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 α-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.Acute rheumatic fever (ARF) is an inflammatory disease that can follow group A streptococcal pharyngitis. The most serious clinical manifestation is rheumatic carditis; however, arthritis, chorea, erythema marginatum, or subcutaneous nodules may be present (40, 41). The pathogenesis of ARF is thought to be mediated by autoimmune mechanisms activated during a streptococcal infection (40). The autoimmune hypothesis is supported by a number of previous observations, including a time interval of at least 3 weeks between the initial streptococcal throat infection and the onset of ARF (40, 41), the identification of heart-reactive immunoglobulin (Ig) and complement deposits in the myocardium of patients with fatal rheumatic carditis (25–27, 30), and the elevation of heart-reactive antibodies in the sera of patients with ARF (46). Cardiac myosin has been identified as one of the cardiac antigens recognized by these heart-reactive antistreptococcal autoantibodies (13, 29).Streptococcal M protein, an α-helical coiled-coil protein, structurally and immunologically mimics host tissue antigens, particularly the rod region of myosin (12, 14, 15, 17, 34, 35). Sequence analysis has revealed that streptococcal M proteins contain blocks of internally repeated amino acid sequences referred to as A, B, and C repeat regions (19). The NH2-terminal nonrepeat and A repeat regions contain determinants of type specificity, while epitopes found in the B and more highly conserved C repeat regions may be common to different M serotypes (19). While there are nearly 100 different serological types of group A streptococcal M protein, epidemiological studies indicate that only a limited number of M protein serotypes are associated with ARF outbreaks (6). This finding suggests that certain M protein serotypes may be more rheumatogenic than others. In a previous attempt to classify streptococcal serotypes according to their rheumatogenic capacity, Widdowson identified human antisera directed to a non-type-specific protein moiety of M protein known as M-associated protein (44, 45). However, a more recent classification scheme has been proposed by Bessen and colleagues, in which streptococcal serotypes were grouped based on the expression of a conserved surface-exposed M protein epitope (4). It was demonstrated that the M serotypes associated with the majority of ARF outbreaks possessed an epitope (class I) defined by monoclonal antibody (MAb) probes 10B6 and 10F5. The sequence of the 10B6 and 10F5 epitope was localized to a 15-amino-acid fragment within the C repeat region of the type 6 M protein (23). The remaining serotypes (class II) lack this epitope or the determinant is structurally inaccessible in those strains. There was a close parallel between serotypes designated class I and those serotypes previously classified as M-associated protein I by Widdowson (44, 45). The fact that only certain serotypes within class I streptococci are rheumatogenic implies that these organisms are of a phenotype that is capable of inducing ARF (4). This implication is supported in part by a recent publication in which it was shown that sera of ARF patients contained high levels of antibodies to the class I epitope, suggesting that their disease was the result of an infection by a class I streptococcus (5).Elevated titers of antibodies to many streptococcal antigens (2), including M protein and the self-antigen myosin (12–15, 17, 29), are associated with ARF. While antibodies to M protein are crucial for the opsonization of streptococci, they have also been implicated in the immunological cross-reactions between streptococci and host tissue antigens such as cardiac myosin (12–15, 17, 29). In earlier studies, many of these cross-reactive epitopes have been localized to the N-terminal, hypervariable A and B repeat regions of the M molecule (12, 15, 17). Myosin-reactive antibodies, found to be elevated in almost all cases of ARF (13), have been shown to bind to human heart tissue and to cross-react with streptococcal M protein (12). Previous studies have demonstrated that immunization of animals with the cell walls of certain strains of group A streptococci resulted in the production of heart-reactive antibodies which could be adsorbed with streptococcal extracts containing streptococcal M protein (16, 24, 28). Human MAbs or myosin affinity-purified antibodies produced from patients with ARF cross-reacted with streptococcal M protein and human cardiac myosin and contributed to the presence of heart-cross-reactive antistreptococcal antibodies in ARF (12, 13, 39). More recent studies have identified cytotoxic antistreptococcal/antimyosin MAbs from rheumatic carditis patients (1). Antimyosin antibody has been shown to deposit in the heart tissues of susceptible mice (31), and a cytotoxic mouse antistreptococcal/antimyosin antibody which binds to the surface of heart cells and to the α-helical coiled coil molecule laminin has been described (10).Identification of myosin cross-reactive epitopes of M protein recognized in ARF has been reported for the amino-terminal half of the molecule (12, 15, 17), and a study by Vashishtha and Fischetti demonstrated antimyosin antibody responses to the C repeat region. However, the reactivity was directed only to denatured myosin (43). More recently, studies of the C repeat or carboxy-terminal region of M protein have shown T-cell cross-reactions with myosin (38). The goal of the present study was to investigate the possibility that the class I epitope in the C repeat region of M protein cross-reacts immunologically with myosin. In this study we show that MAb 10B6, which recognizes the class I epitope of M protein, reacts with cardiac and skeletal myosin. This study also demonstrates that ARF and UNC sera react with a site in the conserved C repeat region of M protein within the class I epitope of rheumatogenic M protein serotypes. The new data show that in addition to previously described N-terminal epitopes, the class I epitope of streptococcal M protein is immunologically cross-reactive with myosin.(Portions of this work were presented at the XIII International Lancefield Society Meeting on Streptococci and Streptococcal Diseases at the Pasteur Institute in Paris, France, in September 1996.)     

The inferior alveolar artery in its bony course

Based on the dissection of 30 hemi-mandibles, the authors report a study of the inferior alveolar artery in its intraosseous course. On morphologic considerations they propose a classification of the collaterals into two groups: the principal collaterals destined for the teeth and the bony alveolar tissue and the secondary collaterals destined for the sheath and the nerve as well as the bony tissue around the canal. Loss of the teeth and absorption of the alveolar bone modify the caliber of the inferior alveolar arterial axis, the distribution of its collaterals and possibly its mode of termination. These facts suggest a consideration of the vascularization of the mandible in terms of four sectors. They arrive at practical conclusions that may be drawn from this study in stomatology.     

Osteoclasts contain macrophage and megakaryocyte antigens

The origin and mechanism of formation of the osteoclast remains controversial. Although it is known to be derived from a circulating mononuclear percursor, the identity of this cell is unknown. Using a panel of monoclonal antibodies raised against macrophage and other marrow-derived cells, we determined the immunocytochemical staining of human osteoclasts in both fetal bone metaphyseal imprints and frozen sections. Osteoclasts and marrow mononuclear cells were stained by three broad spectrum antimacrophage antibodies, EBM-11, Y182a and BM2. T310, an antibody which stains macrophages and T helper cells, and C17, an antimegakaryocyte antibody, also stained osteoclasts. EBM-11, Y182a and BM2 also stained megakaryocytes in bone imprints as well as normal bone marrow smears. The presence of macrophage-associated antigens in osteoclasts, megakaryocytes and bone marrow mononuclear cells indicates that they are phenotypically similar to macrophages.     

Immunogenicity of recombinant protective antigen and efficacy against aerosol challenge with anthrax

Immunization with a recombinant form of the protective antigen (rPA) from Bacillus anthracis has been carried out with rhesus macaques. Rhesus macaques immunized with 25 mug or more of B. subtilis-expressed rPA bound to alhydrogel had a significantly increased immunoglobulin G (IgG) response to rPA compared with macaques receiving the existing licensed vaccine from the United Kingdom (anthrax vaccine precipitated [AVP]), although the isotype profile was unchanged, with bias towards the IgG1 and IgG2 subclasses. Immune macaque sera from all immunized groups contained toxin-neutralizing antibody and recognized all the domains of PA. While the recognition of the N terminus of PA (domains 1 to 3) was predominant in macaques immunized with the existing vaccines (AVP and the U.S. vaccine anthrax vaccine adsorbed), macaques immunized with rPA recognized the N- and C-terminal domains of PA. Antiserum derived from immunized macaques protected macrophages in vitro against the cytotoxic effects of lethal toxin. Passive transfer of IgG purified from immune macaque serum into naive A/J mice conferred protection against challenge with B. anthracis in a dose-related manner. The protection conferred by passive transfer of 500 mug macaque IgG correlated significantly (P = 0.003; r = 0.4) with the titers of neutralizing antibody in donor macaques. Subsequently, a separate group of rhesus macaques immunized with 50 mug of Escherichia coli-derived rPA adsorbed to alhydrogel was fully protected against a target dose of 200 50% lethal doses of aerosolized B. anthracis. These data provide some preliminary evidence for the existence of immune correlates of protection against anthrax infection in rhesus macaques immunized with rPA.     

Effects of age on volumes of cortex, white matter and subcortical structures

The effect of age was investigated in and compared across 16 automatically segmented brain measures: cortical gray matter, cerebral white matter, hippocampus, amygdala, thalamus, the accumbens area, caudate, putamen, pallidum, brainstem, cerebellar cortex, cerebellar white matter, the lateral ventricle, the inferior lateral ventricle, and the 3rd and 4th ventricle. Significant age effects were found for all volumes except pallidum and the 4th ventricle. Heterogeneous age responses were seen in that age relationships for cortex, amygdala, thalamus, the accumbens area, and caudate were linear, while cerebral white matter, hippocampus, brainstem, cerebellar white, and gray matter, as well as volume of the lateral, inferior lateral, and 3rd ventricles showed curvilinear relationships with age. In general, the findings point to global and large effects of age across brain volumes.     

The dragon lizard <Emphasis Type="Italic">Pogona vitticeps</Emphasis> has ZZ/ZW micro-sex chromosomes

The bearded dragon, Pogona vitticeps (Agamidae: Reptilia) is an agamid lizard endemic to Australia. Like crocodilians and many turtles, temperature-dependent sex determination (TSD) is common in agamid lizards, although many species have genotypic sex determination (GSD). P. vitticeps is reported to have GSD, but no detectable sex chromosomes. Here we used molecular cytogenetic and differential banding techniques to reveal sex chromosomes in this species. Comparative genomic hybridization (CGH), GTG- and C-banding identified a highly heterochromatic microchromosome specific to females, demonstrating female heterogamety (ZZ/ZW) in this species. We isolated the P. vitticeps W chromosome by microdissection, re-amplified the DNA and used it to paint the W. No unpaired bivalents were detected in male synaptonemal complexes at meiotic pachytene, confirming male homogamety. We conclude that P. vitticeps has differentiated previously unidentifable W and Z micro-sex chromosomes, the first to be demonstrated in an agamid lizard. Our finding implies that heterochromatinization of the heterogametic chromosome occurred during sex chromosome differentiation in this species, as is the case in some lizards and many snakes, as well as in birds and mammals. Many GSD reptiles with cryptic sex chromosomes may also prove to have micro-sex chromosomes. Reptile microchromosomes, long dismissed as non-functional minutiae and often omitted from karyotypes, therefore deserve closer scrutiny with new and more sensitive techniques.     

Effect of silicon carbide whisker-silica heat treatment on the reinforcement of dental resin composites

The strength and fracture resistance of dental resin composites need to be improved to extend their use to large stress-bearing crown and multiple-unit applications. Recent studies showed that the addition of ceramic whiskers significantly reinforced resin composites. The aim of the present study was to use a silicon carbide whisker-silica particle mixture to reinforce resin composites, and to investigate the effect of whisker-silica mixture heat-treatment on composite properties. The whiskers were blended with silica particles and were thermally fused in an attempt to roughen the whiskers for improved retention in the matrix. The mixtures were heat-treated at temperatures of 500, 650, 800, 950, and 1100 degrees C for 10 min, 30 min, and 3 h. The mixtures were then silanized, incorporated into a dental resin, and the paste was placed into 2 x 2 x 25 mm molds and heat-cured at 140 degrees C for 30 min. A 3-point flexural test was used to measure flexural strength and work-of-fracture. Two commercial indirect composites were tested as controls. Two-way ANOVA showed that there was no significant effect from temperature or time. Therefore, all the whisker-silica mixture samples were combined into one group (n = 96), and compared to composites filled with silica only or whisker only, and the two indirect control composites. The whisker-silica mixture group had a flexural strength (mean +/- SD) of (186 +/- 24) MPa, significantly higher than (99 +/- 29) MPa for silica only, (131 +/- 22) MPa for whisker only, and (109 +/- 23) MPa and (114 +/- 18) MPa for the two indirect composites (Tukey's multiple comparison test; family confidence coefficient = 0.95). Similar results were obtained on work-of-fracture. Scanning electron microscopy revealed rough fracture surfaces for the whisker-silica composites, indicating crack deflection and bridging by whiskers as toughening mechanisms. Whisker-silica mixture minimized whisker entanglement and enhanced whisker-resin bonding, resulting in substantially stronger and tougher dental resin composites.     

Comparison of a Multiplexed Fluorescent Covalent Microsphere Immunoassay and an Enzyme-Linked Immunosorbent Assay for Measurement of Human Immunoglobulin G Antibodies to Anthrax Toxins

Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 μg/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 μg of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 μg/ml, while the dynamic range was 0.06 to 1.7 μg/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 μg of anti-PA IgG per ml, the RDL was 0.016 μg/ml, and the whole-serum equivalent MDC was 1.5 μg/ml. The dynamic range was 0.006 to 6.8 μg/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r² = 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.