Human and murine antibodies cross-reactive with streptococcal M protein and myosin recognize the sequence GLN-LYS-SER-LYS-GLN in M protein

Molecular mimicry or epitope similarity between group A streptococcal M proteins and myosin may contribute to the presence of heart reactive antibodies in acute rheumatic fever. In our study overlapping synthetic peptides copying the entire sequence of PepM5 protein were used to map the myosin cross-reactive epitopes of streptococcal M protein recognized by mouse and human mAb and affinity purified myosin-specific antibodies from acute rheumatic fever and rheumatic heart disease sera. Overlapping M protein peptides SM5(164-197)C and SM5(184-197)C inhibited the murine mAb reactions with PepM5 protein. The human mAb and affinity purified myosin-specific antibodies reacted exclusively with SM5(184-197)C. However, one of the five different purified myosin-specific antibodies not only reacted with SM5(184-197)C but also reacted with SM5(84-116)C. The synthetic subpeptides SM5(175-184)C and SM5(188-197C) did not react with any of the antibodies to PepM5 and myosin demonstrating a requirement of the 184-188 amino acid sequence for antibody recognition. A heptapeptide containing the sequence SM5(183-189) was also found to inhibit selected human myosin-specific antibodies and a human antimyosin mAb. Therefore, the majority of mouse and human myosin crossreactive antibodies recognized an epitope within the 14 residue carboxy terminus of PepM5 which appeared to involve the GLN-LYS-SER-LYS-GLN sequence.     

Mimicry in recognition of cardiac myosin peptides by heart-intralesional T cell clones from rheumatic heart disease

Molecular mimicry between Streptococcus pyogenes Ags and human proteins has been considered as a mechanism leading to autoimmune reactions in rheumatic fever and rheumatic heart disease (RHD). Cardiac myosin has been shown as a putative autoantigen recognized by autoantibodies of rheumatic fever patients. We assessed the human heart-intralesional T cell response against human light meromyosin (LMM) and streptococcal M5 peptides and mitral-valve-derived proteins by proliferation assay. Cytokines induced by LMM peptides were also evaluated. The frequency of intralesional T cell clones that recognized LMM peptides was 63.2%. Thirty-four percent of T cell clones presented cross-reactivity with different patterns: 1) myosin and valve-derived proteins; 2) myosin and streptococcal M5 peptides; and 3) myosin, valve-derived proteins and M5 peptides. In addition, several LMM peptides were recognized simultaneously showing a multiple reactivity pattern of heart-infiltrating T cells. Inflammatory cytokines (IFN-gamma and TNF-alpha) were predominantly produced by heart-infiltrating T cells upon stimulation with LMM peptides. The alignment of LMM and streptococcal M5 peptides showed frequent homology among conserved amino acid substitutions. This is the first study showing the cellular response by human heart-infiltrating T cells against cardiac myosin epitopes in RHD patients. The high percentage of reactivity against cardiac myosin strengthens its role as one of the major autoantigens involved in rheumatic heart lesions. T cell reactivity toward myosin epitopes in RHD patients may also trigger the broad recognition of valvular proteins with structural or functional similarities.     

Myosin autoimmunity is not essential for cardiac inflammation in acute Chagas' disease

Infection with the protozoan parasite Trypanosoma cruzi leads to acute myocarditis that is accompanied by autoimmunity to cardiac myosin in susceptible strains of mice. It has been difficult to determine the contribution of autoimmunity to tissue inflammation, because other inflammatory mechanisms, such as parasite-mediated myocytolysis and parasite-specific immunity, are coincident during active infection. To begin to investigate the contribution of myosin autoimmunity to myocarditis, we selectively inhibited myosin autoimmunity by restoring myosin tolerance via injection of myosin-coupled splenocytes. This tolerization regimen suppressed the strong myosin-specific delayed-type hypersensitivity (DTH) that normally develops in infected mice, although it did not affect myosin-specific Ab production. Suppression of myosin autoimmunity had no effect on myocarditis or cardiac parasitosis. In contrast, myosin tolerization completely abrogated myocarditis in mice immunized with purified myosin, which normally causes severe autoimmune myocarditis. In this case, myosin-specific DTH and Ab production were significantly reduced. We also examined the contribution of T. cruzi-specific immunity to inflammation by injection of T. cruzi-coupled splenocytes before infection. This treatment reduced T. cruzi DTH, although there was no effect on parasite-specific Ab production. Interestingly, cardiac inflammation was decreased, cardiac parasitosis was significantly increased, and mortality occurred earlier in the parasite-tolerized animals. These results indicate that myosin-specific autoimmunity, while a potentially important inflammatory mechanism in acute and chronic T. cruzi infection, is not essential for inflammation in acute disease. They also confirm previous studies showing that parasite-specific cell-mediated immunity is important for myocarditis and survival of T. cruzi infection.     

Identification of a streptococcal octapeptide motif involved in acute rheumatic fever

Acute rheumatic fever is a serious autoimmune sequela of pharyngitis caused by certain group A streptococci. One mechanism applied by streptococcal strains capable of causing acute rheumatic fever is formation of an autoantigenic complex with human collagen IV. In some geographic regions with a high incidence of acute rheumatic fever pharyngeal carriage of group C and group G streptococci prevails. Examination of such strains revealed the presence of M-like surface proteins that bind human collagen. Using a peptide array and recombinant proteins with targeted amino acid substitutions, we could demonstrate that formation of collagen complexes during streptococcal infections depends on an octapeptide motif, which is present in collagen binding M and M-like proteins of different beta-hemolytic streptococcal species. Mice immunized with streptococcal proteins that contain the collagen binding octapeptide motif developed high serum titers of anti-collagen antibodies. In sera of rheumatic fever patients such a collagen autoimmune response was accompanied by specific reactivity against the collagen-binding proteins, linking the observed effect to clinical cases. Taken together, the data demonstrate that the identified octapeptide motif through its action on collagen plays a crucial role in the pathogenesis of rheumatic fever. Eradication of streptococci that express proteins with the collagen binding motif appears advisable for controlling rheumatic fever.     

T cell mimicry and epitope specificity of cross-reactive T cell clones from rheumatic heart disease

Mimicry between streptococcal M protein and cardiac myosin is important in the pathogenesis of rheumatic heart disease. M protein-specific human T cell clones derived from rheumatic carditis were cross-reactive with human cardiac myosin, and laminin, a valve protein. Among the 11 CD4(+) and CD8(+) cross-reactive T cell clones, at least 6 different reactivity patterns were distinguished, suggesting different degrees of cross-reactivity and a very diverse T cell repertoire. The latter was confirmed by a heterogeneous Vbeta gene and CDR3 usage. HLA restriction and Th1 cytokine production in response to rM6 protein were preserved when the T cell clones were stimulated by human cardiac myosin or other alpha-helical proteins, such as tropomyosin and laminin. The cross-reactive human T cell clones proliferated to B2 and B3A, dominant peptide epitopes in the B repeat region of streptococcal M protein. In human cardiac myosin, epitopes were demonstrated in the S2 and light meromyosin regions. In our study, T cell mimicry was defined as recognition of structurally related Ags involved in disease and recognized by the same T cell. Mimicry in our study was related to alpha-helical coiled coil proteins which have a repetitive seven-aa residue periodicity that maintains alpha-helical structure and thus creates a high number of degenerate possibilities for recognition by T cells. The study of human T cell clones from rheumatic heart disease revealed potential sites of T cell mimicry between streptococcal M protein and human cardiac myosin and represents some of the most well-defined T cell mimicry in human autoimmune disease.     

Cryptic epitope identified in rat and human cardiac myosin S2 region induces myocarditis in the Lewis rat

Myocarditis is a common cause of dilated cardiomyopathy leading to heart failure. Chronic stages of myocarditis may be initiated by autoimmune responses to exposed cardiac Ags after myocyte damage. Cardiac myosin, a heart autoantigen, induced experimental autoimmune myocarditis (EAM) in susceptible animals. Although cardiac myosin-induced myocarditis has been reported in Lewis rats, the main pathogenic epitope has not been identified. Using overlapping synthetic peptides of the S2 region of human cardiac myosin, we identified an amino acid sequence, S2-16 (residues 1052-1076), that induced severe myocarditis in Lewis rats. The myocarditic epitope was localized to a truncated S2-16 peptide (residues 1052-1073), which contained a sequence identical in human and rat cardiac myosin. The S2-16 peptide was not myocarditic for three other strains of rats, in which the lack of myocarditis was accompanied by the absence of strong S2-16-specific lymphocyte responses in vitro. For Lewis rats, S2-16 was characterized as a cryptic epitope of cardiac myosin because it did not recall lymphocyte and Ab responses after immunization with cardiac myosin. Lymphocytes from S2-16 immunized rats recognized not only S2-16, but also peptides in the S2-28 region. Furthermore, peptide S2-28 was the dominant epitope recognized by T cells from cardiac myosin immunized rats. S2-16 was presented by Lewis rat MHC class II molecules, and myocarditis induction was associated with an up-regulation of inflammatory cytokine production. S2-16-induced EAM provides a defined animal model to investigate mechanisms of EAM and modulation of immune responses to prevent autoimmune myocarditis.     

Characterization of the antibody response against NeuGcGM3 ganglioside elicited in non-small cell lung cancer patients immunized with an anti-idiotype antibody

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.     

Human monoclonal antibodies reactive with antigens of the group A Streptococcus and human heart

Human mAb were produced from tonsillar or PBL of normal individuals or patients infected with group A streptococci. Lymphocytes were purified on Ficoll-Hypaque gradients and stimulated in vitro with purified group A streptococcal membranes or M protein extracts. The mAb were selected for study based on their reaction with group A streptococci, pep M5 protein, and/or M6 Escherichia coli protein. Further analysis by Western immunoblot or competitive inhibition ELISA revealed that there were two types of antibodies: one type that reacted with myosin and DNA and the other type that reacted with myosin, keratin, and/or actin. The specificities of these human mAb are similar to specificities observed in our previous studies of murine mAb reactive with group A streptococci and heart Ag. For comparison, anti-myosin antibodies were affinity purified from the sera of infected or acute rheumatic fever patients and were shown to react with myosin and DNA as well as with group A streptococci and M protein. To affinity purify these antibodies from normal sera, five times the amount of sera was required to obtain detectable quantities. These data suggest that the human mAb reactive with group A streptococci and myosin reflect the antibodies seen in sera from infected patients or acute rheumatics and that the B lymphocyte clones capable of producing these cross-reactive antibodies are also present in normal individuals.     

Structural correlates of an anticarcinoma antibody: identification of specificity-determining residues (SDRs) and development of a minimally immunogenic antibody variant by retention of SDRs only

Clinical utility of murine mAbs is limited because many elicit Abs to murine Ig constant and variable regions in patients. An Ab humanized by the current procedure of grafting all the complementarity determining regions (CDRs) of a murine Ab onto the human Ab frameworks is likely to be less immunogenic, except that its murine CDRs could still evoke an anti-variable region response. Previous studies with anticarcinoma mAb CC49 showed that light chain LCDR1 and LCDR2 of humanized CC49 could be replaced with the corresponding CDRs of a human Ab with minimal loss of Ag-binding activity. The studies reported in this paper were undertaken to dissect the CC49 Ag-binding site to identify 1) specificity determining residues (SDRs), the residues of the hypervariable region that are most critical in Ag-Ab interaction, and 2) those residues that contribute to the idiotopes that are potential targets of patients' immune responses. A panel of variants generated by genetic manipulation of the murine CC49 hypervariable regions were evaluated for their relative Ag-binding affinity and reactivity to sera from several patients who had been immunized with murine CC49. One variant, designated HuCC49V10, retained only the SDRs of CC49 and does not react with the anti-variable region Abs of the sera from the murine CC49-treated patients. These studies thus demonstrate that the genetic manipulation of Ab variable regions can be accomplished by grafting only the SDRs of a xenogeneic Ab onto human Ab frameworks. This approach may reduce the immunogenicity of Abs to a minimum.     

Autoantibodies specific for the cardiac myosin isoform are found in mice susceptible to Coxsackievirus B3-induced myocarditis

Several mouse strains are susceptible to immunopathic myocarditis after infection with Coxsackievirus B3 (CB3). This disease is associated with autoantibodies that are directed against myosin. In this study we characterized sera from CB3-infected mice for their reactivity with three different myosin isoforms (heart, skeletal muscle, and brain myosins) and for autoantibody isotype by using an ELISA. Competitive inhibition assays and absorption studies with various myosins demonstrated the presence of two autoantibody populations in sera of susceptible A.CA and A.SW mice. The first was specific for cardiac myosin and was mainly IgG. The second antibody population cross-reacted with heart, skeletal muscle, and brain myosin and was mainly IgM. B10.PL/SgSf and B10.A/SgSf mice, which do not develop immunopathic myocarditis, produced only the IgM autoantibody population cross-reactive with all three myosin isoforms. Because the heart-specific myosin autoantibodies were found exclusively in the mouse strains that developed immunopathic myocarditis, they can be considered a serologic marker for autoimmune heart disease.     

Cytotoxic antibodies to thymocyte antigens in rheumatism and other diseases

Cytotoxic antibodies reacting with mouse and human thymocytes were detected in rheumatic patients' sera. The level of cytotoxic antibodies was considerably higher in active than in inactive process. A correlation was found between the antibody level and the clinical course of rheumatic fever. The cytotoxic index was the highest in sera of patients with acute rheumatic fever. Thymocytotoxic antibodies were also found in other autoimmune diseases. In sera of normal individuals, antibodies to thymocytes were revealed rarely and in small quantities. A possible role of thymocytotoxic antibodies as a cause of deficit of T suppressors in autoimmune diseases is discussed.     

In vivo deposition of myosin-specific autoantibodies in the hearts of mice with experimental autoimmune myocarditis.

Experimental autoimmune myocarditis (EAM) is elicited in certain strains of mice by immunizing with mouse cardiac myosin. Concomitant with the onset of myocardial inflammation is the induction of circulating IgG antibodies to myosin. To further examine the role of myosin in disease, both EAM-susceptible (A/J) and EAM-resistant (B10.A) mice were immunized with myosin emulsified in CFA and examined for myocardial inflammation and IgG deposition. Myocarditis was common in susceptible, but not resistant strain mice. IgG deposition was extensive in A/J mice, but modest in B10.A mice, when compared to controls given adjuvant alone. Localization was independent of inflammatory or necrotic lesions. A spot ELISA indicated that antimyosin IgG antibody-secreting cells were present in the myocardial infiltrate and likely contributed to antibody localization. Antibody was eluted from the hearts of immunized animals and found to react strongly with normal heart tissue by indirect immunohistochemistry. This reactivity was not completely absorbed by skeletal muscle, indicating that some of the antibody was heart-specific. Western immunostaining demonstrated that eluates from immunized A/J and B10.A mice possessed anti-myosin antibody activity; similar reactivity was not observed in eluates from control mice of either strain. Comparison of heart reactivity with syngeneic and allogeneic tissue suggests that although myosin immunization elicits homologous antibody in both strains, each may recognize distinct epitopes. These findings strongly suggest that cardiac myosin or a myosin-like determinant is expressed on the surface of normal mouse myocytes.     

Identification of aldolase as a target antigen in Alzheimer's disease

Alzheimer's disease (AD) is the most common human neurodegenerative disease, leading to progressive cognitive decline and eventually death. The prevailing paradigm on the pathogenesis of AD is that abnormally folded proteins accumulate in specific brain areas and lead to neuronal loss via apoptosis. In recent years it has become evident that an inflammatory and possibly autoimmune component exists in AD. Moreover, recent data demonstrate that immunization with amyloid-beta peptide is therapeutically effective in AD. The nature of CNS Ags that are the target of immune attack in AD is unknown. To identify potential autoantigens in AD, we tested sera IgG Abs of AD patients in immunoblots against brain and other tissue lysates. We identified a 42-kDa band in brain lysates that was detected with >50% of 45 AD sera. The band was identified by mass spectrometry to be aldolase A. Western blotting with aldolase using patient sera demonstrated a band of identical size. The Ab reactivity was verified with ELISAs using aldolase. One of 25 elderly control patients and 3 of 30 multiple sclerosis patients showed similar reactivity (p < 0.002). In enzymatic assays, anti-aldolase positive sera were found to inhibit the enzyme's activity, and the presence of the substrate (fructose 1,6-diphosphate) enhanced Ab binding. Immunization of rats and mice with aldolase in complete Freund's adjuvant was not pathogenic. These findings reveal an autoimmune component in AD, point at aldolase as a common autoantigen in this disease, and suggest a new target for potential immune modulation.     

Anticardiac myosin immunity and chronic allograft vasculopathy in heart transplant recipients

Chronic allograft vasculopathy (CAV) contributes to heart transplant failure, yet its pathogenesis is incompletely understood. Although cellular and humoral alloimmunity are accepted pathogenic mediators, animal models suggest that T cells and Abs reactive to graft-expressed autoantigens, including cardiac myosin (CM), could participate. To test the relationship between CAV and anti-CM autoimmunity in humans, we performed a cross-sectional study of 72 heart transplant recipients: 40 with CAV and 32 without. Sera from 65% of patients with CAV contained anti-CM Abs, whereas <10% contained Abs to other autoantigens (p < 0.05), and only 18% contained anti-HLA Abs (p < 0.05 versus anti-CM). In contrast, 13% of sera from patients without CAV contained anti-CM Abs (p < 0.05; odds ratio [OR], associating CAV with anti-CM Ab = 13, 95% confidence interval [CI] 3.79-44.6). Multivariable analysis confirmed the association to be independent of time posttransplant and the presence of anti-HLA Abs (OR = 28, 95% CI 5.77-133.56). PBMCs from patients with CAV responded more frequently to, and to a broader array of, CM-derived peptides than those without CAV (p = 0.01). Detection of either CM-peptide-reactive T cells or anti-CM Abs was highly and independently indicative of CAV (OR = 45, 95% CI 4.04-500.69). Our data suggest detection of anti-CM immunity could be used as a biomarker for outcome in heart transplantation recipients and support the need for further studies to assess whether anti-CM immunity is a pathogenic mediator of CAV.     

Myosin-induced acute myocarditis is a T cell-mediated disease

The heart is a target organ in several autoimmune diseases, and therefore it is important to understand more about the effector cells involved in immune-mediated mechanisms of myocardial cell death. Because immune T lymphocytes are central to many immune responses, we wanted to study the role of T cells in causing cardiac specific inflammation. We used purified mouse cardiac myosin to cause acute myocarditis in mice. The adoptive transfer of purified T cells from C.B-17 mice with active myocarditis to SCID recipients successfully transferred the disease into SCID hosts. In contrast, transfer of serum with high-titer antimyosin antibodies to SCID hosts did not cause myocarditis. Using mAb to deplete A/J mice of CD4+ T cells, we showed that these mice were protected against the induction of myocarditis. Depletion of CD8+ T cells reduced the severity of inflammation but did not prevent induction of myocarditis. We were also able to prevent the induction of myocarditis using major histocompatibility class II protein-binding, nonimmunogenic, competitor peptides. These blocking studies also indicated that in H-2k mice, myocarditis is an I-Ak-restricted disease, and provided further evidence that CD4+ T cells are critical to the induction of disease. Together, these studies provide direct evidence that myosin-induced myocarditis is a T cell-mediated disease.     

Quality of recombinant protein determines the amount of autoreactivity detected against the tumor-associated epithelial cell adhesion molecule antigen: low frequency of antibodies against the natural protein

The human epithelial cell adhesion molecule (EpCAM) is expressed on normal epithelial cells and is overexpressed in most carcinomas. EpCAM-targeted immunotherapy has been tried in several clinical studies. High titers of autoantibodies against EpCAM have been reported by different authors. We have generated large amounts of purified protein in S2 Drosophila cells (S2-EpCAM) with a purity of >96%. In contrast, the protein produced in baculovirus-dependent systems (baculo-EpCAM) that has been used in previous studies shows a purity of 79%. (1)H nuclear magnetic resonance spectrum of S2-EpCAM is typical of folded protein, whereas the baculo-EpCAM sample shows a spectrum corresponding to a partially unfolded protein. Using S2-EpCAM, denatured S2-EpCAM, and baculo-EpCAM, we measured EpCAM Abs of different isotypes in the serum of healthy controls and cancer patients. We found Ab titers against EpCAM in a much lower percentage of sera as published previously, and support the hypothesis that Ab reactivity in some published studies might be due to reactivity against denatured protein, to contaminating proteins in the baculovirus preparations, and to reactivity with BSA. Tetanus toxoid-reactive IgG Abs are present in 1000-fold higher titers compared with EpCAM-reactive Abs. Only IgA Abs were found in higher proportions and in higher concentrations than tetanus toxoid-specific Abs. Our study shows that EpCAM only rarely induces autoantibodies against native protein and emphasizes the importance of using extremely purified Ag preparations when evaluating Abs against tumor-associated Ags.     

Effects of IgM from rheumatic fever patients on intracellular calcium levels of neonatal rat cardiac myocytes

Rheumatic fever (RF), a potential sequela of Streptococcus pyogenes pharyngitis, sometimes results in myocarditis and heart failure. Antibodies have been implicated in the pathogenesis of RF and anti-cardiac myosin antibody levels are elevated in RF patients. Since myocarditis is associated with altered cardiomyocyte calcium transients it was of interest to determine the direct effects of RF patient antibodies on calcium transients in cultured myocytes. RF patient polyclonal IgM treatment caused increased calcium retention by neonatal rat heart cells in vitro as determined with isotopically labeled calcium. Therefore, to further characterize this finding, calcium transients were evaluated by real time fluorescence spectroscopy and deconvolution imaging. RF patient polyclonal IgM produced increased calcium retention during the relaxation stage of the contraction cycle leading to a slowing of contraction rate, disorganized calcium transients, and eventual tetany. In contrast, calcium transient studies of cardiomyocytes following treatment with monoclonal anti-myosin antibodies revealed declining intracellular calcium levels, accompanied by disorganized transients and tetany. Treatment with both antibodies led to myocyte dysfunction and these novel findings suggest a role for antibodies in the pathogenesis of the myocarditis associated with rheumatic carditis.     

Autoimmune myocarditis does not require B cells for antigen presentation.

T cells constitute the pathogenic effector cell population in autoimmune myocarditis in BALB/c mice. Using mice rendered deficient for B cells by a targeted disruption to the IgM transmembrane domain or by treatment with anti-IgM Ab from birth, we asked whether B cells are a critical APC in the induction of autoimmune myocarditis. B cell-deficient mice immunized with cardiac myosin develop myocarditis comparable in incidence and severity to that in wild-type mice, suggesting that autoreactive T cells that cause myocarditis in BALB/c mice are activated by macrophages or dendritic cells. Since it does not appear that presentation of cryptic epitopes is critical for the breakdown of self tolerance, potentially pathogenic T cells recognizing dominant myosin epitopes must have escaped tolerization. Either anatomic sequestration of cardiac myosin peptide-MHC complexes or subthreshold presentation of cardiac myosin peptides by conventional APC can explain the survival of these autoreactive T cells.     

Molecular recognition patterns of serum anti-DNA topoisomerase I antibody in systemic sclerosis

Autoreactive anti-DNA topoisomerase I (anti-Topo I) Abs are commonly detected in sera of systemic sclerosis (SSc) patients. Our studies have established a positive correlation between the levels of serum anti-Topo I Abs and both disease severity and activity of SSc. The molecular targets of anti-Topo I Ab on Topo I domains remain to be further defined. In this report, we studied the molecular recognition pattern of serum anti-Topo I Ab in 52 SSc patients. The highest reactivity of serum anti-Topo I Abs was against the core subdomains I and II (aa 207-441) and, to a lesser extent, against the core subdomain III (aa 433-636) of Topo I. The linker domain (aa 636-712) and the C-terminal domain (aa 713-765) had much less reactivity than the core domain (aa 207-636). Strikingly, very little reactivity was directed against the N-terminal domain (aa 1-213) by serum anti-Topo I Ab. This molecular recognition pattern was consistent among all SSc serum samples studied. Results from patients with serial serum samples indicated that this pattern remained unchanged over time. Interestingly, some naive B cells from healthy controls, upon transformation by EBV, produced IgM Abs against Topo I. These Abs had low affinity for Topo I and reacted equally to all domains of Topo I. The molecular recognition pattern of serum anti-Topo I Ab in SSc suggests the presence of a unique antigenic stimulation in vivo in this disease.     

The autoantibodies to alpha 6 beta 4 integrin of patients affected by ocular cicatricial pemphigoid recognize predominantly epitopes within the large cytoplasmic domain of human beta 4

This study was undertaken to characterize the antigenic determinants recognized by the autoantibodies of patients with ocular cicatricial pemphigoid (OCP). OCP is a subepithelial, blistering, autoimmune disease that mainly affects the conjunctiva and other mucous membranes. We previously demonstrated that a cDNA clone, isolated from a keratinocyte expression library by using immunoaffinity-purified OCP autoantibody, encoded the cytoplasmic domain of beta 4 integrin subunit. Our subsequent studies showed that sera from all the OCP patients that were tested recognize the human beta 4 integrin subunit. To identify the prevalent epitopes of the anti-beta 4 autoantibodies of OCP, we have used cell lines transfected with vectors encoding a wild-type beta 4 subunit, a tailless beta 4 subunit, or a beta 4 subunit lacking the extracellular domain. Nontransfected cell lines were used as controls. Lysates from these cell lines were analyzed with OCP sera, IgG fractions from OCP sera, and immunoaffinity-purified OCP autoantibodies. Abs to extracellular and cytoplasmic domains of human beta 4 integrin were used as positive controls, whereas normal human sera and normal human IgG fractions were used as negative controls. The reactivity of OCP Abs was determined by using immunoblotting, immunoprecipitation, and FACS analysis. The results of this study indicate that OCP sera, OCP IgG fractions, and immunoaffinity-purified OCP autoantibodies react with the intracellular and not the extracellular domain of human beta 4 integrin subunit. In vitro cell culture experiments demonstrated that OCP autoantibody binds to the cytoplasm of the cells. The relevance of these findings to the pathogenesis of OCP is discussed.