Association between anti-nucleophosmin and anti-cardiolipin antibodies in (NZW × BXSB)F1 mice and human systemic lupus erythematosus

We showed previously that nucleophosmin (NPM), a nucleolar phosphoprotein, is recognized by sera from (NZW × BXSB)F₁ (WB) mice, a model of systemic lupus erythematosus (SLE) and anti-phospholipid syndrome. In the present study we analysed the prevalence and kinetics of anti-NPM autoantibodies in WB mice by a solid-phase ELISA with recombinant human (rh) NPM as the antigen and showed that most male WB mouse sera had anti-NPM antibodies that were responsible for their indirect immunofluorescence staining pattern on Hep-2 cells. Anti-NPM antibodies were significantly associated with anti-cardiolipin (aCL) antibodies. This antibody profile mirrored that observed in certain human SLE sera because anti-NPM antibodies were detected in 28% of the sera from patients with SLE and were similarly associated with aCL antibodies. The demonstration that rhNPM bound to cardiolipin (CL) in vitro and increased the CL-binding activity of a WB-derived aCL monoclonal antibody indicates that NPM can interact with CL to form SLE-related immunogenic particles that might be responsible for the concomitant production of anti-NPM and aCL antibodies.     

Heterogeneity of anticardiolipin antibodies defined by the anticardiolipin cofactor.

Anticardiolipin antibodies (aCL) found in sera from patients with SLE react with cardiolipin (CL) in the presence of a 50-kDa serum cofactor. The cofactor, which was identified to be beta 2-glycoprotein I by sequencing the N-terminal amino acids, not only enhances CL binding by antibodies in SLE but also depresses it by antibodies associated with syphilis. Cofactor-dependent binding of aCL in SLE to solid phase CL was competitively inhibited by the simultaneous addition of fluid phase CL but was unaffected by either prior or simultaneous addition of a high excess of the cofactor. Binding of aCL in syphilis to solid phase CL was competitively inhibited by either addition of the cofactor or fluid phase CL. aCL in SLE reacted with CL, PS, and PA in the presence of cofactor. In contrast, biotinyl-cofactor bound directly to these anionic phospholipids (PL) and also to PG. These results show that the cofactor-CL complex bears an epitope that confers recognition specificity for aCL in SLE, in contrast with direct CL recognition by syphilitic aCL. The direct binding of the cofactor to PL suggests that the cofactor dependence of aCL binding to PL is due to recognition by aCL of a unique epitope generated upon the formation of the cofactor-CL complex.     

Characterization of mouse and human monoclonal antibodies cross-reactive with SLE serum antibodies to guanosine

Two new monoclonal antibodies, one a mouse IgM and the other a human IgM that reacted with guanosine, were compared to human serum antibodies from patients with systemic lupus erythematosus (SLE). The human monoclonal antibody was polyspecific in its binding to the nucleoside bases, whereas the mouse monoclonal antibody was relatively specific for guanosine when compared by using an enzyme-linked immunosorbent assay (ELISA). Neither antibody bound polyguanylic acid or denatured single-stranded (ss) DNA, however. Serum IgG antibodies from seven patients with SLE cross-reacted with the mouse monoclonal antibody and showed considerable specificity for guanosine. In contrast, the human serum IgG antiguanosine antibodies also bound ssDNA but not dsDNA or polyguanylic acid. Serum IgG antibodies to guanosine measured by ELISA from the seven SLE patients had a decreased response when compared to the total serum IgG response to ssDNA, and most of the antibodies that bound guanosine also bound ssDNA. These studies provide new evidence that there are specific IgG antibodies to guanosine in SLE sera that are a small fraction of the antibodies to ssDNA. Further efforts to define the role of these guanosine antibodies in SLE may provide a better understanding of the basic mechanisms responsible for the development of SLE in man.     

Pathogenic antibodies in systemic lupus erythematosus: anti-LAMP antibodies

We recently demonstrated that a monoclonal anti-DNA antibody, spontaneously produced in lupus B/W mice, recognizes the same protein(s) at the surface of several human cell types involved in lupus pathogenesis including normal human erythrocytes, normal platelets and rat neuronal tissue. This cell-surface protein(s) cross-react(s) with double-stranded DNA. We suggest to call this protein(s) LAMP [lupus associated membrane protein(s)]. Here we show that: immunoglobulins eluted from kidneys of autoimmune MRL/lpr/lpr mice strongly react with LAMP. Anti-LAMP antibodies are present in large amount in MRL/lpr and B/W mice sera. Anti-LAMP are present in 25 out of 25 human SLE sera ranged as SLE on the basis of revised American Rheumatism Associated classification. Interestingly, two of these sera did not display anti DNA anti-body activity. Taken together, these results strongly suggest a role of LAMP in the pathogeny of SLE.     

The role of the idiotypic network in the induction of experimental systemic lupus erythematosus

Systemic lupus erythematosus (SLE) has been induced in C3H.SW mice by their immunization with a human monoclonal anti-DNA antibody that bears a common idiotype-16/6 Id. Following immunization, high levels of murine anti-16/6 and anti-anti-16/6 antibodies were detected in the sera of the immunized mice. Elevated titers of autoantibodies reacting with ssDNA, dsDNA, poly(I), poly(G), RNP, Ro, and La were also observed. The serological findings were associated with significant proteinuria, leukopenia, and elevated erythrocyte sedimentation rate. Immune complex deposition in the glomerular mesangium and sclerosis of the glomeruli were demonstrated. To study whether or not anti-idiotype antibodies are involved in the induction of the disease, a murine monoclonal antibody against the 16/6 Id was prepared and injected into C3H.SW mice. The anti-16/6 Id antibody induced experimental SLE similarly to the 16/6 Id with an accelerated kidney pathology. A study performed on different mouse strains indicated that the susceptibility to the induction of SLE by the 16/6 Id is strain dependent and directly correlates to their ability to produce anti-16/6 Id specific antibodies.     

The reaction of SLE antibodies with native, single stranded RNA: radioassay and binding specificities.

Escherichia coli 3H-tRNA and MS2 phage 125I-RNA were prepared and used in a sensitive nitrocellulose filter assay. Antibodies that bound these RNA ligands occurred in the sera of several patients with SLE, but not in sera of patients with other connective tissue diseases. The antibody populations that bound polyribonucleotides (largely IgG) were distinct from antibody populations that bound polydeoxyribonucleotides. Competition experiments showed that the anti-RNA antibodies preferentially bound native ssRNA as compared with synthetic single and double stranded polyribonucleotides. There was increasing affinity with increasing m.w. of the ssRNA. The anti-tRNA population was of restricted heterogeneity (Sips index 0.83) and bound tRNA with an average association constant (Ko) of 9 x 10(6) l/mole at 4 degrees C. The anti-MS2 RNA population was much more heterogeneous (Sips index 0.67) and bound MS2 RNA with a Ko of about 3 x 10(9) l/mole at 4 degrees C. Whereas NZB/NZW mice spontaneously produce RNA reactive antibodies with conformation specificity for native tRNA, human SLE anti-RNA antibodies appear to have very little of this type of conformation specificity.     

Anticardiolipin antibodies in NZW x BXSB F1 mice. A model of antiphospholipid syndrome.

NZW x BXSB F1 (W/B F1) male mice develop systemic lupus-like disease, and several autoantibodies, circulating immune complexes, and lupus nephritis become apparent. The abnormally high incidence of degenerative coronary vascular disease with myocardial infarction and thrombocytopenia due to the presence of both platelet-associated antibodies and circulating antiplatelet antibodies in this animal has been reported. We found that W/B F1 male mice produced autoantibodies against cardiolipin (aCL) and that the titer of aCL increases with age. aCL from W/B F1 male mice were mainly IgG and binding activity to cardiolipin was aCL-cofactor (beta 2-glycoprotein I (beta 2-GPI)) dependent. We developed monoclonal aCL from these animals and examined specificity of the autoantibodies. All the mAb used reacted with the negatively charged phospholipids, cardiolipin, phosphatidylserine, and phosphatidylinositol, and some reacted with platelets and DNA. The addition of human or mouse beta 2-GPI enhanced the titer for monoclonal aCL from the W/B F1 mice. From the results of competitive inhibition enzyme immunoassay with monoclonal aCL and purified beta 2-GPI, aCL from the W/B F1 mice recognized the complex of CL and beta 2-GPI. The W/B F1 male mouse may be an appropriate model for use in studies on the pathologic significance of aCL in patients with antiphospholipid syndrome.     

Characterization of centrosomal association of nucleophosmin/B23 linked to Crm1 activity

Nucleophosmin (NPM)/B23 is a multifunctional protein, involving in a wide variety of basic cellular processes, including ribosome assembly, DNA duplication, nucleocytoplasmic trafficking, and centrosome duplication. It has previously been shown that NPM/B23 localizes to centrosomes, and dissociate from centrosomes upon phosphorylation by Cdk2/cyclin E. However, detail characterization of centrosomal association of NPM/B23 has been hampered by the lack of appropriate antibodies that efficiently detects centrosomally localized NPM/B23, as well as by apparent loss of natural behavior of NPM/B23 when tagged with fluorescent proteins. Here, by the use of newly generated anti-NPM/B23 antibody, we conducted a careful analysis of centrosomal localization of NPM/B23. We found that NPM/B23 localizes between the paired centrioles of unduplicated centrosomes, suggesting the role of NPM/B23 in the centriole pairing. Upon initiation of centrosome duplication, some NPM/B23 proteins remain at mother centrioles of the parental centriole pairs. We further found that inhibition of Crm1 nuclear export receptor results in both accumulation of cyclin E at centrosomes and efficient dissociation of NPM/B23 from centrosomes.     

Anti-RNP monoclonal antibodies derived from a mouse strain with lupus-like autoimmunity

systemic lupus erythematosus (SLE) and related rheumatic and connective-tissue diseases are often associated with the production of antibodies directed against a variety of specific cellular components. Recent evidence indicates that two such autoantigens, the Sm and RNP antigens recognized by SLE sera, exist in small ribonucleoprotein complexes found in the nuclei of higher eukaryotes. Studies of the structure and function of these autoantigenic particles with human sera used as probes have been limited because of the multiplicity of autoantibodies often found in an individual serum. Through this communication, we report that MRL/Mp-+/+ (MRL/n) mice, which spontaneously develop a disease exhibiting many of the characteristics of human SLE, possess anti-RNP antibodies in addition to anti-Sm and anti-DNA as previously reported. Spleen cells from one such autoimmune mouse were used to produce a stable hybridoma secreting antibodies that react simultaneously with a protein of Mr 40,000 and a doublet of approximately 70,000, a pattern of reactivity identical to and characteristic of human SLE anti-RNP autoantibodies.     

Serum antibodies to human fetal antigens in patients with systemic lupus erythematosus (SLE)

Serum antibodies to human fetal antigens were measured by a radiolabeled anti-immunoglobulin binding assay by using human fetal fibroblasts (Flow cell line No. 1000) as target cells. High titers of IgG antibody to the fetal cells were found in sera of patients with systemic lupus erythematosus (SLE). The antibody reacted with surface membrane antigens shared by various fetal tissues of human and murine origin but not by adult tissues. The reaction of the SLE antibody to the fetal cells was inhibited by heterologous antiserum to the Flow 1000 cells and antiserum to murine embryonic fibroblasts, but not by antiserum to human alpha-fetoprotein or human fibronectin. Absorption of SLE serum with isolated nuclei did not abolish the reaction indicating that these were not anti-nuclear antibodies. The antibody activity was found to reside in the F(ab')2 fragment. The serum titer of the anti-fetal antibody was higher in SLE patients with active disease than those in clinical remission.     

IgG subclasses of anti-tetanus toxoid antibodies in adult and newborn normal subjects and in patients with systemic lupus erythematosus, Sjogren's syndrome, and drug-induced autoimmunity

The IgG subclasses of anti-tetanus toxoid (anti-TT) antibodies were quantitated in normal sera and sera from patients with rheumatic disease. Detection relied on a set of four mouse monoclonal antibodies, each of which showed specificity for the respective isotype, independent of gamma-chain allotype or light chain class of the human antibody. Approximately 90% of the total anti-TT activity in normal adults and patients with Sjogren's syndrome was IgG1. In addition, IgG4 antibodies were detected in one-half the samples, but IgG2 and IgG3 antibodies were observed in only two out of 36 sera. However, antibodies elicited in children immunized with TT were exclusively IgG1 and IgG3, with IgG4 antibodies detectable only at birth (presumably due to transplacental passage of antibody) in three of 12 children. In contrast to normal adults, patients with systemic lupus erythematosus (SLE) and drug-induced autoimmunity (DIA) had a more promiscuous isotype profile. IgG2 and/or IgG3 anti-TT antibodies were detected in 13 of 22 SLE patients and IgG3 antibodies in six of 11 patients with DIA. IgG4 anti-TT antibodies were predominant in seven of these 33 patients. These findings suggest that IgG isotypes may depend on the frequency of the stimulus, but global alterations in immunologic status as reflected in systemic autoimmune disease may override the homeostatic mechanisms that control isotype restriction.     

Cardiac-oxidized antigens are targets of immune recognition by antibodies and potential molecular determinants in chagas disease pathogenesis

Trypanosoma cruzi elicits reactive oxygen species (ROS) of inflammatory and mitochondrial origin in infected hosts. In this study, we examined ROS-induced oxidative modifications in the heart and determined whether the resultant oxidized cardiac proteins are targets of immune response and of pathological significance in Chagas disease. Heart biopsies from chagasic mice, rats and human patients exhibited, when compared to those from normal controls, a substantial increase in protein 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), carbonyl, and 3-nitrotyrosine (3-NT) adducts. To evaluate whether oxidized proteins gain antigenic properties, heart homogenates or isolated cardiomyocytes were oxidized in vitro and one- or two-dimensional gel electrophoresis (2D-GE)/Western blotting (WB) was performed to investigate the proteomic oxidative changes and recognition of oxidized proteins by sera antibodies in chagasic rodents (mice, rats) and human patients. Human cardiomyocytes exhibited LD(50) sensitivity to 30 μM 4-HNE and 100 μM H(2)O(2) at 6 h and 12 h, respectively. In vitro oxidation with 4-HNE or H(2)O(2) resulted in a substantial increase in 4-HNE- and carbonyl-modified proteins that correlated with increased recognition of cardiac (cardiomyocytes) proteins by sera antibodies of chagasic rodents and human patients. 2D-GE/Western blotting followed by MALDI-TOF-MS/MS analysis to identify cardiac proteins that were oxidized and recognized by human chagasic sera yielded 82 unique proteins. We validated the 2D-GE results by enzyme-linked immunosorbent assay (ELISA) and WB and demonstrated that oxidation of recombinant titin enhanced its immunogenicity and recognition by sera antibodies from chagasic hosts (rats and humans). Treatment of infected rats with phenyl-α-tert-butyl nitrone (PBN, antioxidant) resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic rats. We conclude that ROS-induced, cardiac-oxidized antigens are targets of immune recognition by antibodies and molecular determinants for pathogenesis during Chagas disease.     

Autoantibodies recognizing proteins copurified with PCNA in patients with connective tissue diseases

Objective. Proliferating cell nuclear antigen (PCNA), one of the target antigen recognized by lupus sera, has been reported to be present as a subnuclear multi-peptide complex. But autoantibodies reacting with components of PCNA complex are poorly understood. To study the specificity of those autoantibodies, immunoreactivities of autoimmune sera against purified PCNA antigen were studied. Methods. PCNA antigens were purified from rabbit thymus extract by affinity column using murine monoclonal antibodies (mAbs) to PCNA, TOB7, TO17 and TO30. Immunoreactivities of autoimmune sera against purified PCNA were analyzed by WB. Results. PCNA antigen purified by serum AK predominantly showed a 34 kD band specific for PCNA in SDS-PAGE. When antigens were purified by anti-PCNA mAb TOB7 and TO30 which are known to be targeting different epitopes on PCNA antigen, SDS-PAGE analysis showed various mol. wt of proteins in addition to the 34 kD PCNA while both AK and mAbs reacted only with 34 kD PCNA in WB. In WB using PCNA purified by TOB7, various immunoreactivities were observed at 150, 66, 58, 48, 45, 37, 32 and 16 kDa in sera from patients with connective tissue diseases. Conclusions. These results suggested that many of the proteins copurified with PCNA were also targets of autoimmune responses and these autoantibody experssion may be induced through antigen-driven mechanisms.Abbreviations mAb monoclonal antibody - PCNA proliferating cell nuclear antigen - PCNA/AK PCNA affinity purified by antibodies from patient serum AK - PCNA/TO30 PCNA purfied by mAb TO30 - PCNA/TOB7 PCNA purified by mAb TOB7 - SLE systemic lupus erythematosus     

Molecular and antigenic nature of isolated Sm

The Sm antigen was isolated and purified from calf thymus nuclear extract by affinity chromatography. The affinity columns were made with serum antibodies from an SLE patient or an anti-Sm monoclonal antibody derived from a hybridoma cell line. Proteins eluted from these two columns had m.w. of 58,000 and 35,000 by SDS polyacrylamide gel electrophoresis. The natural conformation of this antigen appears to be 95,000 in m.w. with the 58,000 particle containing the Sm antigenic determinant. The affinity column-purified antigen detected by the human anti-Sm antibodies is also recognized by anti-Sm antibodies in murine lupus serum, as shown by solid-phase radioimmunoassay. This study 1) demonstrates the molecular and antigenic nature of the Sm antigen and 2) compares the anti-Sm binding capabilities of antibody populations present in sera from SLE patients and from MRL lpr/lpr mice.     

DNA on membrane receptors: a target for monoclonal anti-DNA antibody induced by a nucleoprotein shed in systemic lupus erythematosus

Antibodies to double stranded (ds) DNA correlate with clinical evolution in systemic lupus erythematosus (SLE) although little is known about the immunogen and target for these antibodies, since ds DNA is poorly immunogenic. We now show that monoclonal anti DNA antibodies similar to those detected in human SLE can be produced by immunization of genetically non-autoimmune mice with a human circulating DNA-protein complex increased in the circulation of SLE patients. One such monoclonal antibody showed antinuclear reactivity, interacting with a 74 kd DNA-binding membrane protein, in reactions prevented by absorption with ds DNA cellulose. Our data suggests that anti ds DNA antibody reactions in SLE may be triggered by circulating nucleoproteins and directed toward membrane receptors capable of interacting with extracellular DNA.     

Antiphospholipid reactivity against cardiolipin metabolites occurring during endothelial cell apoptosis

We have recently shown that cardiolipin (CL) and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis. In this study, we investigate the immunoreactivity to CL derivatives occurring during endothelial apoptosis in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). We compared the serum immunoreactivity to CL with that of its derivatives monolysocardiolipin (MCL), dilysocardiolipin (DCL), and hydrocardiolipin (HCL) by means of both enzyme-linked immunosorbent assay and thin-layer chromatography (TLC) immunostaining. In addition, we investigated the composition of phospholipid extracts from the plasma membrane of apoptotic endothelial cells and the binding of patients' sera to the surface of the same cells by using high-performance TLC and immunofluorescence analysis. The average reactivity to MCL was comparable with that of CL and significantly higher than that for DCL and HCL in patients studied, both in the presence or in the absence of beta2-glycoprotein I. Of relevance for the pathogenic role of these autoantibodies, immunoglobulin G from patients' sera showed an increased focal reactivity with the plasma membrane of endothelial cells undergoing apoptosis. Interestingly, the phospholipid analysis of these light membrane fractions showed an accumulation of both CL and MCL. Our results demonstrated that a critical number of acyl chains in CL derivatives is important for the binding of antiphospholipid antibodies and that MCL is an antigenic target with immunoreactivity comparable with CL in APS and SLE. Our finding also suggests a link between apoptotic perturbation of CL metabolism and the production of these antibodies.     

Identification of a Lipid Peroxidation Product as the Source of Oxidation-specific Epitopes Recognized by Anti-DNA Autoantibodies

Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids, and has been implicated in the pathogenesis of numerous diseases, including systemic lupus erythematosus (SLE). We have found that bovine serum albumin incubated with peroxidized polyunsaturated fatty acids significantly cross-reacted with the sera from MRL-lpr mice, a representative murine model of SLE. To identify the active substances responsible for the generation of autoantigenic epitopes recognized by the SLE sera, we performed the activity-guiding separation of a principal source from 13-hydroperoxy-9Z,11E-octadecadienoic acid and identified 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of ω6 polyunsaturated fatty acids, as the source of the autoantigenic epitopes. When the age-dependent change in the antibody titer against the ONE-modified protein was measured in the sera from MRL-lpr mice and control MRL-MpJ mice, all of the MRL-lpr mice developed an anti-ONE titer, which was comparable with the anti-DNA titer. Strikingly, a subset of the anti-DNA monoclonal antibodies generated from the SLE mice showing recognition specificity toward DNA cross-reacted with the ONE-specific epitopes. Furthermore, these dual-specific antibodies rapidly bound and internalized into living cells. These findings raised the possibility that the enhanced lipid peroxidation followed by the generation of ONE may be involved in the pathogenesis of autoimmune disorders.     

免疫印迹法在实验大鼠汉坦病毒监测中的应用及与间接免疫荧光法之比较

用汉坦病毒汉滩株（７６－１１８）重组核蛋白作为免疫印迹法（ＷｅｓｔｅｒｎＢｌｏｔ以下简称ＷＢ）的诊断抗原,用于实验感染大鼠血清抗体效价测定。同时与用汉城株（ＳＲ－１１）感染的Ｖｅｒｏ－Ｅ６细胞作抗原的间接免疫荧光法（以下简称ＩＦＡ）进行比较。ＷＢ法对３／４标本在大鼠接种病毒后第３天测得血清ＩｇＭ阳性,而ＩＦＡ法仅１／４标本出现阳性,ＩＦＡ效价为１：５１２０的血清,ＷＢ效价为１’：４０９６０,且在血清１：１０稀释时反应带亦清晰。两种方法分别测定６４份大鼠血清。甩ＩＦＡ法,４４份（６８．８％）出现类似阳性的荧光颗粒,而用ＷＢ法测定,无特异的反应带出现。非感染Ｖｅｒｏ－Ｅ６细胞作ＩＦＡ抗原,３０份（４６．９％）与正常细胞抗原有反应,此结果表明ＷＢ法在特异性和敏感性方面均高于ＩＦＡ法。ＩＦＡ法中的非特异性反应系血清与细胞成份之反应。     

Anti-native DNA antibodies from autoimmune sera also bind to DNA in mitochondria

Sera from 20 patients with systemic lupus erythematosus (SLE), selected for elevated titers of antibody to native DNA (nDNA), were examined by indirect immunofluorescence (IF) on tissue culture Hep-2 and rabbit kidney cells. Twelve sera showed a particulate cytoplasmic staining, in addition to nuclear IF. Double IF staining by using a mouse monoclonal anti-nDNA and a human serum containing anti-mitochondrial antibody as probes showed that the cytoplasmic structures recognized by these 12 SLE sera were mitochondria. SLE sera showing mitochondrial staining had high anti-nDNA levels, as assessed by ELISA (3.5 +/- 1.9 O.D.), compared with those not showing this staining pattern (0.8 +/- 0.4 O.D.). Mitochondrial staining was abolished by DNase I pretreatment of the substrates. Liquid phase absorption of serum anti-nDNA with S1 nuclease-treated calf thymus DNA or purified mitochondrial DNA also removed staining. These findings demonstrate that anti-nDNA antibodies from patients with SLE bind to DNA in intact mitochondria. Therefore, mitochondrial IF staining on tissue culture cells in the presence of nuclear staining should be interpreted with caution, because the phenomenon could be entirely related to anti-native DNA. These observations might also provide new insights concerning the nature of immunogenic cellular components stimulating anti-DNA production.     

Characterization of the immune response to collagen-like proteins Scl1 and Scl2 of serotype M1 and M28 group A Streptococcus

Group A Streptococcus (GAS) infections may trigger autoimmune sequelae thought to involve streptococcal antibodies cross-reactive to human antigens. Here, the antigenicity of the streptococcal collagen-like (CL) proteins, Scl1 and Scl2, that exhibit structural similarity to human collagen, was analyzed. Antibodies to Scl1.1 protein were detected in human sera from healthy individuals previously infected with M1 GAS and from patients with various GAS infections, as well as in sera from mice infected with M1 GAS. Linear B-cell epitope mapping identified immunoreactive peptides corresponding to the CL region of Scl1.1. Humoral responses to Scl1.28 and Scl2.28 were also detected in pediatric patients and mice infected with M28 GAS.