staging information was obtained from the mAb imaging which would have altered patient management. There were no reported side-effects. Conclusion This study confirmed the ability of the mAb technique to detect both primary and recurrent invasive bladder tumours and distant metastases. Some lesions shown by mAb imaging were not detected by other methods. The use of mAb imaging has the potential to improve clinical staging and assist in selecting those patients most likely to benefit from radical therapy. [source] Plectin, an unusual target antigen in bullous pemphigoidBRITISH JOURNAL OF DERMATOLOGY, Issue 1 2001E. Laffitte Background Bullous pemphigoid (BP) is a blistering disease associated with autoantibodies directed against two components of hemidesmosomes, BP180 and BP230. Objectives To assess whether BP patients have autoantibodies targeting plectin, another hemidesmosomal component showing extensive homology to BP230. Methods Examination of sera from 16 patients with BP, using immunoprecipitation studies followed by immunoblotting. Results Serum of one of the 16 (6%) patients with BP contain autoantibodies binding to plectin, while no reactivity was found with sera from three control subjects. Sera from all 16 BP patients immunoprecipitated BP230 from extracts of biosynthetically radiolabelled human keratinocytes. Conclusions Our results indicate that sera from BP patients might contain autoantibodies binding to plectin. Although this protein and BP230 are closely sequence-related, the occurrence of autoantibodies binding to plectin is a rare phenomenon in BP. [source] Teicoplanin-dependent antibodies: detection and characterizationBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2005Stephen F. Garner Summary There are only a few reports of thrombocytopenia associated with clinical doses of teicoplanin, a glycopeptide antibiotic used against Gram-positive bacteria. We investigated 39 patients receiving teicoplanin; 31 were thrombocytopenic with platelet counts between 1,105 × 109/l and 8 were not thrombocytopenic. We identified 14 thrombocytopenic cases (45%) and two (25%) non-thrombocytopenic cases with IgG teicoplanin-dependent platelet-reactive antibodies. Use of glycoprotein (GP) capture enzyme-linked immunosorbent assay with platelets and GPIIb/IIIa transfected Chinese Hamster Ovary cells as well as flow cytometry with GP-deficient platelets indicated that the GPIIb/IIIa complex is a major target antigen of these antibodies. [source] Lack of association of ,2-glycoprotein I polymorphisms Val247Leu and Trp316Ser with antiphospholipid antibodies in patients with thrombosis and pregnancy complicationsBRITISH JOURNAL OF HAEMATOLOGY, Issue 6 2003Raymond S. Camilleri Summary. Beta2 -glycoprotein I (,2GPI) is an important target antigen for antiphospholipid antibodies (aPL) and thus ,2GPI polymorphisms may influence aPL production and the development of antiphospholipid syndrome. We have studied the relationship between the Val247Leu and Trp316Ser ,2GPI polymorphisms and the aPL status of 230 patients referred for aPL screening. Sixty-one (26·5%) had persistent aPL [anticardiolipin antibodies (IgG and/or IgM), lupus anticoagulants and/or IgG anti-,2GPI antibodies]. A comparison of the genotypic and allelic frequencies of these two polymorphisms between the Caucasian patient population and an ethnic-matched normal control group (n = 308) showed no significant differences between aPL-positive patients, aPL-negative patients and the normal control group. This suggests that the Val or Leu allele at position 247 and the Trp or Ser allele at position 316 of ,2GPI do not play a role in the production of aPL. There was a significantly decreased prevalence of the Ser316 allele in aPL-negative women (n = 98) when compared with female normal control subjects (n = 249) {0·020 [95% confidence interval (CI) 0·00,0·04]vs 0·060 (95% CI 0·04,0·08), P = 0·0286}. Subgroup analysis showed no significant difference between female patients with thrombosis and female normal control subjects. Thus, the Ser316 allele may protect women from developing pregnancy complications by influencing an anticoagulant function of ,2GPI via a mechanism distinct from aPL production. [source] Immunopathogenesis of cholestatic autoimmune liver diseasesEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 1 2001J. Medina Primary biliary cirrhosis and primary sclerosing cholangitis are well recognized chronic cholestatic liver diseases that are considered to have an autoimmune basis. Recent progress in the study of autoimmune liver diseases has improved the recognition and characterization of these conditions. An important component of this progress has been the identification of liver disease-associated autoantibodies and their respective target antigens, and the development of specific assays for these autoantibodies. In addition, some nonhumoral immunological findings imply an involvement of specific immunopathogenic mechanisms in the development of these conditions. Furthermore, immunogenetic factors associated with increased susceptibility to some of these diseases have been identified. This article reviews the most relevant information relating to the postulated autoimmune pathogenesis of these diseases, with special emphasis on their associated humoral and cellular immunological abnormalities and immunopathogenetic factors. Some of the remaining important unresolved issues relating to the pathogenesis of these diseases, that need to be addressed in further research, are highlighted. [source] Clonally expanded plasma cells in the cerebrospinal fluid of MS patients produce myelin-specific antibodiesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2008Hans-Christian von Büdingen Abstract Clonally expanded plasma cells (cePC) and their presumed products, oligoclonal immunoglobulin,G bands (OCB), are characteristic findings in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS). While cePC and OCB strongly suggest an involvement of B cell-dependent immune mechanisms in the pathogenesis of MS, their actual pathological relevance and target antigens remain unknown. To further understand the potential role played by cePC, we generated a panel of monoclonal antibodies (MS-mAb) from CSF-derived cePC from four patients with early or definite MS. Single-cell RT-PCR of correctly paired heavy and light chain immunoglobulin genes from individual cePC ensured the subsequent resurrection of their original antigen specificity. Immunofluorescence stainings of MS lesion tissue with MS-mAb revealed myelin reactivity in the cePC repertoire of all four patients and intracellular filament reactivity in one patient. While myelin staining by MS-mAb was only rarely detectable in non-MS CNS white matter tissue, it was greatly enhanced at the edge of demyelinating lesions in MS brain tissue. Our findings provide conclusive evidence for the presence of an antigen-driven B cell response in the CSF of MS patients directed against epitopes present in areas of myelin degradation. [source] Novel ELISA systems for antibodies to desmoglein 1 and 3: correlation of disease activity with serum autoantibody levels in individual pemphigus patientsEXPERIMENTAL DERMATOLOGY, Issue 5 2010Enno Schmidt Please cite this paper as: Novel ELISA systems for antibodies to desmoglein 1 and 3: correlation of disease activity with serum autoantibody levels in individual pemphigus patients. Experimental Dermatology 2010; 19: 458,463. Abstract:, Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are intraepidermal blistering skin diseases. PV is characterised by autoantibodies directed against desmoglein (Dsg) 3 and in patients with the mucocutaneous variant also against Dsg 1, whereas in PF, only Dsg 1 is targeted. Here, ectodomains of Dsg 3 and Dsg 1 were recombinantly expressed in a human cell line (HEK293) and applied as authentic solid phases in ELISA test systems. Autoantibodies against Dsg 3 and/or Dsg 1 could be detected in 71 (100%) of 71 PV sera and against Dsg 1 in 48 (96%) of 50 PF sera. Control sera showed reactivity with Dsg 3 and Dsg 1 in 0.2% and 0.7%, respectively, of 401 healthy blood donors and in 2.1% of 48 randomly selected patients with bullous pemphigoid. No reactivity with Dsg 1 and 3 was detected in 21 patients with linear IgA disease. For both pemphigus variants, a statistically significant correlation between clinical severity and autoantibody levels was observed as demonstrated for 10 PV and 5 PF patients. In conclusion, the use of the ectodomains of Dsg 3 and 1 as target antigens expressed in a human cell line resulted in sensitive and specific ELISA systems for both diagnosis and monitoring of PV and PF. [source] Engineering therapeutic monoclonal antibodiesIMMUNOLOGICAL REVIEWS, Issue 1 2008Xiao-yun Liu Summary: During last two decades, the chimerization and humanization of monoclonal antibodies (mAbs) have led to the approval of several for the treatment of cancer, autoimmune diseases, and transplant rejection. Additional approaches have been used to further improve their in vivo activity. These include combining them with other modalities such as chemotherapy and redesigning them for improved pharmacokinetics, effector function, and signaling activity. The latter has taken advantage of new insights emerging from an increased understanding of the cellular and molecular mechanisms that are involved in the interaction of immunoglobulin G with Fc receptors and complement as well as the negative signaling resulting from the hypercrosslinking of their target antigens. Hence, mAbs have been redesigned to include mutations in their Fc portions, thereby endowing them with enhanced or decreased effector functions and more desirable pharmacokinetic properties. Their valency has been increased to decrease their dissociation rate from cells and enhance their ability to induce apoptosis and cell cycle arrest. In this review we discuss these redesigned mAbs and current data concerning their evaluation both in vitro and in vivo. [source] Identification and prevalence of CD8+ T-cell responses directed against Epstein-Barr virus-encoded latent membrane protein 1 and latent membrane protein 2INTERNATIONAL JOURNAL OF CANCER, Issue 1 2002Pauline Meij Abstract Epstein-Barr virus (EBV) is associated with several human malignancies that each show different viral gene expression profiles. In malignancies such as Hodgkin's disease and nasopharyngeal carcinoma only Epstein-Barr nuclear antigen 1 (EBNA1) and varying levels of latent membrane proteins 1 and 2 (LMP1 and -2) are expressed. Since endogenously expressed EBNA1 is protected from CTL recognition, LMP1 and LMP2 are the most likely target antigens for anti-tumor immunotherapy. Therefore, we sought to identify in a systematic way CD8+ T-cell responses directed against eptitopes derived from LMP1 and LMP2. Using IFN,-ELISPOT assays of interferon-, release, peripheral blood mononuclear cells (PBMC) of healthy donors were screened with peptide panels (15 mer overlapping by 10) spanning the LMP1 and LMP2 sequences of the prototype EBV strain B95.8. When positive responses were found, CD4+ or CD8+ T cells were depleted from donor PBMC to determine the origin of the responder population. We detected CD8+ T-cell responses to LMP1 in 9/50(18%) donors and to LMP2 in 15/28 (54%) donors. In addition to the already described epitopes, 3 new LMP1- and 5 new LMP2-derived CD8+ epitopes were identified. In most donors LMP1- and LMP2-specific CD8+ precursor frequencies were low compared with precursors against immunodominant EBV epitopes from latent (EBNA3A, -3B and -3C) and lytic cycle antigens. These results demonstrate that CD8+ memory T cell responses to LMP1 and especially to LMP2 do exist in Caucasians, albeit at low levels and could potentially be exploited for therapeutic use. © 2002 Wiley-Liss, Inc. [source] Chronic sensorimotor polyradiculopathy with antibodies to P2: An electrophysiological and immunoproteomic analysisMUSCLE AND NERVE, Issue 1 2008Ricard Rojas-Garcia MD Abstract In this study we report a patient with chronic progressive sensory ataxia, proximal weakness, immunoglobulin M (IgM) monoclonal gammopathy, and elevated protein levels in the cerebrospinal fluid, who showed a good response to prednisone. Electrophysiological study disclosed abnormalities predominantly of late responses (F waves and H reflexes), with no evidence of demyelination in the peripheral nerves, suggesting motor and preganglionic sensory nerve roots as the site of the lesion. An immune-mediated pathogenesis was considered and, to identify possible target antigens, we performed bidimensional electrophoresis and a Western blot study. Based on the suspected lesion site, we used human anterior and posterior root extracts. We identified IgM reactivity against peripheral nerve myelin protein P2. Enzyme-linked immunosorbent assay confirmed IgM reactivity toward one synthetic peptide from P2. To our knowledge, reactivity against P2 has not been reported previously in a paraproteinemic neuropathy. Furthermore, we demonstrated that bidimensional electrophoresis and Western blot of the tissue involved, as determined by clinical and electrophysiological studies, may be useful to establish clinical,immunological correlations in paraproteinemic neuropathies. Muscle Nerve, 2008 [source] PL1 Subepithelial bullous diseases , topic overviewORAL DISEASES, Issue 2006M Mravak-Stipeti Subepithelial bullous diseases comprise the group of mucocutaneous autoimmune blistering diseases characterized by subepithelial separation and the deposition of immunoglobulin and complement against several antigens along the basement membrane zone (BMZ). This result in spectrum of diseases that affect skin, oral mucosa, and other mucosal membranes and include bullous pemphigoid (BP), mucous membrane (cicatricial) pemphigoid (MMP), linear IgA disease (LAD), and chronic bullous dermatosis of childhood (CBDC). The most common clinical features are oral erosions, desquamative gingivitis and conjunctival fibrosis, as well as skin lesions, predominantly in older female population. The heterogeneity of clinical presentation and diversity of target autoantigens have contributed to difficulties in characterizing this condition immunologically. In addition to the clinical presentation and a subepithelial vesicle or bullae on routine histologic analysis, the diagnosis is based on direct and indirect immunofluorescence studies. The nature of the disease is determined by the target antigens in the epithelium and BMZ such as antigen 180 (BP180), antigen 230 (BP230), laminin 5, and beta 4 integrin. Circulating IgG and IgA antibodies bind to different epitopes of BP180. The use of salt-split skin substrate enables differentiation between epidermal and dermal 'binders'. Since the antigen and the antibody titer appear to have direct relationships with the disease severity, and a combination of clinical finding and antibody titer provides valuable prognostic data, these investigations should be carried out routinely. Clinicians should recognize clinical spectrum of SBD, the histopathologic and immunopathologic characteristics, the differential diagnosis, the treatment, and the natural history of the disease. Involvement of oral medicine specialists, dermatologists, ophthalmologists, otolaryngologists and gastroenterologists contribute to early diagnosis and will aid in providing SBD patients with the highest quality of care. [source] Antiphospholipid antibodies (APLA) in immune thrombocytopenic purpura (ITP) and antiphospholipid syndrome (APS)AMERICAN JOURNAL OF HEMATOLOGY, Issue 6 2006Carlos J. Bidot Abstract Antiphospholipid antibodies (APLA) are associated with anti-phospholipid syndrome (APS), a thrombotic disorder, but they are also frequently detected in immune thrombocytopenic purpura (ITP), a bleeding disorder. To investigate possible differences of APLA between these two disorders, we assayed IgG and IgM APLA by ELISA in 21 patients with ITP and 33 with APS. The APLA reacting against two protein target antigens, ,2 -glycoprotein 1 (,2GP1) and FVII/VIIa, and four phospholipids [cardiolipin (CL), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE)] as well as lupus anticoagulant (LA) were analyzed. We made the following observations: (i) IgG and IgM antibodies to ,2GP1 and IgM antibodies to FVII/VIIa were more common in APS than ITP, P < 0.05, while IgG antibodies against the phospholipids (aCL, aPC, aPS, aPE) were more common in ITP than APS, P < 0.05; (ii) multiple APLA ,3 antigens) were more frequent in APS than ITP, P < 0.05; (iii) LA was frequently associated with APS but was absent in ITP; (iv) APLA is quite common in ITP: two-thirds were positive for at least one APLA. In summary, APLA are prevalent in ITP but their profile differs from APS. In APS, antibodies were predominantly against ,2GP1 and 80% had positive LA, while in ITP the APLA reacted most often with the phospholipids without LA. The difference in APLA may result in opposite clinical manifestations in two disorders. Am. J. Hematol. 81:391,396, 2006. © 2006 Wiley-Liss, Inc. [source] Redirecting lentiviral vectors by insertion of integrin-tageting peptides into envelope proteinsTHE JOURNAL OF GENE MEDICINE, Issue 7 2009Kouki Morizono Abstract Background Targeting gene therapy vectors that can home in on desired cell and tissue types in vivo comprise the ultimate gene delivery system. We have previously developed targeting lentiviral vectors by pseudotyping vectors with modified Sindbis virus envelope proteins. The envelope protein contains the Fc-binding region of protein A (ZZ domain), so the virus can be conjugated with antibodies. The conjugated antibody mediates specific transduction of the cells and tissues expressing the target antigens, both in vitro and in vivo. However, more stable conjugation of targeting molecules would be optimal for use in immunocompetent animals, as well as in humans. Methods We inserted integrin-targeting peptides into two sites of the targeting envelope proteins and determined whether the peptides serve as receptor-binding regions of the envelope proteins and redirect the pseudotyped viruses. Results The integrin-targeting peptides can mediate binding to cells via the interaction with integrins on target cells and transduction. Peptides with a higher binding affinity increase titers of pseudotyped virus. We found two regions on the envelope protein that can accommodate insertion and serve as receptor-binding regions. Combining the peptides in two distinct regions increased the titers of the virus. Conclusions Successful incorporation of targeting molecules into the envelope protein will broaden the application of targeting vectors for a wide variety of experimental and clinical settings. Copyright © 2009 John Wiley & Sons, Ltd. [source] Exploration of target molecules for prostate cancer gene therapyTHE PROSTATE, Issue 11 2007Kazuhiro Suzuki Abstract BACKGROUND Focusing on Adv-FZ33, a modified adenovirus in which a synthetic 33-amino-acid immunoglobulin G-binding domain was inserted into the adenoviral fiber protein, we tried to identify suitable target molecules for prostate cancer-specific gene therapy. METHODS Hybridomas were established from mice immunized with prostate cancer cell lines. The hybridomas were screened using Adv-FZ33 to create monoclonal antibodies (mAbs) that induced high gene transfer efficiency for PC-3 cells. Furthermore, we identified target antigens of the mAbs by immunoprecipitation and mass spectrometry, and investigated the expression of target molecules by flow cytometry and immunocytochemistry. RESULTS Using Adv-FZ33, we established four different mouse mAbs that increased transduction efficiency for PC-3. The target antigens identified were Ep-CAM, CD155, HAI-1, and Na,K-ATPase ,1. These antigens were expressed in several cancer cell lines, including prostate cancer. Human prostatic myofibroblast cells lacked expression of Ep-CAM and HAI-1. CONCLUSIONS We established anti-Ep-CAM mAb and anti- HAI-1 mAbs. Gene transduction via Ep-CAM and HAI-1 may be a novel strategy for treatment of prostate cancer. Prostate 67: 1163,1173, 2007. © 2007 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||