Induction of autoimmune valvular heart disease by recombinant streptococcal m protein.

Rheumatic heart disease is an autoimmune sequela of group A streptococcal infection. Previous studies have established that streptococcal M protein is structurally and immunologically similar to cardiac myosin, a well-known mediator of inflammatory heart disease. In this study, we investigated the hypothesis that streptococcal M protein could produce inflammatory valvular heart lesions similar to those seen in rheumatic fever (RF). Fifty percent (3 of 6) of Lewis rats immunized with recombinant type 6 streptococcal M protein (rM6) developed valvulitis as well as focal lesions of myocarditis. Valvular lesions initiated at the valve surface endothelium spread into the valve. Anitschkow cells and verruca-like lesions were present. T cells from rM6-immunized rats proliferated in the presence of purified cardiac myosin, but not skeletal myosin. A T-cell line produced from rM6-treated rats proliferated in the presence of cardiac myosin and rM6 protein. The study demonstrates that the Lewis rat is a model of valvular heart disease and that streptococcal M protein can induce an autoimmune cell-mediated immune attack on the heart valve in an animal model. The data support the hypothesis that a bacterial antigen can break immune tolerance in vivo, an important concept in autoimmunity.

Reactivity of rheumatic fever and scarlet fever patients' sera with group A streptococcal M protein, cardiac myosin, and cardiac tropomyosin: a retrospective study.

Archived sera (collected in 1946) from acute rheumatic fever (ARF) and untreated scarlet fever and/or pharyngitis patients were reacted with streptococcal M protein, cardiac myosin, and cardiac tropomyosin. Except for very low levels to tropomyosin, antibodies to other antigens were not elevated in the sera of ARF patients relative to those of non-ARF patients, even though there was roughly equivalent exposure to group A streptococci. This suggests that antibodies to these molecules may not play a central role in the induction of ARF.

Cytotoxic mAb from rheumatic carditis recognizes heart valves and laminin.

Anti-streptococcal antibodies cross-reactive with N-acetyl-betaD-glucosamine (GlcNAc) and myosin are present in the sera of patients with rheumatic fever (RF). However, their role in tissue injury is not clear. In this study, we show that anti-GlcNAc/anti-myosin mAb 3.B6 from a rheumatic carditis patient was cytotoxic for human endothelial cell lines and reacted with human valvular endothelium and underlying basement membrane. Reactivity of mAb 3.B6 with the valve was inhibited by human cardiac myosin > laminin > GlcNAc. The mAb 3.B6 epitopes were localized in fragments of human cardiac myosin, including heavy meromyosin (HMM), the S1 subfragment, and two light meromyosin (LMM) peptides containing amino acid sequences KEALISSLTRGKLTYTQQ (LMM 1) and SERVQLLHSQNTSLINQK (LMM 33). A novel feature of mAb 3.B6 was its reactivity with the extracellular matrix protein laminin, which may explain its reactivity with the valve surface. A laminin A-chain peptide (HTQNT) that includes homology to LMM33 inhibited the reactivity of mAb 3.B6 with human valve. These data support the hypothesis that cross-reactive antibodies in rheumatic carditis cause injury at the endothelium and underlying matrix of the valve.

Molecular analysis of cross-reactive anti-myosin/anti-streptococcal mouse monoclonal antibodies.

Nucleotide sequences of VH- and VL-genes of anti-myosin/anti-streptococcal monoclonal antibodies (mAbs) were analyzed and compared with their highly detailed antigen binding reactivities. Antigen-specificities of the cross-reactive mAbs included myosin, streptococcal M-protein, actin, keratin, N-acetyl-beta-D-glucosamine, vimentin, DNA, tropomyosin, troponin, and laminin as previously described. After nucleotide sequence analysis, homology indicated that some of the V gene sequences aligned with antibodies recognizing gangliosides and blood group antigens glycophorin M and N. Therefore, mAb reactivity with gangliosides and glycophorin M and N was identified. The cross-reactive mAbs utilized a heterogeneous group of germline V-heavy genes comprised of nine J558-, four 7183- and two Q52-family VH-genes. Germline V-light genes utilized by the mAbs included six Vkappa4/5-, three Vkappa8-, two Vkappa10-, three Vkappa19- and one Vkappa23-family VL-genes. No preferential VH/VL-chains correlated with any of the 12 different antigen reactivities, even for mAbs with nearly identical cross-reactivities. However, we did find that the cross-reactive mAb germline genes within a V gene family shared more homology among themselves than with other germline genes within their V gene families, suggesting convergent mutation. Cross-reactive mAbs with the highest relative avidity for myosin were found in the VH7183 family which contained two cytotoxic mAbs. Antibodies with V gene sequences most homologous to those of our cross-reactive anti-myosin/anti-streptococcal mAbs had specificities for laminin, DNA, carbohydrates, or blood group antigens and were reported to cause autoimmune disease in mice.

Atrial septal defects in Florida panthers.

Ostium secundum atrial septal defects (ASDs) were observed in six (3 M, 3 F) of 33 (20 M, 13 F) (18%) Florida panthers (Puma concolor coryi) necropsied by veterinary pathologists between 1985 and 1998. A seventh ASD was found in a female panther necropsied in the field and is included in the pathological description but not the prevalence of ASDs in Florida panthers. One panther (FP205) with severe ASD also had tricuspid valve dysplasia (TVD). Atrial septal defects and/or TVD are believed to have caused or contributed to the deaths of three (9%) Florida panthers in this study. Mean diameter +/- SD of ASDs was 9.0 +/- 4.7 mm (range 3 to 15 mm). Gross pathological changes attributed to ASDs/TVD in severely affected panthers (ASD > or = 10 mm) (n = 4) included mild right ventricular dilatation (n = 3) and hypertrophy (n = 2), mild to severe right atrial dilatation (n = 2), and acute pulmonary edema (n = 3). Panthers with mild ASDs (ASD < or = 5 mm) (n = 3) had no other detectable gross pathological changes associated with the ASDs. Histological examination of lungs of three panthers with severe ASDs revealed mild to moderate dilatation with fibrosis and smooth muscle atrophy of the tunica media of medium to large caliber arteries (n = 2), interstitial and/or pleural fibrosis (n = 2), perivascular fibrosis (n = 1), and acute to chronic edema (n = 3). Twenty-six necropsied panthers were examined one or more times while living; medical records were retrospectively evaluated. Antemortem radiographic, electrocardiographic, and echocardiographic examinations were performed on two panthers with severe ASDs (FP20 and FP205). Thoracic radiographic abnormalities in both included right heart enlargement, and in FP205 (severe ASD and TVD), mild pulmonary overperfusion. Electrocardiographic examination of FP205 revealed a right ventricular hypertrophy pattern, while FP205 had a normal electrocardiogram. Echocardiographic examination of FP20 revealed marked right atrial dilatation; a bubble contrast study indicated regurgitation across the tricuspid valve. Echocardiographic abnormalities in FP20 included right atrial and ventricular lilatation, atrial septal drop-out, and severe tricuspid regurgitation; non-selective angiography revealed significant left to right shunting across the ASD. All panthers with severe ASDs ausculted (n = 3) had systolic right or left-sided grade I-V/VI murmurs loudest at the heart base. All male panthers with ASDs (n = 3) (100%) and 9 of 17 (53%) male panthers without ASDs in this study were cryptorchid.

Anti-human cardiac myosin autoantibodies in Kawasaki syndrome.

Kawasaki syndrome (KS) is the major cause of acquired heart disease in children. Although acute myocarditis is observed in most patients with KS, its pathogenesis is unknown. Because antimyosin autoantibodies are present in autoimmune myocarditis and rheumatic carditis, the purpose of the current study was to determine whether anticardiac myosin Abs might be present during the acute stage of KS. Sera from KS patients as well as age-matched febrile controls and normal adults were compared for reactivity with human cardiac myosin in ELISAs and Western blot assays. A total of 5 of 13 KS sera, as compared with 5 of 8 acute rheumatic fever sera, contained Ab titers to human cardiac myosin that were significantly higher than those found in control sera. Both cardiac and skeletal myosins were recognized in the ELISA by KS sera, although stronger reactivity was observed to human cardiac myosin. Only IgM antimyosin Abs from KS sera were significantly elevated relative to control sera. KS sera containing antimyosin Abs recognized synthetic peptides from the light meromyosin region of the human cardiac myosin molecule and had a different pattern of reactivity than acute rheumatic fever sera, further supporting the association of antimyosin Ab with KS. These Abs may contribute to the pathogenesis of acute myocarditis found in patients with KS.

Seroprevalence of selected disease agents from free-ranging black bears in Florida.

Sera obtained from 66 free-ranging Florida black bears (Ursus americanus floridanus) from three geographic areas of Florida (USA) between November 1993 and August 1995 were tested for antibodies to 13 disease agents. Antibody prevalences were 3 positive of 37 tested (8%) Coxiella burnetti, 37 of 66 (56%) Toxoplasma gondii, 3 of 61 (5%) bluetongue virus/epizootic hemorrhagic disease virus (BTV/EHDV), 4 of 66 (6%) canine adenovirus-type 1, 5 of 66 (8%) canine distemper virus (CDV), 10 of 62 (16%) canine parvovirus (CPV), 7 of 66 (11%) eastern equine encephalitis virus, 4 of 66 (6%) western equine encephalitis virus, 2 of 66 (3%) Venezuelan equine encephalitis virus, and 11 of 66 (17%) St. Louis encephalitis virus. No samples had serologic evidence of exposure to Brucella spp. (n = 37), Francisella tularensis (n = 40), or pseudorabies virus (n = 37). This is the first known published report of antibodies to BTV/EHDV, CDV, and CPV in black bears.

Immunological relationship between the class I epitope of streptococcal M protein and myosin.

The class I epitope of streptococcal M protein is an epidemiological marker for acute rheumatic fever (ARF)-associated serotypes of group A streptococci and is recognized by anti-M protein monoclonal antibody (MAb) 10B6. Using MAb 10B6, we determined the relationship between the class I epitope of M protein and the alpha-helical coiled-coil protein myosin. MAb 10B6 reacted by enzyme-linked immunosorbent assay and Western blotting with human cardiac myosin and rabbit skeletal myosin and its heavy meromyosin (HMM) subfragment. Overlapping synthetic peptides of M5 protein were used to identify the region of M5 protein recognized by MAb 10B6. Two C repeat peptides (C2A and C3) containing the amino acid sequence KGLRRDLDASREAK reacted with MAb 10B6. Partial sequence identity, RRDL, was found in the HMM fragment of myosin, which reacted with MAb 10B6. However, not all peptides of M5 protein and myosin containing the RRDL sequence reacted with MAb 10B6. ARF sera and sera from uncomplicated pharyngitis (UNC) reacted with C repeat region peptides of M protein, while acute glomerulonephritis sera were not as reactive. Affinity-purified human antibody to peptide C3 reacted with myosin. The data demonstrate that the class I epitope of M protein is immunologically cross-reactive with myosin and the HMM subfragment, and antibodies to peptide C3 and myosin were present in ARF and UNC sera.

Molecular analysis of polyreactive monoclonal antibodies from rheumatic carditis: human anti-N-acetylglucosamine/anti-myosin antibody V region genes.

Anti-myosin Abs are associated with inflammatory heart diseases such as rheumatic carditis and myocarditis. In this study, human cross-reactive anti-streptococcal/anti-myosin mAbs 1.C8, 1.H9, 5.G3, and 3.B6, produced from peripheral blood lymphocytes of patients with rheumatic carditis, and mAb 10.2.5, produced from a tonsil, were characterized, and the nucleotide sequences of their V(H) and V(H)L genes were analyzed. Human mAbs 1.C8, 1.H9, 10.2.5, and 3.B6 reacted with human cardiac myosin while mAb 5.G3 did not. The mAbs were strongly reactive with N-acetyl-beta-D-glucosamine, the dominant epitope of the group A streptococcal carbohydrate. mAb 1.H9 was moderately cytotoxic to rat heart cells in vitro in the presence of complement. The anti-myosin mAbs from rheumatic carditis were found to react with specific peptides from the light meromyosin region of the human cardiac myosin molecule. Anti-streptococcal/anti-myosin mAbs from normal individuals reacted with distinctly different light meromyosin peptides. The mAbs were encoded by V(H)3 gene segments V3-8, V3-23, and V3-30 and by the V(H)4 gene segment V4-59. The variable region genes encoding the anti-streptococcal/anti-myosin repertoire were heterogeneous and exhibited little evidence of Ag-driven somatic mutation.

Molecular analysis of human cardiac myosin-cross-reactive B- and T-cell epitopes of the group A streptococcal M5 protein.

The group A streptococcal M protein is an important virulence determinant eliciting protective and autoimmune responses against the streptococcus and cardiac myosin, respectively. In this report, the major human cardiac myosin-cross-reactive T-cell epitopes of M5 protein are identified and localized to myosin-like repeats within the M5 molecule. BALB/c mice were immunized with human cardiac myosin, and the dominant myosin-cross-reactive T-cell epitopes of M5 protein were identified with a panel of 23 overlapping peptides spanning the A, B, and C repeat regions of M5 protein. Human cardiac myosin-cross-reactive T-cell epitopes of M5 protein were localized to several sequences in the M5 peptides NT4 (GLKTENEGLKTENEGLKTE), NT5 (KKEHEAENDKLKQQRDTL), B1B2 (VKDKIAKEQENKETIGTL), B2 (TIGTLKKILDETVKDKIA), B3A (IGTLKKILDETVKDKLAK), and C3 (KGLRRDLDASREAKKQ). The NT4 repeated sequence LKTEN was highly homologous with a site conserved in cardiac myosins, the B repeat region peptides were 47% homologous to human cardiac myosin amino acid sequence, and the C3 sequence RRDL was identical to a highly conserved site in skeletal and cardiac myosins. Immunization of BALB/c mice with each of the overlapping M5 peptides revealed myosin-cross-reactive B-cell epitopes throughout the A and C repeat regions and one major epitope in the B repeat region containing the previously reported Gln-Lys-Ser-Lys-Gln (QKSKQ) epitope. The data suggest that the M5 peptides elicited higher antibody titers to cardiac myosin than to skeletal myosin and that several sites in the A and B repeat regions of M5 protein induced myocardial inflammatory infiltrates.

Molecular analysis of V gene sequences encoding cytotoxic anti-streptococcal/anti-myosin monoclonal antibody 36.2.2 that recognizes the heart cell surface protein laminin.

Anti-streptococcal/anti-myosin mAb 36.2.2 is unique among the cross-reactive anti-streptococcal mAbs due to its cytotoxicity for rat heart cells and its ability to strongly label the surface of heart cells in indirect immunofluorescence assays. In this study, cytotoxic mAb 36.2.2 was found to react strongly with the extracellular matrix protein laminin in immunoblots and inhibition assays, while 11 other cross-reactive anti-streptococcal mAbs did not react with laminin and were not cytotoxic. Cytotoxicity appeared to correlate with the presence of laminin on the surface of cells. Heavy and light chain variable region genes encoding mAb 36.2.2 were highly homologous to other V genes encoding anti-carbohydrate and/or autoantibodies. VH, JH, and Jkappa segments of mAb 36.2.2 may be encoded by germline gene segments. The VH segment may be identical with an as yet unidentified VH7183 family germline sequence, and the 36.2.2 Vkappa region gene is encoded by a Vkappa8 family member.

Streptococcal M protein peptide with similarity to myosin induces CD4+ T cell-dependent myocarditis in MRL/++ mice and induces partial tolerance against coxsakieviral myocarditis.

Immunologic similarities have been demonstrated between Coxsackievirus B3 (CVB3), group A streptococcal M protein, and cardiac myosin. Previous studies have also shown that T lymphocytes obtained from CVB3-infected mice expressing the H-2k MHC haplotype gave an immunodominant proliferative response to the NT4 peptide (GLKTENEGLKTENEGLKTE) of the streptococcal M5 protein. We now show that the NT4 peptide can induce inflammatory heart disease in MRL/++ (H-2k) mice and that induction of anergy to this peptide protects against CVB3-induced myocarditis. MRL/++ mice infected with CVB3 for 7 days or immunized twice at 7-day intervals with the streptococcal NT4 peptide in CFA developed myocarditis. Treatment of the immunized mice with either anti-CD4 or anti-IAk mAbs inhibited cardiac inflammation. Injection of MRL/++ mice with NT4 covalently coupled to syngeneic splenocytes tolerized the animals to this peptide as shown by reduction of the proliferative response. NT4-tolerized mice had significantly reduced myocarditis, although virus titers in the heart were elevated. A control peptide, VP1-10 from the CVB3 capsid protein VP1, did not protect the mice from CVB3-induced myocarditis. The results suggest that immunity to NT4 induced during CVB3 infections is important to the development of cardiac inflammation.

Autoantibody germ-line gene segment encodes VH and VL regions of a human anti-streptococcal monoclonal antibody recognizing streptococcal M protein and human cardiac myosin epitopes.

Cross-reactivity of anti-streptococcal Abs with human cardiac myosin may result in sequelae following group A streptococcal infections. Molecular mimicry between group A streptococcal M protein and cardiac myosin may be the basis for the immunologic cross-reactivity. In this study, a cross-reactive human anti-streptococcal/anti-myosin mAb (10.2.3) was characterized, and the myosin epitopes were recognized by the Ab identified. mAb 10.2.3 reacted with four peptides from the light meromyosin (LMM) tail fragment of human cardiac myosin, including LMM-10 (1411-1428), LMM-23 (1580-1597), LMM-27 (1632-1649), and LMM-30 (1671-1687). Only LMM-30 inhibited binding of mAb 10.2.3 to streptococcal M protein and human cardiac myosin. Human mAb 10.2.3 labeled cytoskeletal structures within rat heart cells in indirect immunofluorescence, and reacted with group A streptococci expressing various M protein serotypes, PepM5, and recombinant M protein. The nucleotide sequence of gene segments encoding the Ig heavy and light chain V region of mAb 10.2.3 was determined. The light chain V segment was encoded by a V kappa 1 gene segment that was 98.5% identical with germ-line gene humig kappa Vi5. The V segment of the heavy chain was encoded by a VH3a gene segment that differed from the VH26 germ-line gene by a single base change. VH26 is expressed preferentially in early development and encodes autoantibodies with anti-DNA and rheumatoid factor specificities. Anti-streptococcal mAb 10.2.3 is an autoantibody encoded by VH and VL genes, with little or no somatic mutation.

Cloning and sequence analysis of a gene encoding a 67-kilodalton myosin-cross-reactive antigen of Streptococcus pyogenes reveals its similarity with class II major histocompatibility antigens.

The group A streptococcal sequela acute rheumatic fever (ARF) has been associated with immunological cross-reactivity between streptococcal and heart proteins. To identify Streptococcus pyogenes genes that encode a myosin cross-reactive antigen(s) recognized by ARF sera, a genomic library from an emm deletion strain (T28/51/4) was screened with a single ARF serum. A positively identified lambda EMBL3 clone (T.2.18) produced a protein which reacted with myosin-specific antibodies affinity purified from individual ARF sera. The recombinant protein was initially estimated to be 60 kDa in size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, upon sequence analysis it had a molecular mass equivalent to 67 kDa. Sera from patients with streptococcal infections, acute glomerulonephritis, and ARF were reactive with the recombinant 67-kDa protein. However, individual sera from healthy persons were negative or demonstrated low levels of reactivity with the 67-kDa antigen. The gene encoding the 67-kDa myosin-cross-reactive antigen was subcloned, and its nucleotide sequence was determined by using a combined strategy of DNA sequencing of the cloned gene and N-terminal amino acid sequencing of the protein expressed in Escherichia coli. The amino-terminal sequence deduced from the nucleotide sequence of an open reading frame was identical to that determined from the 67-kDa protein expressed in E. coli. The gene encoded 590 amino acids with a calculated molecular weight of 67,381. No cleavable signal peptide was detected with the 67-kDa protein expressed in E. coli. The deduced amino acid sequence of the 67-kDa protein did not exhibit significant similarity to any known streptococcal proteins. However, it was found to be 19% identical and 62% similar over 151 amino acid residues to the beta chain of mouse major histocompatibility complex class II antigen (I-Au). Similar degrees of homology to the beta chains of other murine and human class II haplotypes were found. Mouse anti-IA sera reacted with the recombinant 67-kDa protein about five times more strongly than normal mouse sera in the enzyme-linked immunosorbent assay. Southern hybridization experiments using a probe for the gene encoding the 67-kDa protein showed that the gene was present and conserved among pathogenic groups A, C, and G of streptococci. These data suggest that the streptococcal protein, which is distinct from the M protein, may have structural features in common with the beta chain of the class II antigens, as well as myosin, and may play an important role in the pathogenesis of streptococcal infections.

Poststreptococcal anti-myosin antibody idiotype associated with systemic lupus erythematosus and Sjögren's syndrome.

Anti-myosin antibodies are found in acute rheumatic fever (ARF), a sequela of group A streptococcal infection. An antiidiotypic serum was produced that was specific for idiotopes expressed by anti-myosin antibodies in ARF (anti-My1). Studies indicated that idiotypic determinants detected with this serum were present in anti-myosin antibodies and absent from normal human immunoglobulins that lacked specificity for myosin. Anti-My1 was tested against sera from patients with other types of autoimmune diseases as well as uncomplicated streptococcal infections. Sera from systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), and poststreptococcal acute glomerulonephritis patients demonstrated idiotypic reactivity with anti-My1. Affinity-purified anti-myosin antibodies from SLE, SS, and ARF sera also reacted strongly with anti-My1, indicating that immunoglobulins produced in these diseases share idiotypic determinants. The data demonstrated an association of the My1 idiotype with poststreptococcal sequelae and the two autoimmune diseases SLE and SS.