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Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of the myocardial cell damage involved in viral myocarditis. Using a murine model of acute myocarditis caused by Coxsackievirus B3 (CVB3), perforin-expressing killer cells have been shown to infiltrate the heart, and intercellular adhesion molecular-1 (ICAM-1) together with major histocompatibility complex (MHC) antigen was induced on myocardial cells in acute viral myocarditis. To clarify the immunological mechanisms in more detail, the expression of vascular cell adhesion molecular-1 (VCAM-1) has been examined in the heart of acute myocarditis and on cultured cardiac myocytes treated with interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). The effects of in vivo antibody treatment to VCAM-1 on myocardial damage involved in acute myocarditis were also analysed. CVB3-induced myocarditis resulted in enhanced expression of VCAM-1 on myocardial cells. VCAM-1 expression was also induced on cultured cardiac myocytes by treatment with IFN-gamma and TNF-alpha. The in vivo antibody treatment to VCAM-1 decreased the myocardial damage to some extent, but the effects were not statistically significant. These data suggest that the expression of VCAM-1 on myocardial cells may play at least a partial role in the myocardial damage involved in acute viral myocarditis.

The function of apolipoprotein J (apoJ) is unknown, but it has been hypothesized to be cytoprotective. In the normal heart, abundant apoJ mRNA and protein are expressed in atrial myocytes; no expression is detected in ventricular myocytes. To provide clues about the role of apoJ in the heart, the response of apoJ to heart disease, including three models of myocarditis and two models of in vivo pressure overload hypertrophy, were examined. In the disease model studied extensively, myosin-induced myocarditis, in situ hybridization detected induction of apoJ mRNA in ventricular myocytes immediately before histological evidence of injury. ApoJ message in ventricular myocytes reached high levels as myocarditis became more severe. Evidence of early apoJ induction, before inflammation and injury, also occurred in viral myocarditis. ApoJ mRNA was not present in the inflammatory or interstitial cells during myocarditis. In areas of severe inflammation and myocardial fiber degeneration, apoJ showed a gradient of expression, with highest levels in myocytes immediately surrounding the lesion and diminishing with increasing distance. ApoJ protein also accumulated in myocytes at the interface between degenerated myocardial tissue and the surrounding cardiac tissue. During cardiac hypertrophy that occurred without associated inflammation or cell damage, ventricular apoJ mRNA was not detected. When ischemic damage accompanied hypertrophy, apoJ was induced in the ventricular myocytes near the lesion borders. The correlation of apoJ induction with ventricular tissue damage, but not hypertrophy, suggests that apoJ is a repair response protein. We propose that apoJ functions to limit tissue injury and/or promote tissue remodeling.

Two variants of coxsackievirus B3 have been used to investigate the pathogenesis of myocarditis in BALB/c mice. H3 virus induces moderate myocarditis and H310A1 virus induces minimal myocarditis, although both viruses infect and replicate in the heart. Cells expressing the gamma delta+ T-cell receptor composed 5 to 13% of the lymphocytes infiltrating the hearts of H3 virus-infected mice and belonged to either the CD4- CD8+ gamma delta+- or CD4- CD8- gamma delta+-cell population. Giving 5,000 gamma delta+ cells isolated from the hearts of H3 virus-infected mice to H310A1 virus-infected recipients restored myocarditis susceptibility in the recipient animals and shifted the pattern of cytokine production in the virus-immune CD4+-cell population from being predominantly interleukin-4 producing to being predominantly gamma interferon producing in the H310A1 virus-infected mice. Apoptosis was evident in the infiltrating lymphocyte population in the myocardia of H3 virus-infected mice by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay and in splenic lymphocytes by DNA fragmentation in agarose gel electrophoresis and was confined to the CD4+ population. No apoptosis was observed in H310A1 virus-infected mice, but apoptosis was induced subsequent to gamma delta +-T-cell transfer. These results are consistent with the hypothesis that gamma delta+ T cells may help modulate cytokine responses during virus infections in vivo and that apoptosis might be involved in this modulation.

To establish the existence of predominant right leg involvement in Coxsackievirus B1-induced myositis (CB1 myositis) 189 neonatal CD1 Swiss mice were inoculated with 300 pfu CB1, and regularly observed for posture, mobility, and gait. After 2 and 4 weeks, quantitative comparison of motor dysfunction of right and left leg yielded an asymmetry score; on light microscopy mononuclear cell infiltration and muscle fiber necrosis were quantified in bilateral hamstring muscles, using a five-grade scale (0-4). Motor asymmetries were seen during acute viral myositis as soon as hind leg dysfunction appeared, and animals with a predominant dysfunction of one leg preserved that preference throughout the observation period. At 2 weeks, mice with predominant right leg dysfunction (n = 34) significantly outnumbered those with predominant left leg dysfunction (n = 11) (p = 0.01). At 2 and 4 weeks, infiltration and necrosis in hamstrings from legs with predominant dysfunction were not higher than in those from contralateral legs, and infiltration in right-sided hamstrings was not higher than in left-sided ones, nor was infiltration at 4 weeks. At 4 weeks right-sided muscles were more necrotic (mean +/- SD, 1.8 +/- 1.5) than left-sided muscles (1.1 +/- 1.2; p = 0.03). In the absence of predominant inflammatory disease of the right leg, we interpret the hind leg asymmetry as a preferential use of the left leg, due to left-leggedness, and suggest that in CD1 Swiss mice left-leggedness is associated with increased susceptibility to CB1 myositis.

The pathogenesis of CVB3-induced chronic myocarditis remains unknown. Activated monocytes and macrophages may maintain ongoing inflammation during a persistent CVB3 infection and possibly represent the major mechanism leading to chronic myocarditis. We decided to study the activation status of cells by studying TNF alpha secretion in vitro using whole blood culture in CVB3-induced murine chronic myocarditis. Seven DBA/2 +/+ mice and 18 NMRI nu/nu mice were inoculated intraperitoneally with 5x10(5) pfu of CVB3, and mice were mock-infected. Thirty-one days post-infection, all mice were sacrificed, blood samples were obtained from the heart, and the heart was removed. Enteroviral genomic detection by RT-PCR, virus isolation and histological analysis of heart samples were performed. Heparinized whole blood (25 microliters) was cultured for 4 hr and 24 hr in sterile 96 well-plate containing 225 microliters RPMI in the presence or the absence of activators (LPS + PHA). The TNF alpha levels in the whole blood from mock-infected DBA/2 (n = 4) and NMRI nu/nu mice (n = 5) were not different. A moderate increase of TNF alpha was observed in three out of five DBA/2 mice with negative CVB3 that had no histological abnormalities in myocardium. An increased level of TNF alpha was found in the sole DBA/2 mouse with positive CVB3 detection and chronic myocarditis. An increased level of TNF alpha was found in one out of nine NMRI nu/nu mice with positive CVB3 detection and chronic myocarditis and in one out of seven mice with positive CVB3 detection exempt of lesions in myocardium. In other infected mice, the level of TNF alpha was normal. Enteroviral genome was not detected in the blood from infected mice at 31 days post-infection. The increased TNF alpha level in some mice may be designed for a beneficial inflammatory and immune response, however, an exaggerated release may be associated with an adverse effect. The normal TNF alpha level in whole blood cultures from mice with chronic myocarditis does not exclude enhanced cytokine production at infected loci such as myocardial tissue. This is the first report to use whole blood cultures to study the production of cytokines in virus-induced disease in a small animal model.

Molecular hybridization studies have demonstrated that human enteroviruses, including group B coxsackieviruses (CVB), are detectable not only in endomyocardial biopsies of patients with acute enterovirus myocarditis but also in those with chronic disease. Such infections are observed in some patients with end-stage dilated cardiomyopathy, indicating the possibility of persistent heart muscle infection. Enterovirus persistence in the human heart is supported by the recent discovery in various murine models of enterovirus myocarditis that chronic inflamed heart muscle lesions are consistently associated with enterovirus persistence. Application of in-situ hybridization in a multiorgan study of CVB3-infected immunocompetent mice now reveals that, in addition to the myocardium, spleen and lymph nodes are persistently infected. During acute myocarditis, the majority of infected spleen cells was found to be located within the follicles of spleen and lymph nodes. At later stages of the disease, enteroviral infection was shown to be restricted to cells of the germinal centre in secondary follicles of spleen and lymph nodes. Thus, infected immunocompetent cells may play an important role in dissemination of the virus in the host and maintenance of a non-cardiac viral reservoir.

We used right ventricular papillary muscles to study cellular dysfunction in acute murine coxsackievirus myocarditis. We measured unloaded sarcomere shortening velocity (V0) with laser diffraction (HeNe, lambda = 623.8 nm) 7 days after coxsackievirus infection (M) (n = 7) and after infection + monoclonal antibodies to eliminate T cells (T) (n = 4) and in normals (N) (n = 8). A servomotor rapidly shortened a muscle until slack early in contraction and V0 was measured at the onset of zero force. V0 in N was 4.14 +/- 0.84 microns.s-1 at Sl = 2.08 +/- 0.09 microns, 1.70 +/- 0.33 microns.s-1 at 2.06 +/- 0.08 microns in M and, in preliminary experiments, 4.75 +/- 0.96 microns.s-1 at 2.06 +/- 0.07 microns in T. Resting force and stiffness were normal in M. Ventriculor weights in M and T were the same as N. There was an increase in mononuclear cells in M papillary muscles, but no fibrosis or necrosis. Thus, V0 was markedly reduced in acute viral myocarditis in the absence of tissue disruption or hypertrophy, but not if T cells were absent. Pyrophosphate gel electrophoresis showed a shift from predominantly fast in N to a slow myosin isoform in M. Myosin remodelling and reduced unloaded sarcomere shortening velocity occur early in acute coxsackievirus myocarditis and are dependent on immune responses to the virus, but are not a result of histopathological changes.

The effects of macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF) on murine coxsackievirus B3 myocarditis were investigated. A total of 4 x 10(6) U/kg/day M-CSF and 20 micrograms/kg/day G-CSF were injected subcutaneously every day on day 0 to day 14 starting simultaneously with virus inoculation. Serum interferon-alpha was measured periodically. The survival rate of the M-CSF group was higher than that of the untreated control group (p < 0.05). On days 7 and 14 cardiac disease was significantly lower in the M-CSF group than in the untreated control group. Myocardial virus titers on day 7 in the M-CSF group were lower compared with those of the untreated control group. No significant difference was seen in the survival, cardiac disease, or myocardial virus titers between the G-CSF and the control groups. Monocyte counts on days 7 and 14 in the M-CSF group were increased compared with those in the control group. Serum interferon-alpha titers in the M-CSF and G-CSF groups on day 4 and those in the M-CSF group on day 7 were significantly increased in comparison with those of the untreated control group. We conclude that M-CSF but not G-CSF has the potency to limit myocardial virus titers and to reduce cardiac disease in the acute coxsackievirus B3 myocarditis. This ability is associated with an elevated interferon-alpha. Thus macrophages may play a defensive role in this model.

Granulocyte colony-stimulating factor is a potent activator of mature granulocytes, and subsequently enhances superoxide release. The purpose of this study was to investigate the effects of granulocyte colony-stimulating factor upon murine coxsackievirus B3 myocarditis in relation to free radical-mediated cardiac damage. Two-week-old, male, C3H/He mice were inoculated intraperitoneally with coxsackievirus B3. Granulocyte colony-stimulating factor 20 micrograms.kg-1.day-1, polyethylene glycol-conjugated superoxide dismutase (an enzyme catalyzing the conversion of O2- to H2(O2)) 1 x 10(3) U.kg-1.day-1 and granulocyte colony-stimulating factor 20 micrograms.kg-1.day-1, plus polyethylene glycol-conjugated superoxide dismutase 1 x 10(3) U. kg-1. day-1, were injected subcutaneously daily on days 0 to 14. Treated groups were compared to the infected, untreated group. The survival rate in the polyethylene glycol-conjugated superoxide dismutase group was higher than that of the untreated group on day 14, but on day 7, cardiac pathology was not significantly different among the four groups. On day 14, the scores of cellular infiltration, myocardial necrosis and calcification were lower in the polyethylene glycol-conjugated superoxide dismutase group and in the granulocyte colony-stimulating factor plus polyethylene glycol-conjugated superoxide dismutase group than in the untreated group. The myocardial virus titres on days 7 and 14 did not differ significantly among the four groups. The number of total white blood cell and neutrophil counts were significantly greater in the granulocyte colony-stimulating factor group than in the untreated group on day 7. Taken altogether with the previous reports and present evidence that the administration of granulocyte colony-stimulating factor did not exacerbate coxsackievirus B3 myocarditis, it may be that oxygen-free radicals appeared to be derived not from leukocytes but from other components in this experimental model of myocarditis, whereas the myocardium was inflamed with leukocytes.

Challenge of several murine strains with two highly myocarditic variants of coxsackievirus B3 (CVB3) induced acute and chronic myocarditis, detectable at 21 and 45 days post-inoculation (p.i.). In-situ hybridization of coronal heart sections showing chronic inflammation with a radiolabelled CVB3 probe detected viral genomic RNA at day 7 p.i. but rarely at 21 or 45 days p.i., suggesting few murine heart cells actively replicate virus during chronic myocardial inflammation. Data will be presented that favour an alternative hypothesis, i.e. autoimmune responses to shared epitopes among CVB3 proteins, cardiac myosin and myocardial cell surface proteins (molecular mimicry) can affect the severity of chronic inflammation. Mice inoculated with human cardiac myosin (HM) prior to a CVB3m challenge develop less myocarditis than mice inoculated with virus only, suggesting that antibodies stimulated by HM bind virus, reduce the virus burden and provide protection. Mice inoculated with HM only develop non-neutralizing antibodies against purified CVB3m particles. Several strains of mice inoculated with specific synthetic peptides of HM produce antibodies against CVB3m and/or develop cardiomyopathy. Thus antigen-challenged mice can produce antibodies which cross-react among CVB3m HM or cardiac cells to protect or exacerbate heart disease.

The human enteroviruses, especially the coxsackie B viruses, have been established as aetiologic agents of human inflammatory heart disease, a condition which may lead to dilated cardiomyopathy and heart failure. It is clear from murine models of coxsackievirus B3-induced inflammatory heart disease that not all strains of the virus are cardiovirulent (able to cause disease). Here, we present preliminary data mapping the site in a coxsackievirus B3 genome which determines a cardiovirulent phenotype.

The most extensively studied animal model of coxsackievirus B3 (CVB3)-induced inflammatory heart muscle disease is the murine model. In the acute and chronic phase of the disease, it has been suggested that autoimmune mechanisms play a major role in the pathogenesis of the disease. In this study, C3H mice without functional T- and B-lymphocytes (C3H SCID) were inoculated either with a cardiovirulent (CVB3/20) or a non-cardiovirulent (CVB3/0) strain of coxsackievirus B3. Both viruses caused myocarditis in SCID mice. Furthermore, it could be demonstrated, that CVB3/0 had mutated to a cardiovirulent phenotype, able to cause myocarditis in immunocompetent mice.

In susceptible DBA/2 mice infection with coxsackievirus B3 leads to severe inflammatory and necrotic lesions in the heart. There is a temporal discrepancy of peak concentrations of replicative virus in the heart and maximal cardiac inflammation. Aims of this study were, first, to determine whether haemodynamic changes occur in coxsackievirus B3-induced murine myocarditis and, second, the time frame in which those alterations may be apparent. By puncture of the left ventricle, pressures and the first derivative dp/dt as parameters of left ventricular function could be obtained on several days of the infection. Haematoxylin-eosin stains of cross-sections of the heart showed the course of inflammatory lesions in the heart; a plaque forming assay assessed virus titres in the heart. Cardiac concentrations of replicative virus peaked on day 3, inflammatory lesions in the heart were maximal on day 7. Left ventricular function was almost preserved until day 5 of the infection, then dropped significantly until day 10. The study suggests that either a cumulative virus-mediated destruction of the myofibres or virally triggered immune reactions to heart cells lead to impairment of left ventricular function.

In analyzing the molecular determinants of virulence of coxsackievirus B4, chimeric viruses were constructed from avirulent and virulent viruses. The vCB424 recombinant contained a single nucleotide substitution on an avirulent genetic background, resulting in replacement of Ser-16 of VP4 with Arg-16. Mice infected with vCB424 displayed an intermediate phenotype.

The ability of two coxsackievirus B3 (CBV3) variants to induce myocarditis in BALB/c mice was studied and plaque-forming assay, polymerase chain reaction (PCR), in situ hybridization, and immunohistochemistry were compared for detecting viruses and viral components in the myocardium. The virological findings were related to histopathologic and ultrastructural changes in the myocardium. CBV3-W induced severe myocarditis characterized by massive myocyte necrosis. Widely distributed myocyte damage clearly preceded modest inflammatory infiltrates in the myocardium. In contrast, CBV3-M1 induced mild myocardial injury. Both variants caused fulminant pancreatitis with nearly complete necrosis of the exocrine pancreas. CBV3 RNA was identified by PCR in the myocardium of CBV3-W-infected mice until the end of the follow-up period of 14 days. Moreover, semiquantitative results were obtained when the PCR/hybridization results were analyzed by a phosphor imaging system. Immunohistochemistry and in situ hybridization from formaldehyde-fixed, paraffin-embedded specimens were highly similar in detecting viral components during the early stages of the myocardial injury. The results indicate that: (i) direct viral damage plays an essential role in acute murine CBV3-induced myocarditis, (ii) PCR appears a useful and sensitive diagnostic method in acute myocarditis, and (iii) immunohistochemistry as a specific and relatively rapid method might be practicable also in studying the early stages of acute myocarditis from archival clinical material.

Coxsackievirus infections have previously been shown to cause acute or chronic myocarditis in humans, and several mouse models have been established to study the pathology of this disease. Myocardial injury may result from direct viral effects and/or may be immune mediated. To determine the relative roles of these processes in pathogenesis, we have compared coxsackievirus B3 (CVB3) infections of normal and immuno-compromised transgenic knockout (ko) mice. CVB3 was able to infect all strains used (C57BL/6, CD4ko, and beta-microglobulin ko [beta 2Mko]), and following intraperitoneal injection, two disease processes could be distinguished. First, the virus caused early (3 to 7 days postinfection) death in a viral dose-dependent manner. Immunocompetent C57BL/6 mice were highly susceptible (50% lethal dose = 70 PFU), while immunodeficient transgenic ko mice were less susceptible, showing 10- and 180-fold increases in the 50% lethal dose (for CD4ko and beta 2Mko mice, respectively). Second, a histologic examination of surviving CD4ko mice at 7 days postinfection revealed severe myocarditis; the inflammatory infiltrate comprised 40 to 50% macrophages, 30 to 40% NK cells, and 10 to 20% CD8+ T lymphocytes. The infiltration resolved over the following 2 to 3 weeks, with resultant myocardial fibrosis. In vivo depletion of CD8+ T lymphocytes from these CD4ko mice led to a marked reduction in myocarditis and an increase in myocardial virus titers. beta 2Mko mice, which lack antiviral CD8+ T cells, are much less susceptible to early death and to the development of myocarditis. We conclude that our data support a strong immunopathologic component in CVB3-induced disease and implicate both CD4+ and CD8+ T cells. Compared with immunocompetent animals, (i) mice lacking CD4+ T cells (CD4ko) were more resistant to virus challenge, and (ii) mice lacking CD8+ T cells (beta 2Mko and in vivo-depleted CD4ko) showed enhanced survival and a reduced incidence of the later myocarditis. Nevertheless, the picture is complex, since (iii) removal of the CD4+ component, while protecting against early death, greatly magnified the severity of myocarditis, and (iv) removal of the CD8+ cells from CD4ko mice, although protecting against early death and later myocarditis, led to markedly increased virus titers in the heart. These data underscore the complex balance between the costs and benefits of effective antiviral immune responses.

Objective: Cardiomyopathy is usually associated with marked alterations in myocardial transmembrane signalling. Although acute viral myocarditis may result in chronic cardiomyopathy in some cases, the possible consequences of viral infection on function of the myocardial signal-transducing complex have not been explored. Therefore, the present study was designed to investigate the G-protein-regulated adenylyl cyclase signalling system in murine myocardium during myocarditis induced by coxsackievirus B3 (CVB3) infection., Methods: We examined the functional characteristics of adenylyl cyclase as well as the function and distribution of beta-adrenoceptors, m-cholinoceptors and G-proteins in myocardial plasma membranes isolated from the hearts of mice with acute (7 days pi) or late phase (21 days pi) myocarditis and the obtained results were compared with the corresponding data determined in age-matched controls., Results: While the basal adenylyl cyclase activity was not significantly altered, the ability of forskolin, sodium fluoride and GTP gamma S to activate adenylyl cyclase was lowered by about 20% in samples from virus-infected animals. The level of Gs alpha in myocardial plasma membranes as well as the functional activity of Gs alpha was not affected by viral infection, but the Gi alpha content was increased by about 20%. The total number of beta-adrenoceptors in myocardial plasma membranes increased by about 12-15% due to higher content of the beta 2-adrenoceptor subtype. Although the agonist-binding parameters of beta-adrenoceptors were not significantly altered, the ability of these receptors to mediate stimulation of adenylate cyclase was markedly diminished (by 56-80%). The total number of m-cholinoceptors in samples derived from virus-infected mice increased considerably (by 29-59%) and a significant proportion of the receptors shifted to a higher affinity status, but their ability to transduce agonist signals was impaired., Conclusions: These data are the first to demonstrate that several different sites of the myocardial G-protein-regulated adenylyl cyclase signalling complex are significantly altered in acute as well as in late phase of CVB3-induced myocarditis.

Background: The treatment of some inflammatory diseases (eg, Kawasaki disease) with immunoglobulin has been demonstrated to be effective. Accordingly, to elucidate the mechanism underlying such actions of immunoglobulin, we examined its effects on murine coxsackievirus B3 (CB3) myocarditis., Methods and Results: An in vitro study showed dose-dependent suppression of CB3 by immunoglobulin. Immunoglobulin 1 g.kg-1.d-1 IP was administered to CB3-infected C3H/He mice daily for 2 weeks, beginning simultaneously with virus inoculation in experiment 1 and on day 14 after virus inoculation in experiment 2. In both experiments, survival was higher in treated than in control mice; at the time of death, inflammatory also were reduced. Notably, in experiment 1, immunoglobulin administration completely suppressed the development of myocarditis. Serum-neutralizing antibody titers in the treated mice were significantly higher than those in untreated mice in experiment 1 but not in experiment 2. The circulating antibodies of the treated mice were primarily of exogenous origin in experiment 1 and of exogenous and endogenous origins in experiment 2. The analysis of splenic lymphocyte subsets revealed a marked decrease of the B cell population in the treated mice., Conclusions: Immunoglobulin therapy completely suppressed acute CB3 myocarditis by transferring the neutralizing antibody into the host in the acute viremic stage and induced an anti-inflammatory effect in the subsequent aviremic stage; the reduction of the splenic B-cell population may be closely associated with an anti-inflammatory effect.

Male CD-1 mice were challenged with a diabetogenic strain (E2) of coxsackievirus B4 (CVB4). At 7 weeks, 6 months, and 1 year after inoculation, mean histopathologic scores, postprandial blood glucose levels, and serum levels of antibody to islet cells were significantly elevated and mean fasting serum insulin levels significantly reduced in infected mice versus uninfected controls (P < .001 for each). At 7 weeks after infection, viral RNA, but not protein or infectious virus, was demonstrated in the pancreases of most infected mice. The pancreases of 4 of 12 and 0 of 10 infected animals were positive for viral RNA at 6 months and at 1 year after infection, respectively. Interferon-gamma-stimulated peritoneal macrophages were cytotoxic to islet cells with and without sera with high titers of islet cell autoantibody (ICA). Thus, islet cell destruction in mice infected with CVB4 strain E2 is associated with chronic islet cell inflammation, elevation of islet cell antibody, and prolonged presence of viral RNA in the pancreas. Stimulated peritoneal macrophages lyse islet cells directly and by an antibody-dependent mechanism.

An epidemic of exanthematic illness in a day care center is described. Ten children aged 7 to 13 months were affected by the illness. The exanthem was characterized by nonconfluent macular or maculopapular lesions that appeared on the face, body and limbs. Fifty percent of the infected children had fever of up to 39 degrees C at the beginning of the disease. Coxsackievirus B3 (CB3) was isolated from the stool of one ill child. Paired serum samples were obtained from eight ill children and six of them presented seroconversion to CB3. Antibodies to CB3 were detected at titers higher than 16 in a single serum sample collected from the other two patients. Neutralizing antibodies to CB3 were detected in 71.0% of the contact children.

Objective: The aim was to test the therapeutic efficacy of naloxone, an opiate receptor antagonist, upon murine coxsackievirus B3 myocarditis with the analysis of neurohumoral kinetics., Methods: Two week old C3H/He mice were inoculated with 10(3) plaque forming units of coxsackie B3 virus. Naloxone, 1 mg.kg-1.d-1, was given intraperitoneally daily on days 0-14 in experiment I, and on days 14-28 in experiment II. The treated groups were compared to infected controls in each experiment. For the analysis of the endogenous opiate and neurohumoral system, plasma beta endorphin and catecholamines were measured., Results: In experiment I, survival rate did not differ significantly between naloxone treated and untreated groups (11/15 v 8/15, p = NS). Pathological scores (infiltration and necrosis), myocardial virus titres, and plasma beta endorphin and catecholamine concentrations did not differ significantly between the two groups. In experiment II, survival rate (13/16 v 6/14, P < 0.05) was higher and cardiac pathology was less severe, with a lower incidence of congestive heart failure, in naloxone than in controls groups. In addition, beta endorphin and noradrenaline were significantly increased in the naloxone group compared to the control., Conclusions: The endogenous opiate system is activated in congestive heart failure caused by severe myocardial damage in murine coxsackie B3 myocarditis, where an associated limitation of the sympathetic system may be present. Naloxone is beneficial in congestive heart failure caused by coxsackie B3 myocarditis because of its neurohumoral modulating effect.

We investigated the role of humoral factors in the pathogenesis of Coxsackie B1 virus-induced murine myositis (CB1-myositis). At 2, 4 and 8 weeks after inoculation, serum was studied for circulating immune complexes (CIC) (Raji-cell assay), haemolytic complement activity (CH50 titre) and anticytoplasmic autoantibodies (Western blotting, immunoprecipitation) in relation to degree of mononuclear cell infiltration and muscle fibre necrosis. At 2, 4 and 8 weeks, cell infiltration correlated positively to muscle fibre necrosis. From 2 weeks on, moderate quantities of CIC were found in nearly all CB1-inoculated mice, but without correlation to histological changes. Except for a positive correlation of CH50 titre to muscle necrosis at 4 weeks (r = 0.60; P = 0.02), CH50 titres did not correlate to muscle lesions. Anticytoplasmic and other known autoantibody specificities were absent. In conclusion, first, in CB1-myositis CIC occurred from 2 weeks on, but no correlative evidence was found for their involvement in pathogenesis, neither for that of complement nor for anticytoplasmic autoantibodies. Secondly, cell infiltration correlated positively to muscle necrosis, underscoring the importance of cellular mechanisms. Thus, our data do not support, or conclusively exclude, a role for humoral processes in CB1-myositis.

Coxsackievirus B3 (CVB3)-induced myocarditis was studied in euthymic (nu/+) and athymic (nu/nu) C3H/HeN (H-2k) mice. Mice were inoculated intraperitoneally with 10(6) p.f.u. of CVB3 (Nancy strain) and sacrificed at intervals up to 92 days post-inoculation (p.i.). Viraemia peaked at day 2 to 3 p.i. and ceased at day 5 to 7 p.i. in a synchronized manner in both sets of mice. Very few infectious particles were detected in the blood of nu/nu mice after day 14 p.i. In nu/nu mice, CVB3 persisted in myocardial tissue with constant titres between 2.7 +/- 1.9 x 10(4) and 7.6 +/- 5.2 x 10(4) p.f.u./mg from day 3 to 92 p.i., which were comparable to those of nu/+ mice in the acute phase. In nu/+ mice, the virus was recovered from all animals examined by day 11 p.i. and from three out of 13 mice between days 14 and 21 p.i., yet no virus was recovered from nu/+ mice at day 42 p.i. In nu/nu mice, sense and antisense RNA for CVB3 was detected in the myocardial tissue up to day 42 p.i. by in situ hybridization and up to day 92 p.i. by reverse transcriptase-PCR. Neither sense nor antisense RNA was detected after day 21 p.i. in nu/+ mice with the same techniques. Myocardial tissue damage was analysed morphologically. At day 92 p.i., the area of myocardial injury peaked at 23% of the section in nu/nu mice. In contrast, less than 0.6% of tissue sections contained lesions in nu/+ mice. A neutralizing antibody response to CVB3 was observed in both nu/nu and nu/+ mice. The mean titre of neutralizing antibody was significantly higher at day 21 p.i. in nu/+ mice, but similar at day 42 p.i. with nu/nu and nu/+ mice. Perforin-producing natural killer-like cells, which are considered to play an important role in causing acute myocarditic lesions in immunocompetent mice, were found in the lesions of nu/nu mice persistently infected with CVB3. Prolonged tumour necrosis factor-alpha mRNA synthesis detected in nu/nu mice appears to reflect the continuous activation of macrophages, which extend phagocytic reactions to virus-infected myocytes. These immunological results suggested that the host immune response devoid of antigen-specific T cell function is not sufficient to terminate CVB3 infection in nu/nu mice. Also, it appears that competent cellular immunity, on the whole, plays a role in curing rather than in aggravating myocarditis in nu+mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Using Balb/c mice infected with Coxsackie virus B3 (CVB3) as a model, the effect of Astragalus membranaceus (AM) on CVB3-RNA has been observed in myocardial tissues of mice by RNA-RNA in situ hybridization with negative-strand RNA probes leballing with 35S and quantitative imaging analysis of positive hybridization signals. The mechanism of its effect on CVB3-RNA has also been investigated by induction with AM and detection of beta-interferon (beta-IFN). Results showed that the copy numbers of CVB3-RNA as well as the histologic necrotic sizes in myocardial tissues of AM treated infected mice were significantly smaller than that in infected normal saline treated mice (P < 0.01, P < 0.05) respectively, suggesting that AM could inhibit the replication of CVB3-RNA, but its effect on CVB3-RNA was not correlated with induction of beta-IFN.

We developed a mouse myocardial preparation to study cellular dysfunction in acute coxsackievirus myocarditis. Thin right ventricular papillary muscles from normal mice (n = 8) were compared with muscles from mice 7 days after coxsackievirus infection (n = 7). Sarcomere shortening was studied with laser diffraction (HeNe, lambda = 623.8 nm). A servomotor was used to shorten a muscle until slack early in isometric contraction. Unloaded sarcomere shortening velocity (Vo) was measured at the start of zero force at slack length. Vo was independent of the extent of slack release and was the same as that estimated with an isotonic force-sarcomere shortening velocity relation. Resting muscle stiffness was calculated from shortening perturbations in resting muscles. The histology of some papillary muscles (normal, n = 4; infected, n = 3) was studied. There was no ventricular hypertrophy. Resting sarcomere length (SL) in infected preparations (2.11 +/- 0.07 micron) (mean +/- 1 SD) was the same as in normal preparations (2.11 +/- 0.08 micron). In isometric twitches in normal and infected muscles, total peak force (4.31 +/- 1.07 and 3.77 +/- 1.86 g/mm2, respectively) resting force (0.81 +/- 0.37 and 0.81 +/- 0.35 g/mm2, respectively), and time to peak force (129.5 +/- 20.3 and 125.2 +/- 13.0 milliseconds, respectively) were not significantly different. Vo was 4.14 +/- 0.84 micron/s in normal muscles at an SL of 2.08 +/- 0.09 micron and 1.70 +/- 0.33 micron/s in infected muscles at an SL of 2.06 +/- 0.08 micron. Resting stiffness was the same for normal and infected muscles. There was inflammation but no fibrosis or necrosis. Thus, Vo was depressed early in acute viral myocarditis without hypertrophy, myocyte necrosis, fibrosis, or altered resting stiffness. Pyrophosphate gel electrophoresis showed a shift from predominantly fast to slow myosin isoforms. Apparently, there is remodeling of the contractile apparatus early in acute coxsackievirus myocarditis that is caused either by the direct effects of the virus or the immune response.

Coxsackievirus B3 (CVB3/20)-induced myocarditic lesions occurred more quickly and were more severe and virus titers in heart and liver were higher in selenium (Se)-deficient than Se-adequate mice. NK cell activity and serum neutralizing antibody titers were similar in both Se-adequate and -deficient CVB3/20-infected mice; however, lymphocyte proliferation to both mitogen and antigen was decreased in Se-deficient mice. CVB3/20 isolated from Se-deficient donor mice and inoculated into Se-adequate recipient mice induced severe myocarditis. In contrast, CVB3/20 isolated from Se-adequate donor mice and inoculated into Se-adequate recipient mice induced only moderate myocarditis, similar to that caused by the original virus stock. Thus, the general population of CVB3/20 virions, as a consequence of replicating in an Se-deficient host, underwent a phenotypic change to increased virulence. These results have important implications for the emergence of virulent viruses.

Coxsackievirus B (CB) 4 causes transmural myocarditis in suckling mice with ensuing development of focal ventricular thinning or aneurysms. We studied whether subsequent infection with another cardiotropic virus influences the expression of CB4 disease. CB4 infection was established in 2-day-old CD1 mice by intraperitoneal (IP) inoculation. Three weeks later, surviving animals were randomized to receive CB3 or saline IP. They were then killed over a 45-day period. CB4 neutralizing antibody (NA) titres were comparable in both groups (31 +/- 23 vs 37 +/- 19). CB3 NA were detected in CB3 infected animals only (72 +/- 86 versus 0). The incidence of myocarditis was comparable (67.4% vs 55.2%). The indices of histopathological changes (assessed according to a semiquantitative grading scale from 0-4) were greater among CB3 recipients on day 9 post CB3 challenge (1.38 +/- 0.43 vs 0.46 +/- 0.4, P < 0.001) and to a lesser extent, on day 13 (0.56 +/- 0.56 vs 0.19 +/- 0.38, P > 0.1). On days 30, and 45, these indices became similar in both groups. Focal thinning was noted on days 45 in 6/11 animals with CB4 infection alone and in 0/11 mice with subsequent CB3 infection (P = 0.006). These findings show that CB3 myocarditis can be expressed in mice with prior CB4 disease, that sequential infections do not lead to cumulative cardiac injury, and that subsequent CB3 infection suppresses the formation of CB4 induced ventricular aneurysms.

The studies of the therapeutic and prophylactic action of a compound from the group of nonsteroid antiinflammatory substances, iodantipyrine-4, in newborn white mice infected with Coxsackie B3 virus showed the drug to possess both therapeutic and prophylactic activity in the acute period of infection.

In order to address the question whether the virus or immune reactions to the heart induce cardiac damage in the murine model of coxsackievirus B3 induced myocarditis, calcium-resistant cardiac myocytes of a myocarditis susceptible strain of mice were isolated and exposed to myocarditic coxsackievirus B3. The tetrazolium salt MTT was used to visualize the effect of the virus upon cell viability by inverse light microscopy and by an EA test. Coxsackievirus B3 infected isolated adult cardiac myocytes as well as infected cultured cardiac fibroblasts were examined. In vitro the virus killed the myocytes within 16 h of infection whereas the fibroblasts survived the infection. Since coxsackievirus B3 is able to kill cardiac myocytes by itself, immunosuppressive treatment of acute coxsackievirus B3 induced myocarditis may be harmful by eliminating host immune defence mechanisms and, therefore, may lead to an enhanced viral spread and virus induced myocyte necrosis in the heart.

Background: It has been shown that the development of coxsackievirus B3 (CB3) myocarditis is regulated by T cells and not by B cells. Interleukin-2 (IL-2) is a T-cell-derived cytokine that stimulates the growth of T cells. This study was carried out to determine the effects of IL-2 on CB3-infected BALB/c mice., Methods and Results: In two separate experiments, recombinant human IL-2 (5 x 10(4) U) was administered subcutaneously to 30 mice early (days 0 to 7) and 30 mice late (days 7 to 14) after infection with CB3. Each experiment had a control group of infected animals that did not receive IL-2. On days 7 and 10, splenic natural killer (NK) cell activity determined by 51Cr release assay and the distribution of myocardial lymphocyte subsets were compared in the treated and untreated groups. In the early treatment experiment, survival at 7 days was higher in treated compared with control animals, myocardial virus titers were lower, inflammatory cell infiltration was less (as was the severity of necrosis at the time the mice were killed), and NK cell activity was higher. However, in the late treatment experiment, survival at 14 days was lower in treated compared with control animals, and there was more infiltration, more severe necrosis, and more T-cell infiltration, but the NK cell activity did not differ significantly. In a third experiment similar to the late experiment described above but involving infected athymic nude mice, we confirmed the lack of effect of late in vivo administration of IL-2 on outcome., Conclusions: IL-2 has the capacity to limit CB3 myocarditis by enhancing NK cell activity in the acute viremic stage, resulting in a reduction of cardiac pathology. However, in the subacute aviremic stage, in contrast, IL-2 exacerbates the course and severity of the disease by increasing the number of T cells infiltrating the myocardium. That is, IL-2 has differential effects on acute CB3 myocarditis. IL-2 is beneficial if treatment is given early but later in murine CB3 myocarditis.

A transfection-reactivated Coxsackievirus B3 (rCVB3), from a full-length cDNA clone of Nancy strain, has previously been shown to be as cardiovirulent as the wild-type virus. Myocarditis induced by this genetically defined virus was compared in SWR mice with the traditional Balb/c model. SWR mice inoculated with rCVB3 developed more severe myocarditis but less severe pancreatitis than Balb/c mice. In contrast to the poor general health and frequent mortality of Balb/c mice following CVB3 infection, the body weight of SWR mice was not affected by CVB3 inoculation and no mortality occurred at titres of 10(2)-10(7) plaque forming units (PFU). Typical myocarditis developed in SWR mice 7 days post infection. Myocarditic foci consisting of necrotic myocardial fibres and mononuclear cell infiltrates resolved by day 30, similar to that observed in Balb/c. However, SWR mice were more sensitive to rCVB3-induced myocarditis than were Balb/c mice: mild myocarditis was induced (4/4) by as low as 10(2) PFU of the virus (ID50 < 10(1.5) PFU), and more severe myocarditis was seen at higher PFU of virus in a dose-dependent manner. The SWR model was tested with attenuated variants derived from cardiovirulent rCVB3. The ID50 for myocarditis was 10(7) PFU for a large plaque-size attenuant and 10(6) PFU for a minute plaque-size attenuant, indicating loss of cardiovirulence by a factor of more than 10(4)-10(5). rCVB3-induced SWR mouse is a sensitive and reliable model for myocarditis. It is useful in assessing the cardiovirulence of different CVB3 variants and evaluating the efficacies of anti-viral therapies. It will allow follow-up study after high dose infection with cardiovirulent rCVB3.

Feeding a vitamin E-deficient diet increases pathology in hearts of mice infected with a myocarditic coxsackievirus B3 (CVB3/20). Hearts from infected mice fed a vitamin E-deficient diet rich in highly unsaturated fat (menhaden oil) exhibited more severe pathology than hearts from infected mice fed a vitamin E-deficient diet based largely on saturated fat (lard). Furthermore, a cloned and sequenced amyocarditic coxsackievirus B3 (CVB3/0), which caused little or no pathology in the hearts of vitamin E-supplemented mice, induced extensive cardiac pathology in vitamin E-deficient mice. In infected mice, both mitogen and antigen responses were depressed by vitamin E deficiency, although neutralizing antibody responses were unaffected. Natural killer cell responses were comparable in infected mice fed a lard-based diet with or without supplemented vitamin E. However, a menhaden oil-based diet, whether supplemented with vitamin E or not, significantly depressed natural killer cell activity in infected mice compared with mice fed the lard-based diet. Coxsackievirus B3/0 recovered from the heart of a vitamin E-deficient donor mouse, passaged one time onto HeLa cells, caused significant heart damage when passed back into vitamin E-supplemented recipient mice, demonstrating that the amyocarditic CVB3/0 had changed to a virulent phenotype. Enhanced virulence was also seen with CVB3/20 virus similarly passaged in a vitamin E-deficient donor. Our work demonstrates the important role of host nutritional antioxidant status in determining the severity of certain viral infections.

Two variants of coxsackievirus B3 (CVB3) which differ dramatically in the ability to induce myocarditis in BALB/c mice were studied. H3 virus infection of murine monocytes in vitro resulted in release of concentrations of interleukin 1 (IL-1) and alpha/beta interferon that were high compared with those of cells infected with the H310A1 virus variant. In vivo, H3 virus infection caused substantial inflammatory cell infiltration of the myocardium, and lymphocytes from these animals gave predominantly Th1-cell responses to either whole H3 virus or overlapping peptides of the CVB3 vp1 capsid protein, as determined by IL-2 production. In contrast, H310A1 virus infection produced minimal myocarditis and Th1-cell responses, but Th2-cell activation was more pronounced than in H3 virus-infected mice (as determined by IL-4 concentrations). Exogenous treatment of H310A1 virus-infected mice with either IL-1 or IL-2 restored both myocarditis susceptibility and Th1-cell responses to whole virus and vp1 peptides. Furthermore, H310A1 virus-infected mice given exogenous IL-1 showed substantial in situ IL-2 deposition in the myocardium. These results indicate that CVB3-induced myocarditis may depend upon release of specific cytokines during infection and that activation of Th1 cells may be an important factor in pathogenesis.

Inoculation of Coxsackie B1 virus (CB1) in newborn CD1 Swiss mice induces a chronic myositis of proximal hindlimb muscles (CB1 myositis). To study the possible role of the virus dose, and of antiviral antibodies in the development of CB1 myositis, we infected groups of newborn mice with six CB1 doses, ranging from 30 to 10,000 plaque forming units (pfu); after 4 and 8 weeks we determined morbidity and antiviral antibody titer, and quantified histopathological changes. At 4 weeks, morbidity and mononuclear cell infiltration differed significantly for the various groups, with the most prominent changes in 300 pfu animals. At 4 and 8 weeks diseased animals had significantly higher antibody titers than clinically unaffected animals, and at 4 weeks myopathic and infiltrative changes correlated positively with the serum antibody titer. Our data indicate that the virus dose plays a pathogenic role in CB1 myositis, and they suggest further study on the role of humoral immune mechanisms in the early phase of CB1 myositis.

The persistence of coxsackievirus B1 in the muscles of mice with coxsackievirus B1-induced chronic myositis was investigated. Neonatal CD1 Swiss mice were inoculated with a myositis-causing variant of coxsackievirus B1 (Tucson strain). Hamstring muscle samples of diseased mice obtained at various times after inoculation were examined for the presence of infectious virus, viral RNA and histological abnormalities. Viral RNA was detected up to 4 weeks after initiation of infection, whereas virus could be isolated from hamstring muscles for up to 2 weeks. Thereafter no sign of infection was demonstrated although histological abnormalities remained present for the entire observation period of 16 weeks. That viral RNA was detectable for only 2 weeks after tissues became negative for infectious virus suggests that the infection slowly waned rather than the viral RNA persisting. Hence, it is concluded that coxsackievirus B1 plays an essential role in the initiation of myositis but not in the maintenance of the chronic phase.

Cardiotropic virus infection of mice typically produces florid myocardial inflammation and, in certain strains, elicits a chronic postinfectious autoimmune myocarditis. In humans, myocarditis may progress into cardiomyopathy manifested by interstitial fibrosis and myocyte hypertrophy in the absence of myocardial inflammation. Because of their pleiotropic effects, cytokines may provide a common link between connective tissue changes, myocardial injury, and cardiac autoimmunity. To explore such possibilities, histopathological and serological studies were performed on virus-infected mice which were or were not subject to cytokine manipulation. Mice infected with low doses of encephalomyocarditis virus exhibited little myocardial inflammation although myocyte hypertrophy and reactive fibrosis were common. Profound ultrastructural changes in the interstitium were observed through much of the noninflamed myocardium. B10.A mice which are resistant to the development of postinfectious autoimmune myocarditis developed severe autoimmune myocarditis when infected with Coxsackie virus B3 (CB3) and treated with bacterial lipopolysaccharide (LPS), tumor necrosis factor (TNF), or interleukin 1 (IL-1). B10.A mice given CB3, LPS, TNF, or IL-1 alone did not develop myocarditis and there was no evidence of autoimmune recognition of the heart. When CB3-infected A/J mice, which typically develop autoimmune sequelae to the infection, were treated with an IL-1 receptor antagonist, myocardial injury was diminished and heart antibody activity was reduced. These results suggest that TNF, IL-1, and possibly other cytokines contribute to myocardial pathogenesis and the induction of heart-specific autoimmunity. Further studies may lead to the development of cytokine-based therapeutic approaches to treating myocarditis and cardiomyopathy.

To identify the molecular determinants of virulence for coxsackievirus B4, a panel of recombinant, chimeric viruses were constructed from cDNA clones of a virulent virus, CB4-V, and a nonvirulent virus, CB4-P. Initial studies mapped a major determinant of virulence to the 5' end of the viral genome, which contained the 5' untranslated and the P1 regions (A. Ramsingh, A. Hixson, B. Duceman, and J. Slack, J. Virol. 64:3078-3081, 1990). To determine whether the 5' untranslated region contributed to the virulent phenotype, a chimeric virus (vCB403) containing this region of the virulent virus on an avirulent background was tested in mice. The vCB403 construct displayed a phenotype similar to that of CB4-P, suggesting that the 5' untranslated region did not significantly contribute to virulence. Analysis of the sequence data of the P1 regions of both CB4-V and CB4-P revealed five mutations that resulted in amino acid substitutions in VP1, VP2, and VP4 (A. Ramsingh, H. Araki, S. Bryant, and A. Hixson, Virus Res. 23:281-292, 1992). Analysis of individual mutations in both VP1 and VP2 revealed that a single residue (Thr-129 of VP1) determined the virulent phenotype.

Objective: To assess the expression of coxsackievirus B3 (CB3) myocarditis in mice with pre-existing CB4 myocardial disease., Design: Double blind comparative study of CB3 myocarditis in CD1 mice with or without prior CB4 induced cardiac damage., Interventions: Antecedent myocardial injury was produced by CB4 infection intraperitoneally at age two days. Two to three weeks later, when CB4 myocarditis was established, infected and control animals were inoculated intraperitoneally with CB3. They were then sacrificed over a 45-day period. Virus and neutralizing antibody titres were measured on days 3 and 13 after CB3 infection, respectively. The incidence of myocarditis and the intensity of histopathological changes (assessed according to a semiquantitative grading scale from 0 to 4) over a 45-day period were compared., Main Results: Among animals with prior CB4 disease, CB3 titres were lower (2.3 +/- 1.7 versus 3.6 +/- 0.8, tissue culture infective dose 50, P = 0.05) and neutralizing antibody response was slightly higher. The incidence of myocarditis was diminished (59.1 versus 89.3%, P = 0.01) and the indices of pathological changes were lower but the differences were not significant (0.68 +/- .54 versus 1.10 +/- 0.20, 1.38 +/- 0.43 versus 1.50 +/- 0.25, 0.56 +/- 0.56 versus 1.26 +/- 0.75, 0.38 +/- 0.58 versus 1.30 +/- 0.78, 0.12 +/- 0.28 versus 0.47 +/- 0.2 on days 3, 9, 13, 30 and 45 post infection, respectively, P > 0.1)., Conclusion: These results demonstrate that prior exposure to CB4 offers some protection from subsequent CB3 infection. Moreover, they show that antecedent CB4 myocardial damage does not predispose to a worsened expression of CB3 myocarditis.

Objective: The aim was to test the role of oxygen derived free radicals in the development of myocarditis. This involved investigating the effects of polyethylene glycol conjugated superoxide dismutase (PEG-SOD, an enzyme catalysing the conversion of O2.- to H2O2) and polyethylene glycol conjugated catalase (PEG-catalase, accelerating the reaction of H2O2 to H2O and O2) upon coxsackievirus B3 (CB3) myocarditis., Methods: Two week old male C3H/He mice were inoculated intraperitoneally with 10(3) plaque forming units of CB3. PEG-SOD, 1 x 10(3) U.kg-1 x d-1, and PEG-SOD, 1 x 10(3) U.kg-1 x d-1, plus PEG-catalase, 1 x 10(3) U.kg-1 x d-1, were injected subcutaneously daily on days 0 to 14. Treated groups were compared to the infected control., Results: On day 7, there were no significant differences in pathological scores among the three groups. On day 14, the cellular infiltration, myocardial necrosis, and calcification scores were significantly lower in the PEG-SOD group and the PEG-SOD plus PEG-catalase group than in the control. There were no significant differences in pathological scores between the PEG-SOD group and the PEG-SOD plus PEG-catalase group. There were no differences in the myocardial virus titres on day 7 among the three groups. On day 14, virus was not detected from the myocardium in any of the three groups., Conclusions: The results suggest that superoxide anion is mostly responsible for myocyte injury in CB3 myocarditis in mice, and that hydrogen peroxide formed as a result of dismutation of superoxide anion may not play a significant role in the development of myocarditis. Superoxide anion is one of the most important factors in free radical mediated injury in CB3 myocarditis in mice and the administration of PEG-SOD alone has therapeutic potential in clinical CB3 myocarditis.

We have recently demonstrated that killer cells expressing a cytolytic factor, perforin, infiltrate the hearts of mice with acute viral myocarditis and may play an important role in the mechanism of myocardial damage. To clarify the mechanism of in vivo cardiac myocyte injury mediated by perforin, we investigated the release of perforin molecules from killer cells by immunoelectron microscopy and examined the circular lesions formed by perforin on the membrane of cardiac myocytes. We found that there was massive release of perforin molecules from the killer cells directly onto the surface of the cardiac myocytes. Furthermore, electron microscopy of ultrathin ventricular sections treated with trypsin revealed numerous circular lesions with the characteristics of perforin pores, in the membranes of cardiac myocytes. These findings provide the first direct evidence that killer cells injure cardiac myocytes by releasing perforin and may play a critical role in the myocardial damage occurring in acute viral myocarditis.

Interstitial oedema in chronic inflammation and ischaemia is related to an accumulation of hyaluronan (hyaluronic acid; HA) in the interstitium. As interstitial oedema will affect the oxygen transport in the interstitium we have evaluated whether accumulation of HA is related to signs of myocardial energy depletion in virus induced myocarditis. Myocarditis was induced in Balb/c mice by inoculation of Coxsackie B3 virus. The extractable HA content of the myocardium increased progressively from a baseline value of 153 +/- 26 micrograms g-1 dry weight to a maximum of 286 +/- 78 micrograms g-1 dry weight at day 7, whereafter there was a slight decline. Affinity histochemistry visualized HA in the endo-perimysium in healthy myocardium. At day 5 after Coxsackie B3 inoculation there was a general widening of the endomysium, which exhibited a positive staining for HA. In conjunction with focal inflammatory infiltrates the staining for HA was even more pronounced. The energy rich adenylates were reversibly affected by the Coxsackie B3 infection. There was a slight, but significant decline in EC from 0.74 +/- 0.05 in the control group to a minimum value of 0.64 +/- 0.10 at day 5, whereafter a restitution was observed at days 7 and 10. The total adenine nucleotide pool was similarly decreased from 27.8 +/- 2.9 mumol g-1 dry weight in controls to 24.6 +/- 2.1 micrograms g-1 dry weight at day 5, and normalized at days 7 and 10. The data suggest that virus induced myocarditis is associated with a local accumulation of HA in the myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of cardiac muscle damage in virus-induced myocarditis remains to be fully understood. In particular, a direct role for viral replication has been obscured by evidence of immune-mediated injury. The combined use of severe combined immunodeficient (scid) and doubly mutant scid/beige mice of different genetic backgrounds has provided a unique opportunity to study the consequences of viral proliferation apart from the influence of immunologic responses. In these immunodeficient animals, inoculation with coxsackievirus B3 caused severe myocardial injury, indicating a profound and direct pathogenic potential of viral infection. These studies, in turn, serve to illustrate the value of the scid mutation in experimental biology, as defined among different genetic backgrounds and combined with other mutations.

Objective: The aim was to test the efficacy of the immune system modulator lobenzarit disodium in the treatment of coxsackievirus B3 myocarditis., Methods: Two week old C3H/He mice were inoculated with 10(3) plaque forming units of coxsackievirus B3. Lobenzarit disodium, 25 mg.kg-1.d-1, was given subcutaneously daily on days 0-14 (experiment I; group 2) and days 14-28 (experiment II; group 4). Both treated groups were compared to infected controls for each experiment (groups 1 and 3). For the analysis of splenic lymphocyte subsets, additional mice in untreated and treated groups were killed on d 7, and the percentages of Thy 1.2 (CD3), L3T4 (CD4), Ly 2 (CD8) subsets were analysed by laser flow cytometry (experiment III)., Results: In experiment I, the survival rate in the lobenzarit treated group was significantly lower than in the controls (2/11 v 8/11). Cellular infiltration and myocardial necrosis in the lobenzarit group were more severe. Myocardial virus titres and serum neutralising antibody titres did not differ significantly between the two groups. In experiment II, the survival rate (7/9 v 13/13) and cardiac pathology between the two groups did not differ significantly. In experiment III, the percentage of the Thy 1.2 subset (CD3) in the treated group was significantly lower (p < 0.05) than in the control group, at 36.0(SD 2.9)% v 42.8(5.8)%., Conclusions: Lobenzarit disodium decreased splenic pan T cells and aggravated both clinical course and cardiac pathology in acute murine coxsackievirus B3 myocarditis.

Electrophysiological action of right ventricular myocardium examined by standard intracellular microelectrode technique and real-time microcomputer data processor system and histological and ultrastructural changes of myocardium in BALB/c mice infected with coxsackie B-3 virus from 3 days to 9 months were observed. It was found that electrophysiologic parameters of action potential changed very quickly at the early stage (3 days to 1 month) of the disease. Those abnormalities became most apparent by the 5-30th day, and 7 patterns of abnormal action potential occurred frequently within the same period. These changes were basically parallel to the myocardial lesions. At the late stage (3-9 months) the electrophysiological parameters were nearly normal, while the myocardial lesions decreased gradually. However, the abnormal patterns of action potential were still detected, even though they were improved gradually. The results suggest that myocardial damages caused by viral infection may lead to changes of cardiac electric action, which may be one of the factors in arrhythmias in the episode of viral myocarditis.