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Autoantigen
Kinds of Autoantigen Selected AbstractsHemidesmosome protein dynamics in live epithelial cellsCYTOSKELETON, Issue 2 2003Daisuke Tsuruta Abstract Hemidesmosomes mediate stable anchorage of epithelial cells to laminin-5 in the basement membrane zone and have been likened to spot-welds. Indeed, it has been assumed that hemidesmosomes are not dynamic, at least when compared to other matrix adhesion sites including focal contacts. We tested this notion by monitoring the fate of green fluorescent protein (GFP)-tagged human integrin ,4 subunit (GFP-h,4) and GFP-tagged 180-kD human bullous pemphigoid (BP) autoantigen (GFP-BP180) in live cultures of 804G cells that assemble numerous mature hemidesmosomes. In subconfluent 804G cells, both GFP-h,4 and GFP-BP180 protein clusters are not stable but assemble into and disassemble out of cat paw,like arrays at a relatively rapid rate. In confluent populations of 804G cells, although some cat paw,like clusters of both GFP-h,4 and GFP-BP180 are stable over periods of >60 min, other GFP-h,4 and GFP-BP180 protein arrays form and/or disappear during the same time period. Moreover, individual labeled particles show considerable motility in the plane of the membrane. Fluorescence recovery after photobleaching analyses provide a further indication of the dynamics of hemidesmosome proteins. In particular, bleached GFP-h,4 protein clusters in confluent cells recover signal within about 30 min, indicating that there is a relatively rapid turnover of hemidesmosome components in protein arrays clustered along the substratum attached surface of a cell. The rate of recovery is dependent on an intact microfilament system. In sharp contrast, bleached GFP-BP180 protein clusters in confluent cells fail to recover signal even when observed for longer than 60 min. To evaluate hemidesmosome protein dynamics in motile cells, we monitored GFP-h,4 and GFP-BP180 in 804G cells populating scrape wound sites in vitro. In these migratory cells, which lack mature hemidesmosomes, integrin ,4 subunit and BP180 protein clusters progressively assemble and disassemble into linear and cat-paw arrays. In summary, hemidesmosome protein clusters, like their counterparts in focal contacts, are dynamic. We discuss these results in relation to hemidesmosome functions. Cell Motil. Cytoskeleton 54:122,134, 2003. © 2003 Wiley-Liss, Inc. [source] Malondialdehyde modification of myelin oligodendrocyte glycoprotein leads to increased immunogenicity and encephalitogenicityEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2007Maja Wållberg Abstract Self proteins may become autoantigenic through structural modification. We studied malondialdehydation of recombinant rat (rr) myelin oligodendrocyte glycoprotein (MOG), an autoantigen in multiple sclerosis. Malondialdehyde (MDA) modification changed protein weight and charge, the location of these adducts being mapped by Fourier transform ion cyclotron resonance. Molecular modelling revealed significant differences in the MDA-rrMOG three-dimensional structure. DBA/1 mice immunised with MDA-rrMOG developed greater proliferative responses and more severe experimental autoimmune encephalomyelitis than mice immunised with unmodified rrMOG. MDA-rrMOG was taken up more effectively by antigen-presenting cells (APC), at least partially through scavenger receptors. Exposure to MDA-rrMOG led to increased expression of IL-23, IL-12 and IL-12R, indicating a role not only for increased antigen uptake but also for activation of APC. We thus provide biochemical, structural, immunological and clinical data that suggest that the post-translationally modified form of this myelin autoantigen is a more relevant form of the molecule. [source] Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin lociEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2005Evdokia Protopapadakis Abstract The isolation of human antibodies against muscle acetylcholine receptor (AChR), the autoantigen involved in myasthenia gravis (MG), is important for the development of therapeutically useful reagents. Monovalent antibody fragments from monoclonal antibodies against the main immunogenic region (MIR) of AChR protect the receptor from the destructive activity of MG autoantibodies. Human anti-AChR ,-subunit antibody fragments with therapeutic potential have been isolated using phage display antibody libraries. An alternative approach for obtaining human mAb has been provided by the development of humanized mice. In this report, we show that immunization of transgenic mouse strains with the extracellular domain of the human AChR ,-subunit results in antibody responses and isolation of hybridomas producing human mAb. Four specific IgM mAb were isolated and analyzed. mAb170 recognized the native receptor the best and was capable of inducing AChR antigenic modulation, suggesting its specificity for a pathogenic epitope. Moreover, the recombinant antigen-binding (Fab) fragment of this mAb competed with an anti-MIR mAb, revealing that its antigenic determinant lies in or near the MIR. Finally, Fab170 was able to compete with MG autoantibodies and protect the AChR against antigenic modulation induced by MG sera. This approach will be useful for isolating additional mAb with therapeutic potential against the other AChR subunits. [source] Alpha B-crystallin is not a dominant peripheral T-cell autoantigen in multiple sclerosis amongst SardiniansEUROPEAN JOURNAL OF NEUROLOGY, Issue 5 2003S. Sotgiu The heat shock protein alpha B-crystallin appears to be the dominantly recognized autoantigen in the early demyelinative process of multiple sclerosis (MS) in brain of patients. In Sardinia, MS is linked to human leucocyte antigen (HLA)-DR alleles that might influence the production of cytokines from peripheral lymphocytes. We tested the nature of peripheral anti-alpha B-crystallin-specific T-cell response in the context of predisposing HLA haplotypes both in MS patients and healthy controls. The alpha B-crystallin specific T-cell lines were generated by using the ,split-well' technique. The results indicate that the presence of short-term T-cell lines towards alpha B-crystallin is numerically comparable between the two groups and not restricted to MS-predisposing HLA-DR alleles. As for the T-cell characterization, CD4+ anti-alpha B-crystallin T cells secreting high levels of interferon- , are similarly identified in MS and healthy donors. In conclusion, the peripheral response towards the myelin antigen alpha B-crystallin is neither quantitatively nor qualitatively peculiar to MS, in contrast to the theoretical paradigm suggesting peripheral activation of myelin-reactive T cells to be the prerequisite for MS induction. [source] Desmoglein-3 is a target autoantigen in spontaneous canine pemphigus vulgarisEXPERIMENTAL DERMATOLOGY, Issue 2 2003Thierry Olivry Abstract: Pemphigus vulgaris (PV) is an autoimmune blistering skin disease of humans and companion animals. In human patients, PV is associated with the production of IgG autoantibodies specific for keratinocyte desmosomal glycoproteins of the cadherin family. The purpose of this study was to determine whether antikeratinocyte IgG autoantibodies were present in the skin and serum of dogs with PV, and also to identify the canine PV autoantigen(s) targeted by circulating autoantibodies. Eleven dogs were selected because of the microscopic demonstration of suprabasal epithelial acantholysis. Direct immunofluorescence revealed the presence of IgG autoantibodies bound to the membrane of keratinocytes in skin biopsy specimens of 8/9 dogs (89%). Using indirect immunofluorescence, serum-circulating IgG autoantibodies were found in 10/11 (91%) and 5/11 (45%) dogs, using normal canine gingiva and cultured canine oral keratinocytes, respectively. By immunoblotting using cultured canine oral keratinocyte protein lysates, IgG autoantibodies from 7/9 (78%) tested dogs recognized a 130-kDa antigen that comigrated with that identified by rabbit polyclonal antibodies raised against desmoglein-3. This 130 kDa antigen was confirmed to represent the canine equivalent of human desmoglein-3 by immunoprecipitation-immunoblotting. The results of these studies provide evidence that the canine desmoglein-3 homologue is a major autoantigen in dogs with PV. These observations further establish spontaneous canine PV as a natural model for research on pathogenesis, etiology and novel therapeutic approaches for this disease of humans. [source] The analysis of the fine specificity of celiac disease antibodies using tissue transglutaminase fragmentsFEBS JOURNAL, Issue 21 2002Daniele Sblattero Celiac disease is an intestinal malabsorption characterized by an intolerance to cereal proteins accompanied by immunological responses to dietary gliadins and an autoantigen located in the endomysium. The latter has been identified as the enzyme tissue transglutaminase which belongs to a family of enzymes that catalyze protein cross-linking reactions and is constitutively expressed in many tissues as well as being activated during apoptosis. In a recent paper, we described the selection and characterization of anti-transglutaminase Igs from phage antibody libraries created from intestinal lymphocytes from celiac disease patients. In this work, using transglutaminase gene fragments, we identify a region of tissue transglutaminase recognized by these antibodies as being conformational and located in the core domain of the enzyme. This is identical to the region recognized by anti-transglutaminase Igs found in the serum of celiac disease patients. [source] Helicobacter pylori, T cells and cytokines: the "dangerous liaisons"FEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2005Mario Milco D'Elios Abstract Helicobacter pylori infection is the major cause of gastroduodenal pathologies, but only a minority of infected patients develop chronic and life threatening diseases, as peptic ulcer, gastric cancer, B-cell lymphoma, or autoimmune gastritis. The type of host immune response against H. pylori is crucial for the outcome of the infection. A predominant H. pylori -specific Th1 response, characterized by high IFN-,, TNF-,, and IL-12 production associates with peptic ulcer, whereas combined secretion of both Th1 and Th2 cytokines are present in uncomplicated gastritis. Gastric T cells from MALT lymphoma exhibit abnormal help for autologous B-cell proliferation and reduced perforin- and Fas,Fas ligand-mediated killing of B cells. In H. pylori -infected patients with autoimmune gastritis cytolytic T cells infiltrating the gastric mucosa cross-recognize different epitopes of H. pylori proteins and H+K+ ATPase autoantigen. These data suggest that peptic ulcer can be regarded as a Th1-driven immunopathological response to some H. pylori antigens, whereas deregulated and exhaustive H. pylori -induced T cell-dependent B-cell activation can support the onset of low-grade B-cell lymphoma. Alternatively, H. pylori infection may lead in some individuals to gastric autoimmunity via molecular mimicry. [source] Cellular and humoral autoimmunity directed at bile duct epithelia in murine biliary atresia,,HEPATOLOGY, Issue 5 2006Cara L. Mack Biliary atresia is an inflammatory fibrosclerosing lesion of the bile ducts that leads to biliary cirrhosis and is the most frequent indication for liver transplantation in children. The pathogenesis of biliary atresia is not known; one theory is that of a virus-induced, subsequent autoimmune-mediated injury of bile ducts. The aim of this study was to determine whether autoreactive T cells and autoantibodies specific to bile duct epithelia are present in the rotavirus (RRV)- induced murine model of biliary atresia and whether the T cells are sufficient to result in bile duct inflammation. In vitro analyses showed significant increases in IFN-,,producing T cells from RRV-diseased mice in response to bile duct epithelial autoantigen. Adoptive transfer of the T cells from RRV-diseased mice into naïve syngeneic SCID recipients resulted in bile duct,specific inflammation. This induction of bile duct pathology occurred in the absence of detectable virus, indicating a definite response to bile duct autoantigens. Furthermore, periductal immunoglobulin deposits and serum antibodies reactive to bile duct epithelial protein were detected in RRV-diseased mice. In conclusion, both cellular and humoral components of autoimmunity exist in murine biliary atresia, and the progressive bile duct injury is due in part to a bile duct epithelia,specific T cell,mediated immune response. The role of cellular and humoral autoimmunity in human biliary atresia and possible interventional strategies therefore should be the focus of future research. (HEPATOLOGY 2006;44:1231,1239.) [source] Bacterial motif DNA as an adjuvant for the breakdown of immune self-tolerance to pyruvate dehydrogenase complexHEPATOLOGY, Issue 3 2002David E. J. Jones Bacterial DNA containing unmethylated CpG dinucleotide motifs is immunostimulatory to mammals, skewing CD4+ T-cell responses toward the Th1 phenotype. Autoreactive T-cell responses seen in primary biliary cirrhosis (PBC) are typically of the Th1 phenotype, raising the possibility that bacterial DNA might play a role in the generation of pathologic autoimmunity. We therefore studied the effects of CpG motif-containing oligodeoxynucleotides (ODN) on responses to pyruvate dehydrogenase complex (PDC, the autoantigen in PBC) in a murine model. Sensitization of SJL/J mice with non,self-PDC has been shown to result in induction of autoreactive T-cell responses to PDC sharing characteristics with those seen in patients with PBC. Administration of CpG ODN to SJL/J mice at the time of sensitization with PDC resulted in a significant skewing of splenic T-cell response to self-PDC, with significant augmentation of the Th1 cytokine response (interleukin [IL] 2 and interferon [IFN] gamma) and reduction of the Th2 response (IL-4 and IL-10). In fact, CpG ODN seemed to be more effective at biasing the response phenotype and as effective at inducing liver histologic change as complete Freund's adjuvant (CFA), the standard adjuvant used for induction of Th1 responses in murine autoimmune and infectious immunity models. In conclusion, our findings raise the possibility that bacteria play a role in the development of autoimmunity (in PBC at least) through the potential of their DNA to shift the T-cell responses toward the phenotype associated with autoimmune damage. Moreover, this study suggests caution in the therapeutic use of CpG ODN as vaccine adjuvants. [source] Genetic analysis of collagen-induced arthritis in rats: a polygenic model for rheumatoid arthritis predicts a common framework of cross-species inflammatory/autoimmune disease lociIMMUNOLOGICAL REVIEWS, Issue 1 2001Marie M. Griffiths Summary: Collagen-induced arthritis (CIA) is a useful model for dissecting the genetic patterns underlying susceptibility to rheumatoid arthritis (RA) and related chronic/inflammatory autoimmune diseases. CIA exhibits three phenotypes characteristic of autoimmune disease pathogenesis: abnormal levels of immune reactivity to self antigens; chronic inflammation of target organs expressing that specific autoantigen; activation and direct participation of invading mononuclear cells and resident tissue fibroblasts in organ damage. Over 25 different quantitative trait loci (QTL) regulating arthritis severity and autoantibody in rats with CIA are mapped. QTL-congenic strains show that certain CIA,QTLs can modulate arthritis independently. These monogenic models are proving to be highly informative for fine mapping and function studies, revealing gender effects and evidence of gene clusters. Recent genome scans of RA populations identified RA-susceptibility loci in chromosome regions homologous to rat chromosomal segments housing CIA,QTLs. Also, CIA,QTLs frequently co-localize with susceptibility QTLs mapped in other rat arthritis models induced with non-immunogenic adjuvant oils and/or in rat autoimmune models of multiple sclerosis and diabetes. Common autoimmunity genes and inflammation genes important to several human diseases are likely being detected in the various rat disease models. Continued dissection of the genetic underpinnings of rat arthritis models should provide candidate genes for investigation in human patients and lead to a clearer understanding of the complex genetics of RA. [source] Induction and mechanism of action of transforming growth factor-,-secreting Th3 regulatory cellsIMMUNOLOGICAL REVIEWS, Issue 1 2001Howard L. Weiner Summary: Th3 CD4+ regulatory cells were identified during the course of investigating mechanisms associated with oral tolerance. Different mechanisms of tolerance are induced following oral antigen administration, including active suppression, clonal anergy and deletion. Low doses favor active suppression whereas high doses favor anergy/deletion. Th3 regulatory cells form a unique T-cell subset which primarily secretes transforming growth factor (TGF)-,, provides help for IgA and has suppressive properties for both Th1 and Th2 cells. Th3 type cells are distinct from the Th2 cells, as CD4+ TGF-,-secreting cells with suppressive properties have been generated from interleukin (IL)-4-deficient animals. In vitro differentiation of Th3 cells from Th precursors from T-cell antigen receptor (TCR) transgenic mice is enhanced by culture with TGF-,, IL-4, IL-10, and anti-IL-12. Th3 CD4+ myelin basic protein regulatory clones are structurally identical to Th1 encephalitogenic clones in TCR usage, MHC restriction and epitope recognition, but produce TGF-, with various amounts of IL-4 and IL-10. Because Th3 regulatory cells are triggered in an antigen-specific fashion but suppress in an antigen-non-specific fashion, they mediate "bystander suppression" when they encounter the fed autoantigen at the target organ. In vivo induction of Th3 cells and low dose oral tolerance is enhanced by oral administration of IL-4. Anti-CD86 but not anti-CD80 blocks the induction of Th3 cells associated with low dose oral tolerance. Th3 regulatory cells have been described in other systems (e.g. recovery from experimental allergic encephalomyelitis) but may be preferentially generated following oral antigen administration due to the gut immunologic milieu that is rich in TGF-, and has a unique class of dendritic cells. CD4+CD25+ regulatory T-cell function also appears related to TGF-,. [source] Central and peripheral autoantigen presentation in immune toleranceIMMUNOLOGY, Issue 2 2004Alberto Pugliese Summary Recent studies in both humans and experimental rodent models provide new insight into key mechanisms regulating tolerance to self-molecules. These recent advances are bringing about a paradigm shift in our views about tolerance to self-molecules with tissue-restricted expression. There is, indeed, mounting evidence that selected antigen-presenting cells (APCs) have the ability to synthesize and express self-molecules, and that such expression is critical for self-tolerance. Insulin is a key hormone produced exclusively by pancreatic ,-cells and a critical autoantigen in type 1 diabetes. It provides an excellent example of a molecule with tissue-restricted expression that is expressed ectopically by APCs. The fact that APCs expressing insulin have been demonstrated in both thymus and peripheral lymphoid tissues suggests that they may play a role in insulin presentation in both the central and peripheral immune system. Experimental mice, in which insulin expression was altered, provide functional data that help to dissect the role of insulin presentation by APCs of the immune system. This review addresses recent literature and emerging concepts about the expression of self-molecules in the thymus and peripheral lymphoid tissues and its relation to self-tolerance. [source] Studies on the association between immunoglobulin E autoreactivity and immunoglobulin E-dependent histamine-releasing factorsIMMUNOLOGY, Issue 2 2002Ilona Kleine Budde Summary It has been reported that serum immunoglobulin E (IgE) from certain atopic patients can sensitize basophils to release histamine in response to IgE-dependent histamine-releasing factors (HRFs). It has also been shown that patients suffering from severe forms of atopy may contain IgE autoantibodies. It was investigated whether HRF-responsive sera contained IgE autoantibodies and if there was an association between IgE autoreactivity and IgE-dependent responsiveness to HRF. The presence of HRF-responsive IgE (IgE+) in serum of patients with respiratory atopy was determined by stimulating stripped human basophils sensitized by serum with peripheral blood mononuclear cell (PBMC)-derived HRF, and measuring the release of histamine. In parallel, these sera were screened for the presence of IgE autoantibodies to nitrocellulose-blotted human cellular extracts. The capacity of IgE autoantigen-containing preparations to induce histamine release was tested in the stripped basophil assay. Eleven out of 52 sera contained IgE autoantibodies to blotted cellular extracts of human PBMCs or of the human epithelial cell line A431. No significant association was found between IgE autoreactivity and IgE-dependent responsiveness to HRF: 7/26 IgE+ sera contained IgE to human cellular extracts, and 4/26 of the sera without IgE+ did also. IgE autoantigen-containing extracts did not induce histamine release of appropriately sensitized basophils. By size-exclusion chromatography it was shown that a 32,000 MW autoantigen eluted in the >55,000 MW fraction, which indicates that this protein forms polymers or complexes with other macromolecules. This might explain the discrepancy between binding and histamine-releasing activity. A 20,000 MW IgE-defined autoantigen cross-reacted with a shrimp allergen. Our results indicate that IgE-reactivity to immunoblotted human protein and IgE-dependent HRF activity are distinct entities that may co-occur in atopic patients. [source] Identification of a novel staining pattern of bile duct epithelial cells in primary sclerosing cholangitisINFLAMMATORY BOWEL DISEASES, Issue 2 2010Brita Ardesjö PhD Abstract Background: Primary sclerosing cholangitis (PSC) is an inflammatory disease of the bile ducts with an unknown etiology. A number of autoantigens have been proposed, but an early diagnostic marker is still lacking. Our aim was to identify such an autoantigen. Methods: Immunostaining was performed on normal human bile duct with sera from patients with PSC and controls. To identify an autoantigen a cDNA library from normal human choledochus was constructed and immunoscreened with patient sera. Using in vitro transcription and translation and immunoprecipitation we examined the immunoreactivity against PDZ domain containing 1 (PDZK1) in 35 patients with PSC, 198 control patients, and 94 healthy controls. Results: We observed a previously unpublished staining pattern in which cytoplasmatic granules and apical cell membranes of biliary epithelial cells were stained by PSC sera. Strong immunoreactivity to these structures was obtained with 12 out of 35 PSC sera (34%) but not with sera from healthy controls. By screening the cDNA library we identified PDZK1 as a candidate antigen. Immunoreactivity against PDZK1 was detected in 9% of PSC patients, 2% of inflammatory bowel disease (IBD) patients, 8% of autoimmune pancreatitis patients, 18% of Grave's disease patients, and 1% of healthy controls. Conclusions: Previously unpublished, specific, and strong autoantibodies against epithelial cells of the bile duct in PSC sera were identified. Furthermore, PDZK1 is suggested as a potential new autoantigen. Inflamm Bowel Dis 2009 [source] SP100B is a repressor of gene expressionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2005Kent W. Wilcox Abstract Mammalian cell nuclei exhibit discrete sites where specific proteins characteristically localize. PML nuclear bodies (PML NBs) (nuclear domain 10s (ND10s)) are the primary localization site for the promyelocytic leukemia (PML) protein and the SP100 autoantigen. The observations that some PML and SP100 isoforms can function as transcriptional regulators, that both the size and number of PML bodies increase in response to interferon treatment, and that many mammalian viruses encode proteins that mediate disruption of PML bodies suggest that these sites suppress viral infection, perhaps by repressing viral gene expression. We hypothesized that a component of PML NBs functions as a repressor of gene expression. To test this hypothesis, we characterized the effect of PML or SP100 isoforms on expression of transfected reporter genes. PML-I, PML-VI, and SP100A did not repress reporter gene expression. In contrast, SP100B repressed reporter gene expression, especially under conditions in which the reporter gene expression was elevated by a viral transactivator or addition of trichostatin A to the culture medium. The SP100B DNA binding domain was required for repression. SP100B had no detectable effect on the amount, methylation pattern, or topological form of plasmid DNA in the nuclei of transfected cells. The demonstrated repressive activity of SP100B supports the hypothesis that SP100B is a component of an innate immune response that represses expression of ectopic DNA. © 2005 Wiley-Liss, Inc. [source] Innate and adaptive immune activation in the brain of MPS IIIB mouse modelJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2009Julianne DiRosario Abstract Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to ,- N -acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and real-time quantitative reverse transcriptase,polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect. © 2008 Wiley-Liss, Inc. [source] Co-localization of IgA and TG3 on healthy skin of coeliac patientsJOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY & VENEREOLOGY, Issue 4 2007C Cannistraci Abstract Background, Dermatitis herpetiformis (DH), the skin's expression of coeliac disease (CD), is induced by the presence of IgA antibodies and epidermal transglutaminase (TG3) as the main autoantigen, stored in the papillary dermis and on the vessel walls. Aims, To evaluate the presence of IgA and TG3 deposits, considered to be the first step in inducing DH, in healthy skin of coeliac patients without cutaneous manifestations. Methods, Punch biopsies were taken from 11 consecutive coeliac patients, two with DH and nine without cutaneous manifestations, three of whom were adhering to a gluten-free diet (GFD), and evaluated for the presence of deposits in the upper dermis and vessel walls by immunofluorescence and confocal microscopy. Results, In coeliac patients affected by DH we found the presence of IgA and TG3 deposits mainly on the upper dermis, but also in vessel walls. In all coeliac patients without DH and also in those patients who were following a strict GFD, we found widely variable deposits of IgA and TG3 in both the papillary dermis and the vessel walls, although a lower intensity of the fluorescence signal was detected than with coeliac patients affected by DH. Double immunostaining with anti-IgA and anti-TG3 antibodies showed a strong co-localization in the upper dermis in patients with DH and a weaker co-localization in those without DH. Conclusions, We have demonstrated the presence of IgA and TG3 deposits in the healthy skin of coeliac patients, which are considered to play a central role in the pathogenesis of DH. [source] Acetylcholine receptor-, subunit expression in myasthenia gravis: A role for the autoantigen in pathogenesis?MUSCLE AND NERVE, Issue 2 2009Jian Rong Sheng PhD Abstract Previous studies have shown increased expression of acetylcholine receptor-alpha (AChR-,) subunit transcripts in myasthenia gravis (MG) and experimental MG (EAMG), but none examined the functional properties of this overexpression. In this study we examined the mRNA and protein expression of AChR-, as well as the pattern of ,-bungarotoxin labeling in muscle tissue from EAMG mice with varying disease severity. AChR-, expression was increased considerably in endplates from mice with severe EAMG, but it was distinct and greatly in excess of ,-bungarotoxin labeling. This "aberrant expression" occurred in mice with morphologic endplate damage, and the pattern of complement and immunoglobulin deposition in muscle from these mice appeared to mirror the pattern of AChR-, expression. The loss of functional AChR in severe MG increases transcription of AChR-, mRNA, but the expressed protein is "functionally inert," failing to compensate for loss of AChR. This enhanced expression of AChR may play a role in driving the ongoing autoimmune response. Muscle Nerve 40: 279,286, 2009 [source] Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutantPLANT BIOTECHNOLOGY JOURNAL, Issue 8 2010Linda Avesani Summary The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM. [source] MS analysis of rheumatoid arthritic synovial tissue identifies specific citrullination sites on fibrinogenPROTEOMICS - CLINICAL APPLICATIONS, Issue 5 2010Monika Hermansson Abstract Purpose: Citrullination is a post-translational modification of arginine residues to citrulline catalyzed by peptidyl arginine deiminases. Induced expression of citrullinated proteins are frequently detected in various inflammatory states including arthritis; however, direct detection of citrullination in arthritic samples has not been successfully performed in the past. Experimental design: Citrullination of human fibrinogen, a candidate autoantigen in arthritis, was studied. Accurate identification of citrullinated fibrinogen peptides from rheumatoid arthritis synovial tissue specimens was performed using accurate mass and retention time analysis. Results: A peptide with the sequence ESSSHHPGIAEFPSRGK corresponding to amino acids 559,575 of fibrinogen ,-chain was identified to be citrullinated with an occupancy rate between 1.4 and 2.5%. Citrullination of the peptide KREEAPSLRPAPPPISGGGYRARPAK corresponding to amino acids 52,77 of the fibrinogen ,-chain was identified with an occupancy rate of 1.2%. Conclusions and clinical relevance: We report a proof of principle study for the identification of citrullinated proteins and within them, identification of citrullination sites and quantification of their occupancies in synovial tissue from rheumatoid arthritis patients using high-resolution MS. Detailed studies on which molecules are citrullinated in arthritis can provide information about their role in immune regulation and serve as novel biomarkers and potentially even as therapeutic targets. [source] Transplantation of bone marrow genetically engineered to express proinsulin II protects against autoimmune insulitis in NOD miceTHE JOURNAL OF GENE MEDICINE, Issue 11 2006James Chan Abstract Background Type 1 diabetes (T1D) is a T-cell-dependent autoimmune disease resulting from destructive inflammation (insulitis) of the insulin-producing pancreatic ,-cells. Transgenic expression of proinsulin II by a MHC class II promoter or transfer of bone marrow from these transgenic mice protects NOD mice from insulitis and diabetes. We assessed the feasibility of gene therapy in the NOD mouse as an approach to treat T1D by ex vivo genetic manipulation of normal hematopoietic stem cells (HSCs) with proinsulin II followed by transfer to recipient mice. Methods HSCs were isolated from 6,8-week-old NOD female mice and transduced in vitro with retrovirus encoding enhanced green fluorescent protein (EGFP) and either proinsulin II or control autoantigen. Additional control groups included mice transferred with non-manipulated bone marrow and mice which did not receive bone marrow transfer. EGFP-sorted or non-sorted HSCs were transferred into pre-conditioned 3,4-week-old female NOD mice and insulitis was assessed 8 weeks post-transfer. Results Chimerism was established in all major lymphoid tissues, ranging from 5,15% in non-sorted bone marrow transplants to 20,45% in EGFP-sorted bone marrow transplants. The incidence and degree of insulitis was significantly reduced in mice receiving proinsulin II bone marrow compared to controls. However, the incidence of sialitis in mice receiving proinsulin II bone marrow and control mice was not altered, indicating protection from insulitis was antigen specific. Conclusions We show for the first time that ex vivo genetic manipulation of HSCs to express proinsulin II followed by transplantation to NOD mice can establish molecular chimerism and protect from destructive insulitis in an antigen-specific manner. Copyright © 2006 John Wiley & Sons, Ltd. [source] Na+ -H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3THE JOURNAL OF PHYSIOLOGY, Issue 2 2001Xuhang Li 1Rabbit ileal Na+ -absorbing cell Na+ -H+ exchanger 3 (NHE3) was shown to exist in three pools in the brush border (BB), including a population in lipid rafts. Approximately 50 % of BB NHE3 was associated with Triton X-100-soluble fractions and the other ,50 % with Triton X-100-insoluble fractions; ,33 % of the detergent-insoluble NHE3 was present in cholesterol-enriched lipid microdomains (rafts). 2The raft pool of NHE3 was involved in the stimulation of BB NHE3 activity with epidermal growth factor (EGF). Both EGF and clonidine treatments were associated with a rapid increase in the total amount of BB NHE3. This EGF- and clonidine-induced increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the total detergent-insoluble fraction, but there was no change in the detergent-soluble pool. In agreement with the rapid increase in the amount of NHE3 in the BB, EGF also caused a rapid stimulation of BB Na+ -H+ exchange activity. 3Disrupting rafts by removal of cholesterol with methyl-,-cyclodextrin (M,CD) or destabilizing the actin cytoskeleton with cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically, NHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibody-coated magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and M,CD treatment. 4We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3; (iii) lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent-soluble NHE3 pool, are involved in the EGF- and clonidine-induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles in the basal endocytosis of BB NHE3. [source] AMPA receptor antibodies in limbic encephalitis alter synaptic receptor location,ANNALS OF NEUROLOGY, Issue 4 2009Meizan Lai MD Objective To report the clinical and immunological features of a novel autoantigen related to limbic encephalitis (LE) and the effect of patients' antibodies on neuronal cultures. Methods We conducted clinical analyses of 10 patients with LE. Immunoprecipitation and mass spectrometry were used to identify the antigens. Human embryonic kidney 293 cells expressing the antigens were used in immunocytochemistry and enzyme-linked immunoabsorption assay. The effect of patients' antibodies on cultures of live rat hippocampal neurons was determined with confocal microscopy. Results Median age was 60 (38,87) years; 9 were women. Seven had tumors of the lung, breast, or thymus. Nine patients responded to immunotherapy or oncological therapy, but neurological relapses, without tumor recurrence, were frequent and influenced the long-term outcome. One untreated patient died of LE. All patients had antibodies against neuronal cell surface antigens that by immunoprecipitation were found to be the glutamate receptor 1 (GluR1) and GluR2 subunits of the ,-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Human embryonic kidney 293 cells expressing GluR1/2 reacted with all patients' sera or cerebrospinal fluid, providing a diagnostic test for the disorder. Application of antibodies to cultures of neurons significantly decreased the number of GluR2-containing AMPAR clusters at synapses with a smaller decrease in overall AMPAR cluster density; these effects were reversed after antibody removal. Interpretation Antibodies to GluR1/2 associate with LE that is often paraneoplastic, treatment responsive, and has a tendency to relapse. Our findings support an antibody-mediated pathogenesis in which patients' antibodies alter the synaptic localization and number of AMPARs. Ann Neurol 2009;65:424,434 [source] Autoantigen diversity in the opsoclonus-myoclonus syndromeANNALS OF NEUROLOGY, Issue 3 2003Luis Bataller MD Despite circumstantial evidence that opsoclonus-myoclonus (OM) is often immune mediated, no specific autoantigen has been identified. Using sera of 21 patients with several types of OM (idiopathic, associated to small cell lung cancer, and associated to neuroblastoma), we probed a brainstem cDNA library to isolate target neuronal antigens. Thirty-seven clones coding for 25 proteins were isolated, with two groups of autoantigens emerging: (1) proteins of the postsynaptic density, among them the adenomatous polyposis coli, and 2) proteins with expression or function restricted to neurons, including RNA or DNA-binding proteins and zinc-finger proteins. Usually, each patient's serum recognized a different autoantigen, except for adenomatous polyposis coli that was recognized by sera of two patients with idiopathic OM and two control patients with nystagmus, diplopia, and paraneoplastic brainstem dysfunction. Overall, in the indicated types of OM, (1) we found frequent and heterogeneous immunity to neuronal autoantigens without a single specific antibody marker of OM, (2) the occasional detection of antibodies to known onconeuronal antigens (ie, Hu proteins) probably is related to cancer-induced immunity rather than to OM, and (3) the postsynaptic density is a frequent source of novel autoantigens, with several proteins of this complex targeted by antibodies of OM patients. Ann Neurol 2003 [source] The MAZ protein is an autoantigen of Hodgkin's disease and paraneoplastic cerebellar dysfunctionANNALS OF NEUROLOGY, Issue 1 2003Luis Bataller MD Probing a cerebellar expression library with TrAb sera from patients with Hodgkin's disease and paraneoplastic cerebellar degeneration resulted in the isolation of MAZ (myc -associated zinc-finger protein). Eleven of 19 TrAb sera and 16 of 131 controls reacted with MAZ, indicating a significant, although not specific, association between Tr and MAZ immunities (p < 0.001). Interestingly, 9 of 16 positive control patients also had cerebellar dysfunction. Purified MAZ antibodies reacted with Purkinje cells. In neuronal cells, MAZ interacts with DCC (Deleted in Colorectal Cancer product), the receptor for netrin-1, a neuronal survival factor. These findings suggest epitope spreading between the Tr antigen and the MAZ,DCC complex and offer a possible model of immune-mediated cerebellar disease. Ann Neurol 2003;53:000,000 [source] Reactivity with dichotomous determinants of Ro 60 stratifies autoantibody responses in lupus and primary Sjögren's syndromeARTHRITIS & RHEUMATISM, Issue 5 2010Joanne H. Reed Objective Analysis of B cell determinants of Ro 60 exposed on the surface of apoptotic cells (apotopes) or intracellular epitopes provides insight into the structural forms of the autoantigen that break immune tolerance. This study was initiated to compare anti,Ro 60 responses in systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (SS) against membrane-bound and intracellular forms of Ro 60. Methods The reactivity of autoantibodies from patients with SLE and primary SS to Ro 60 apotopes and epitopes was assessed by multiparameter flow cytometry and solid-phase immunoassay. Anti,Ro 60 IgG was eluted from early apoptotic cells or recombinant Ro 60 immobilized on nitrocellulose, and binding to membrane-bound and intracellular forms of Ro 60 was quantitated by flow cytometry. Results An immunodominant apotope, which was recognized by IgG from a subset of SLE patients with anti-Ro, but not anti-La, autoantibodies, was mapped to a region forming a helix-loop-helix at the apical tip of the Ro 60 molecule. Immobilization of this region to the solid phase exposed an epitope that was recognized by IgG from primary SS and SLE patients whose sera had both anti-Ro and anti-La autoantibodies. Autoantibodies eluted from either the surface of apoptotic cells or the Ro 60 epitope on the solid phase were non,cross-reactive and specifically recognized membrane-bound or cytoplasmic forms of Ro 60. Conclusion This is the first example of a dichotomy of human autoantibody responses against mutually exclusive determinants linked to a single domain of a systemic autoantigen and supports a model in which tolerance is broken by different immunogenic forms of Ro 60. [source] Functional autoantibodies against serpin E2 in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 1 2010H. Maciejewska-Rodrigues Objective To search for novel autoantibodies in patients with rheumatoid arthritis (RA) in an effort to better understand the processes of joint destruction in this disease. Methods Using a modified SEREX technique and complementary DNA derived from RA synovium, serpin E2 was identified as a novel autoantigen and was analyzed by immunohistochemistry. Levels of anti,serpin E2 autoantibodies in serum and synovial fluid from patients with RA, osteoarthritis (OA), psoriatic arthritis, and ankylosing spondylitis, and/or from healthy individuals were assessed by enzyme-linked immunosorbent assay. Since serpin E2 is an inhibitor of serine proteases, we studied the inhibitory activity of serpin E2 toward its target, urokinase plasminogen activator (uPA), in vitro in the presence of isolated anti,serpin E2 autoantibodies and in vivo using the uPA activity assay. Results We identified autoantibodies against serpin E2 by the SEREX technique. Serpin E2 was overexpressed in RA synovial tissues as compared with OA synovial tissues. Significantly higher levels of anti,serpin E2 autoantibodies were present in samples of synovial fluid (28%) and serum (22%) from RA patients as compared with OA patients (0 and 6%, respectively) or with healthy individuals (6% of sera). Most importantly, anti,serpin E2 autoantibodies isolated from RA sera reversed the inhibitory activity of serpin E2 by 70%. Furthermore, the levels of anti,serpin E2 autoantibodies correlated with the uPA activity in vivo. Conclusion This study characterizes a functional property of a novel autoantibody in RA. Since anti,serpin E2 autoantibodies interfere with the inhibitory activity of serpin E2 toward serine proteases, they might facilitate the joint destruction in RA. [source] Plasmin immunization preferentially induces potentially prothrombotic IgG anticardiolipin antibodies in MRL/MpJ miceARTHRITIS & RHEUMATISM, Issue 10 2009Kaleo Ede Objective To test the hypothesis, utilizing 2 experimental mouse models, that plasmin is an important autoantigen that drives the production of certain IgG anticardiolipin (aCL) antibodies in patients with the antiphospholipid syndrome. Methods BALB/cJ and MRL/MpJ mice were immunized with Freund's complete adjuvant in the presence or absence of human plasmin. The mouse sera were analyzed for production of IgG antiplasmin, IgG aCL, and IgG anti,,2 -glycoprotein I (anti-,2GPI) antibodies. IgG monoclonal antibodies (mAb) were generated from the plasmin-immunized MRL/MpJ mice with high titers of aCL, and these 10 mAb were studied for their binding properties and functional activity in vitro. Results Plasmin-immunized BALB/cJ mice produced high titers of IgG antiplasmin only, while plasmin-immunized MRL/MpJ mice produced high titers of IgG antiplasmin, IgG aCL, and IgG anti-,2GPI. Both strains of mice immunized with the adjuvant alone did not develop IgG antiplasmin or IgG aCL. All 10 of the IgG mAb bound to human plasmin and cardiolipin, while 4 of 10 bound to ,2GPI, 3 of 10 bound to thrombin, and 4 of 10 bound to the activated coagulation factor X (FXa). Functionally, 4 of the 10 IgG mAb inhibited plasmin activity, 1 of 10 hindered inactivation of thrombin by antithrombin III, and 2 of 10 inhibited inactivation of FXa by antithrombin III. Conclusion Plasmin immunization leads to production of IgG antiplasmin, aCL, and anti-,2GPI in MRL/MpJ mice, but leads to production of only IgG antiplasmin in BALB/cJ mice. IgG mAb generated from plasmin-immunized MRL/MpJ mice bind to various antigens and exhibit procoagulant activity in vitro. These results suggest that plasmin may drive potentially prothrombotic aCL in genetically susceptible individuals. [source] Nuclear autoantigen CENP-B transactivation of the epidermal growth factor receptor via chemokine receptor 3 in vascular smooth muscle cellsARTHRITIS & RHEUMATISM, Issue 9 2009Geneviève Robitaille Objective We have previously found that the CENP-B nuclear autoantigen, which is specifically targeted by autoantibodies in the limited cutaneous form of systemic sclerosis, behaved as a potent migratory factor for human pulmonary artery smooth muscle cells (PASMCs). Other recent studies have shown that several disease-associated autoantigens induced cell migration by interacting with various chemokine receptors. Prompted by this hypothesis, we undertook this study to determine whether CENP-B interacts with chemokine receptors on the surface of human PASMCs, to explore the relevant signaling pathways, and to characterize the effects of anti,CENP-B binding on SMC stimulation. Methods To demonstrate the expression of specific chemokine receptors by human PASMCs at both the messenger RNA and protein levels, reverse transcription,polymerase chain reaction, immunoblotting, and flow cytometry analyses were performed. Desensitization studies and specific inhibitors were used to further identify the CENP-B target on the surface of human PASMCs. Results Our data strongly suggested that CENP-B used chemokine receptor 3 (CCR3) to mediate human PASMCs signaling. Moreover, several lines of evidence indicated that CENP-B binding subsequently stimulated the cross-talk between CCR3 and epidermal growth factor receptor (EGFR) via a matrix metalloprotease,dependent mechanism that involved the processing of heparin-binding EGF-like growth factor. Transactivation of the EGFR through CCR3 was found to be a critical pathway that elicits MAP kinase activation and secretion of cytokines such as interleukin-8. Finally, anti,CENP-B autoantibodies were found to abolish this signaling pathway, thus preventing CENP-B from transactivating EGFR and exerting its cytokine-like activities toward vascular smooth muscle cells. Conclusion The identification of CENP-B as a CCR3 ligand opens up new perspectives for the study of the pathogenic role of anti,CENP-B autoantibodies. [source] RNA helicase encoded by melanoma differentiation,associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis: Association with rapidly progressive interstitial lung disease,ARTHRITIS & RHEUMATISM, Issue 7 2009Shinji Sato Objective To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C-ADM) and rapidly progressive interstitial lung disease (ILD). Methods An anti,CADM-140 antibody,positive prototype serum sample was used to screen a HeLa cell,derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro,transcribed and in vitro,translated products using anti,CADM-140 antibody,positive and anti-CADM-140 antibody,negative sera. The lysates of COS-7 cells transfected with the putative antigen were similarly tested. An enzyme-linked immunosorbent assay (ELISA) to detect the anti,CADM-140 antibody was established using a recombinant CADM-140 antigen, and its specificity and sensitivity for C-ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue diseases. Results By cDNA library screening and immunoprecipitation of in vitro,transcribed and in vitro,translated products, we obtained a cDNA clone encoding melanoma differentiation,associated gene 5 (MDA-5). The anti,CADM-140 antibodies in patients' sera specifically reacted with MDA-5 protein expressed in cells transfected with full-length MDA-5 cDNA, confirming the identity of MDA-5 as the CADM-140 autoantigen. The ELISA, using recombinant MDA-5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the "gold standard" immunoprecipitation assay, and was useful for identifying patients with C-ADM and/or rapidly progressive ILD. Conclusion Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA-5 protein makes detection of the anti,CADM-140 antibody routinely available. [source] | |||||||||||||||||||||||||||||||||||||