Cardiac myosin induces myocarditis in genetically predisposed mice

After infection with coxsackie virus B3 (CB3), H-2 congenic mice on an A- background develop immunologically mediated myocarditis associated with an increased titer of myosin autoantibody, part of which is specific for the cardiac myosin isoform. The present study demonstrates that cardiac myosin itself induces severe myocarditis and high titers of myosin autoantibodies in A/J, A.SW/SnJ, and A.CA/SnJ mice. As in CB3-induced myocarditis, one population of these autoantibodies was specific for cardiac myosin. A.BY/SnJ and B10.A/SgSnJ mice also developed the disease after immunization, but the prevalence and the myosin autoantibody titers were lower. In contrast, C57BL/6J and C57BL/10J mice were resistant to myocarditis induced by cardiac myosin and did not develop increased myosin autoantibodies or cardiac myosin-specific autoantibodies. Immunization with skeletal muscle myosin had no effect compared with controls injected with complete Freund's adjuvant, thereby suggesting that the immunogenic epitopes are unique to the cardiac myosin isoform. Furthermore, we found that susceptibility to myocarditis induced by cardiac myosin is influenced by the major histocompatibility complex and by genes not closely linked to the major histocompatibility complex. Because there are parallels between myocarditis induced by cardiac myosin and that induced by CB3, this new animal model can be used to analyze the pathologic mechanisms in autoimmune heart disease.     

Variations in the susceptibility to Coxsackievirus B3-induced myocarditis among different strains of mice

This study was undertaken to examine the inherent predisposition of different inbred strains of mice to develop Coxsackievirus B3-induced myocarditis. A time course study established the pertinent, differential parameters of the disease and their corresponding genetic control. The A.BY/SnJ (H-2b), A.SW/SnJ (H-2s), A.CA/SnJ (H-2f), B10.S/SgSf (H-2s), B10.PL/SgSf (H-2u), and C3H.NB/SnJ (H-2p) strains were found to vary widely in the extent and duration of viremia, in the temporal appearance and titer of neutralizing antibody, and in the prevalence, severity, and duration of myocardial disease. The A.BY/SnJ (H-2b), A.SW/SnJ (H-2s), A.CA/SnJ (H-2f), and C3H.NB/SnJ (H-2p) mice developed continuing, chronic myocardial disease, whereas B10.S/SgSf (H-2s) and B10.PL/SgSf (H-2u) did not. The four strains that displayed prolonged myocarditis also produced heart-specific myocardial autoantibodies. Heart-specific autoantibodies were not found in the B10.S/SgSf and B10.PL/SgSf animals. Differences in prevalence and titer of these heart-specific autoantibodies were noted among the three A strain H-2 congenic lines. The influence of the major histocompatibility complex (MHC) on disease production was demonstrated by comparison of the three A strain and two B10 strain H-2 congenics. Differences between A.SW/SnJ (H-2s) and B10.S/SgSf (H-2s) suggested non-MHC control of disease. These studies additionally indicate that the genetic regulation of susceptibility to CB3 infection and the direct virus-induced inflammation differ from the later immunopathic myocarditis.     

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.     

Cardiac myosin-induced myocarditis. Heart autoantibodies are not involved in the induction of the disease

We recently demonstrated that cardiac myosin is capable of inducing autoimmune myocarditis in genetically predisposed mice. This disease parallels coxsackievirus B3-induced autoimmune myocarditis in many respects and is associated with high-titer autoantibodies specific for cardiac myosin. The following lines of evidence suggest that these autoantibodies are not involved in the induction of autoimmune myocarditis: 1) immunoperoxidase staining of heart sections from cardiac myosin-immunized A/J and A.SW mice revealed IgG depositions only along damaged muscle fibres in infiltrated areas, but not in intact tissue; 2) myosin autoantibodies did not bind to the surface of viable cardiac myocytes isolated from mice, but only reacted with myocytes permeabilized with detergent; 3) mice treated with a single high dose of cyclophosphamide, which reduces the humoral immune response, still developed severe myocarditis, despite the fact that their autoantibody titers were reduced to the level of adjuvant-injected controls; and 4) passive transfer of high-titer myosin autoantibodies failed to induce myocarditis, although the titers in the recipients were comparable to those found in mice with cardiac myosin-induced disease. Together, the results suggest that high-titer myosin autoantibodies are secondary rather than primary to the disease.     

LPS promotes CB3-induced myocarditis in resistant B10.A mice.

Coxsackie virus B3 (CB3) infection of A/J or A.SW mice results in autoimmune myocarditis characterized by a diffuse mononuclear cell infiltrate and heart-specific autoantibodies. C57BL/10 congenic mice that are identically treated are resistant to this disease. CB3-infected resistant B10.A mice were treated with LPS to determine if this immunomodulator alters disease susceptibility. In contrast to mice infected only with CB3 or treated only with LPS. CB3-infected/LPS-treated (CB3/LPS) B10.A mice developed autoimmune myocarditis similar to that observed in susceptible A/J or A.SW mice. By Day 14, CB3/LPS-induced disease was characterized by significant mortality, myocardial immunoglobulin deposition, and mononuclear cell infiltration of the heart. Immunohistochemical examination revealed deposits of IgG in the heart tissue and serum IgG autoantibodies reactive with sarcolemmal and fibrillary antigens in normal heart tissue. This serum IgG reacted with normal mouse cardiac antigens of a wide range of molecular weights by Western immunoblotting. Because LPS treatment is capable of increasing cytokine levels as well as MHC Class I and Class II expression in heart tissue, it suggests that these factors may contribute to susceptibility to autoimmune myocarditis in CB3-infected mice.     

Comparison between autoantibodies arising during Trypanosoma cruzi infection in mice and natural autoantibodies

The autoantibodies induced in (C57BL/6 x BALB/c)F1 mice during Trypanosoma cruzi (CL strain) infection were analyzed and compared with natural autoantibodies present in healthy mice. Mice were killed at intervals after infection and their sera were tested by enzyme immunoassay against a panel of self- and non-self-Ag: actin, myoglobin, myosin, tubulin, DNA, and TNP-OVA. The level of IgM and IgG autoantibodies against all Ag started to increase from day 15 until 6 wk after the parasite infection. The high level of all autoantibodies persisted 3 mo postinfection, and 1 yr later, half of the mice still had elevated levels of IgM and IgG autoantibodies, particularly antitubulin IgG antibodies. IgM and IgG were isolated from pools of normal and infected mouse sera and their binding capacity to all Ag was compared. The titers of infected mouse sera were increased and the slopes of both IgM and IgG binding curves of autoantibodies to actin, myosin, and tubulin were greater than those of control mouse sera, indicating higher affinities. The average dissociation constant of the IgG2a autoantibody to mouse tubulin was 5 times lower than that of natural antitubulin IgG2a antibodies. Furthermore, absorption of the IgG from infected mouse sera onto a tubulin immunoadsorbent removed half the reactivity with tubulin and also with myosin, actin and parasite extracts. The eluted antibodies bound the same Ag. When IgG were further analyzed by Western blot on proteolytic fragments of tubulin, we found that antibodies from both groups bound to the same broad spectrum of polypeptide bands. However, additional fragments were recognized by antibodies from infected mice. All these results indicate that the autoantibodies naturally present in mice are significantly affected after infection with T. cruzi, in quantity as well as in specificity and affinity.     

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.     

Subcellular compartmentalization of myosin isoforms in embryonic chick heart ventricle myocytes during cytokinesis

Embryonic chick heart ventricle myocytes retain the ability to alternate between proliferation and functional differentiation. A cytoplasmic isoform of myosin is present in cleavage furrows of various nonmuscle cells during cytokinesis, whereas one or more of the cardiac myosin isoforms are localized in sarcomeres of beating cardiomyocytes. Antibodies were employed to reveal the subcellular localizations of cytoplasmic and cardiac myosin isoforms in embryonic chick ventricle cardiomyocytes during cytokinesis. Monoclonal anticytoplasmic myosin antibodies were prepared against myosin purified from brains of 1-day-posthatched chickens and shown to react with chick brain myosin heavy chain by Western blots and/or ELISA tests. One monoclonal antibrain myosin antibody also cross-reacted with chick cardiac myosin but not with skeletal or smooth muscle myosins. Two antichick cardiac myosin monoclonal antibodies and one antichick skeletal myosin polyclonal antibody that cross-reacts with cardiac myosin were employed to identify cardiac sarcomeric myosin. Cells were isolated from day 8 embryonic chick heart ventricles, enriched for myocytes, grown in vitro for 3 days, and then examined by immunofluorescence techniques. Monoclonal antibodies against cytoplasmic myosin preferentially localized in the cleavage furrows of both cardiofibroblasts and cardiomyocytes in all stages of cytokinesis. In contrast, antibodies that recognize cardiac myosin were distributed throughout cardiomyocytes during early stages of cytokinesis, but became progressively excluded from the furrow area during middle and late stages of cytokinesis. These data suggest that in cells that contain both cytoplasmic and sarcomeric myosin isoforms, only cytoplasmic myosin isoforms are mobilized to from the contractile ring for cytokinesis.     

Tissue-specific and non-tissue-specific heavy-chain isoforms of myosin in the brain as revealed by monoclonal antibodies.

Four types of monoclonal antibody (BM-1, BM-2, BM-3 and BM-4) each having distinctive tissue specificity were obtained by immunizing mice with purified bovine cerebrum myosin. Both BM-1 and BM-2 reacted most efficiently with cerebrum myosin and less efficiently with myosins from other limited nonmuscle tissues, the tissue specificity of BM-1 being much narrower than that of BM-2. BM-3 reacted more efficiently with several other nonmuscle myosins than with cerebellar or cerebral myosin. BM-4 recognized various nonmuscle and smooth muscle myosins with a nearly equal efficiency. Cerebral myosin as well a cerebellar myosin contained two or more electrophoretic variants of the heavy chains. BM-1 and BM-3 as well as BM-2 and BM-3 were found to recognize selectively these distinct heavy-chain isoforms. The antigenic sites of the three tissue-specific antibodies (BM-1, BM-2 and BM-3) were all localized near the head/tail junction of the myosin molecules, while that of non-tissue-specific antibody BM-4 was near the center of the tail. These and additional results indicate that mammalian brain tissues as well as several other nonmuscle tissues contain multiple heavy-chain isoforms of myosin, the levels of which differed considerably from one tissue to another.     

Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system

Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.     

Association of iron-protoporphyrin-IX (hemin) with myosins

Addition of myosins isolated from guinea pig heart and rabbit skeletal muscle to hemin solutions resulted in the appearance of new absorption spectra indicating association of hemin and the myosins. Binding stoichiometry based on absorption changes was found to be two hemin sites per myosin molecule. The binding constants calculated from quenching of the intrinsic fluorescence of the myosins by hemin are Ka = 7 (+/- 2) 10(6) M-1 for skeletal muscle myosin, and Ka = 3 (+/- 1) x 10(7) M-1 for heart muscle myosin. Based on these findings, myosins are suggested as potential transporters of free hemin between cell organelles.     

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.     

Genetic analysis of myosin assembly inCaenorhabditis elegans

The established observations and unresolved questions in the assembly of myosin are outlined in this article. Much of the background information has been obtained in classical experiments using the myosin and thick filaments from vertebrate skeletal muscle. Current research is concerned with problems of myosin assembly and structure in smooth muscle, a broad spectrum of invertebrate muscles, and eukaryotic cells in general. Many of the general questions concerning myosin assembly have been addressed by a combination of genetic, molecular, and structural approaches in the nematode Caenorhabditis elegans. Detailed analysis of multiple myosin isoforms has been a prominent aspect of the nematode work. The molecular cloning and determination of the complete sequences of the genes encoding the four isoforms of myosin heavy chain and of the myosin-associated protein paramyosin have been a major landmark. The sequences have permitted a theoretical analysis of myosin rod structure and the interactions of myosin in thick filaments. The development of specific monoclonal antibodies to the individual myosins has led to the delineation of the different locations of the myosins and to their special roles in thick filament structure and assembly. In nematode body-wall muscles, two isoforms, myosins A and B, are located in different regions of each thick filament. Myosin A is located in the central biopolar zones, whereas myosin B is restricted to the flanking polar regions. This specific localization directly implies differential behavior of the two myosins during assembly. Genetic and structural experiments demonstrate that paramyosin and the levels of expression of the two forms are required for the differential assembly. Additional genetic experiments indicate that several other gene products are involved in the assembly of myosin. Structural studies of mutants have uncovered two new structures. A core structure separate from myosin and paramyosin appears to be an integral part of thick filaments. Multifilament assemblages exhibit multiple nascent thick filament-like structures extending from central paramyosin regions. Dominant mutants of myosin that disrupt thick filament assembly are located in the ATP and actin binding sites of the heavy chain. A model for a cycle of reactions in the assembly of myosin into thick filaments is presented. Specific reactions of the two myosin isoforms, paramyosin, and core proteins with multifilament assemblages as possible intermediates in assembly are proposed.     

Neonatal and adult myosin heavy chains form homodimers during avian skeletal muscle development

Myosin isoforms contribute to the heterogeneity and adaptability of skeletal muscle fibers. Besides the well-characterized slow and fast muscle myosins, there are those isoforms that appear transiently during the course of muscle development. At a stage of development when two different myosins are coexpressed, the possibility arises for the existence of heterodimers, molecules containing two different heavy chains, or homodimers, molecules with two identical heavy chains. The question of whether neonatal and adult myosin isoforms can associate to form a stable heterodimer was addressed by using stage-specific monoclonal antibodies in conjunction with immunological and electron microscopic techniques. We find that independent of the ratio of adult to neonatal myosin, depending on the age of the animal, the myosin heavy chains form predominantly homodimeric molecules. The small amount of hybrid species present suggests that either the rod portion of the two heavy chain isoforms differs too much in sequence to form a stable alpha-helical coiled coil, or that the biosynthesis of the heavy chains precludes the formation of heterodimeric molecules.     

Structural characterization of myosin from bovine brain

Myosins isolated from bovine brain, rabbit skeletal muscle, and chicken gizzard smooth muscle and their heavy meromyosin and light meromyosin fractions were studied in the electron microscope by negative staining with uranyl acetate. Under similar conditions of preparation and polymerization, the three myosins formed paracrystals of different structures. The light meromyosin portion of the skeletal muscle myosin also assembled in a different fashion than the brain or smooth muscle light meromyosins; the latter two assembled similarly. The heavy meromyosin portion from each of the three myosins was shown to interact with the actins isolated from each of the three tissue sources by the formation of the characteristic arrowhead patterns with similar periodicities. The brain heavy meromyosin attachment to both skeletal and brain actins was dissociated by ATP. It is suggested that differences in the light meromyosin portions of the three myosins may account in part for their differences in assembly in vivo.     

Order-disorder structural transitions in synthetic filaments of fast and slow skeletal muscle myosins under relaxing and activating conditions

In the previous study (Podlubnaya et al., 1999, J. Struc. Biol. 127, 1-15) Ca2+-induced reversible structural transitions in synthetic filaments of pure fast skeletal and cardiac muscle myosins were observed under rigor conditions (-Ca2+/+Ca2+). In the present work these studies have been extended to new more order-producing conditions (presence of ATP in the absence of Ca2+) aimed at arresting the relaxed structure in synthetic filaments of both fast and slow skeletal muscle myosin. Filaments were formed from column-purified myosins (rabbit fast skeletal muscle and rabbit slow skeletal semimebranosusproprius muscle). In the presence of 0.1 mM free Ca2+, 3 mM Mg2+ and 2 mM ATP (activating conditions) these filaments had a spread structure with a random arrangement of myosin heads and subfragments 2 protruding from the filament backbone. Such a structure is indistinguishable from the filament structures observed previously for fast skeletal, cardiac (see reference cited above) and smooth (Podlubnaya et al., 1999, J. Muscle Res. Cell Motil. 20, 547-554) muscle myosins in the presence of 0.1 mM free Ca2+. In the absence of Ca2+ and in the presence of ATP (relaxing conditions) the filaments of both studied myosins revealed a compact ordered structure. The fast skeletal muscle myosin filaments exhibited an axial periodicity of about 14.5 nm and which was much more pronounced than under rigor conditions in the absence of Ca2+ (see the first reference cited). The slow skeletal muscle myosin filaments differ slightly in their appearance from those of fast muscle as they exhibit mainly an axial repeat of about 43 nm while the 14.5 nm repeat is visible only in some regions. This may be a result of a slightly different structural properties of slow skeletal muscle myosin. We conclude that, like other filaments of vertebrate myosins, slow skeletal muscle myosin filaments also undergo the Ca2+-induced structural order-disorder transitions. It is very likely that all vertebrate muscle myosins possess such a property.     

柯萨奇病毒B3型诱导的免疫偏离与心肌炎发病关系的研究

目的 研究2种近交系小鼠在柯萨奇病毒B3型(CVB3)感染后辅助性T细胞(Th)免疫偏离对心肌炎发病的影响。方法 用CVB3腹腔感染BALB/c和C57BL/62种近交系小鼠,感染后7d通过检测小鼠血清肌酸激酶(CK)活性,观察心脏外观变化以及心脏石蜡切片H.E染色观察心脏病理改变,比较2种小鼠心肌炎的发病情况;通过体外感染心肌细胞观察病毒复制情况以及体内心脏组织病毒载量的分析,比较2种小鼠对病毒感染和复制的差异;通过检测感染小鼠细胞因子白细胞介素-4(IL-4)、IL-12和γ干扰素(IFN-γ)的表达,抗CVB3VP1抗体的亚型以及T-bet和Gata-3的表达,比较2种小鼠Th免疫偏离的情况。结果 CVB3在体外和体内都可以感染BALB/c和C57BL/6小鼠心肌细胞,但仅BALB/c小鼠感染后可发生明显的病毒性心肌炎,C57BL/6小鼠则不能;BALB/c小鼠感染后表现为Th1型免疫反应而C57BL/6小鼠则偏向于Th2型免疫反应。结论 CVB3感染2种品系小鼠表现为不同的心肌炎发生率,与其诱导了不同类型的免疫偏离密切相关。     

A combined histochemical and immunohistochemical study on the dynamics of fast-to-slow fiber transformation in chronically stimulated rabbit muscle

Summary Chronically stimulated fast-twitch muscles of the rabbit were histochemically and immunohistochemically analyzed in serial cross sections (1) for percentages of fiber types, and (2) for the presence of myosin heavy chain isoforms during fast-to-slow transformation. By four weeks of stimulation the number of type-I fibers had increased more than fourfold, while only about 6% of the original IIB fibers remained. Type-IC and -IIC fibers transiently rose to 20% of the total fiber population. After 16 weeks, the number of type-I fibers had increased to 42%. With prolonged stimulation fewer fibers reacted with antibodies against embryonic and neonatal myosins and more with the antibody against slow myosin. The reaction for embryonic myosin was most often detected in the C fibers (IC, IIC). Immunohistochemical subtypes were observed for each fiber type in the stimulated muscles. The greatest number was seen in type-IIC fibers, which, in addition to their reaction for fast/neonatal and slow myosins, might also react with the antibodies against neonatal/embryonic and embryonic myosins. These findings indicated that the transforming fibers temporarily expressed myosin heavy chain isoforms normally not detectable in adult skeletal muscle. Myotubes reacted strongly with the antibodies against fast/neonatal and embryonic myosins, and some of them also with the antibody against slow myosin. Thus, it appears that under the influence of the low frequency stimulus pattern some of the newly formed myotubes developed into type-I fibers.     

Receptor editing can lead to allelic inclusion and development of B cells that retain antibodies reacting with high avidity autoantigens

Receptor editing is a major B cell tolerance mechanism that operates by secondary Ig gene rearrangements to change the specificity of autoreactive developing B cells. In the 3-83Igi mouse model, receptor editing operates in every autoreactive anti-H-2K(b) B cell, providing a novel receptor without additional cell loss. Despite the efficiency of receptor editing in generating nonautoreactive Ag receptors, we show in this study that this process does not inactivate the autoantibody-encoding gene(s) in every autoreactive B cell. In fact, receptor editing can generate allelically and isotypically included B cells that simultaneously express the original autoreactive and a novel nonautoreactive Ag receptors. Such dual Ab-expressing B cells differentiate into transitional and mature B cells retaining the expression of the autoantibody despite the high avidity interaction between the autoantibody and the self-Ag in this system. Moreover, we find that these high avidity autoreactive B cells retain the autoreactive Ag receptor within the cell as a consequence of autoantigen engagement and through a Src family kinase-dependent process. Finally, anti-H-2K(b) IgM autoantibodies are found in the sera of older 3-83Igi mice, indicating that dual Ab-expressing autoreactive B cells are potentially functional and capable of differentiating into IgM autoantibody-secreting plasma cells under certain circumstances. These results demonstrate that autoreactive B cells reacting with ubiquitous membrane bound autoantigens can bypass mechanisms of central tolerance by coexpressing nonautoreactive Abs. These dual Ab-expressing autoreactive B cells conceal their autoantibodies within the cell manifesting a superficially tolerant phenotype that can be partially overcome to secrete IgM autoantibodies.     

Pancreatic expression of interferon-gamma protects mice from lethal coxsackievirus B3 infection and subsequent myocarditis

Cardiovascular disease is one of the leading causes of death worldwide, and has been associated with many environmental risk factors. Recent evidence has indicated the involvement of pathogens such as viruses as causative agents, and specifically identified the coxsackievirus B serogroup as the leading culprit. Not only has coxsackievirus B3 (CB3) been identified from patients with cardiovascular disease, but also infection of mice with CB3 strains can reproduce human clinical heart disease in rodents. Several mechanisms have been proposed in an attempt to distinguish between pathology mediated by direct viral destruction of cardiac muscle cells or by the virus-induced immune response directed at infected myocytes or at 'mimicked' epitopes shared between viral and cardiac antigens. To distinguish between these mechanisms, we infected a unique mouse that diminishes the extent of infection and spread of the virus, but allows complete immunity to the virus. Transgenic mice expressing interferon-gamma in their pancreatic beta cells failed to develop CB-3-induced myocarditis. This work challenges the idea of the function of the immune response and 'molecular mimicry' in the CB-3-induced autoimmune myocarditis model, and instead favors the idea of virus-mediated damage. These results emphasize the benefit of reducing the level of viremia early during infection, thereby reducing the incidence of virus-mediated heart damage and autoimmunity.