Coxsackievirus B3 Myocarditis Research Articles

Generation of an infectious cDNA of a highly cardiovirulent coxsackievirus B3(CVB3 m) and comparison to other infectious CVB3 cDNAs

An infectious cDNA of a highly myocarditic coxsackievirus B3 (CVB3 m; Nancy strain) was cloned. Sequence data revealed 43 extra non-viral nucleotides upstream of the initial 5′ sequence. However, the authentic 5′ end sequence was maintained during replication of viral RNA transfected into HeLa cells, suggesting the RNA synthesizing complex edits the picornaviral 5′ terminus sequence. Nucleotide sequences of the 5′ nontranslated region and the capsid protein gene sequence of CVB3 m were compared with the published sequences of five other CVB3 Nancy strains and two main lineages were found. In comparative assays for cardiovirulence, three of four CVB3 tested were cardiovirulent in adolescent male CD-1 mice. Only one of the three available CVB3 strains was neutralized with several anti-CVB3 m monoclonal antibodies, suggesting that mutations in the surface epitopes of the capsid polypeptides contribute to antigenic drift within the serotype, perhaps in part through immunoselective pressures. Thus, phenotypic diversity of CVB3 within the prototype Nancy strain is an example of RNA viruses adapting to changing environments (cells, mice and humans) through mutations and selective pressure.

Experimental CVB3-induced chronic myocarditis in two murine strains: evidence of interrelationships between virus replication and myocardial damage in persistent cardiac infection.

In order to analyse the relationships between enteroviral replication and the myocardial damage at the onset of chronic cardiac infection, 2 mouse strains with different degrees of immunological competence (NMRI nu/nu, DBA/2) were infected by a myocarditic Coxsackie virus B3 (CVB3-M1) variant. At 31 days post-inoculation, plaque-forming assay, polymerase chain reaction (RT-PCR), and immunohistochemistry were carried out for detecting viruses and viral components in the myocardium. The virological findings were related to histopathological changes in the myocardium as well to the dilatation of both cardiac ventricles. Chronic myocardial lesions characterized by large fibrosis areas and interstitial inflammatory infiltrates were detected together with cardiomegalia in 52.6% (10/19) of athymic mice and in 9% (2/22) of euthymic mice. Viral replication foci were located and were found only in myocarditic cells adjacent to myocardial inflammatory lesions by immunostaining myocardial tissue sections with anti-serum to VP1 virus capsid protein. Using PCR followed by microwell capture hybridization assay, a large excess of viral positive strand RNA over negative strand was semiquantified in heart tissue from mice with chronic myocarditis, whereas approximately equal amounts of plus and minus strand RNA were detected in cases of persistent cardiac infection without chronic myocardial injuries. These findings provide evidence of the major role of viral replication in the pathogenesis of chronic murine CVB3-induced cardiomyopathy. The results indicate that the cardiac persistence of enteroviral RNAs can be observed without chronic cardiomyopathy, which could be explained by a defective viral positive RNA replication.

Captopril suppresses interstitial fibrin deposition in coxsackievirus B3 myocarditis.

The effects of captopril on murine coxsackievirus B3 (CB3) myocarditis were investigated, with focus on interstitial fibrin deposition and changes in the connective tissue matrix. Captopril was administered intraperitoneally at a dose of 0.1 mg/g to CB3-infected mice daily on days 10-30 in experiment I (inflammatory phase) and on days 30-60 in experiment II (fibrotic phase). In experiment I, mouse survival was higher in the captopril-treated group than in the untreated group. Histological improvement, including prevention of extravasated fibrin deposition, maintenance of connective tissue architecture, suppression of myocyte hypertrophy, and prevention of myosin isoform shift from alpha to beta, was observed in captopril-treated mice in experiment I, but not in experiment II; in experiment II, captopril administration suppressed thickening of the interstitial reticulin fibers. Captopril inhibited inflammatory fibrin deposition, postmyocarditic myocyte hypertrophy, and ventricular remodeling during the inflammatory phase, but not during the fibrotic phase, of CB3 myocarditis in mice.

Effects of methyl mercury on cytokines, inflammation and virus clearance in a common infection (Coxsackie B3 myocarditis)

A myocarditic coxsackievirus B3 (CB3) infection in Balb/c mice was used to investigate the effects of 12 weeks of methyl mercury (MeHg) exposure (3.69 mg/g diet) on inflammatory heart lesions, virus in the heart, the cytokine response i.e. cachectin/TNF-α and γ-interferon (IFN-γ) levels in plasma, and on disease complications and mortality. This dose of MeHg did not influence mortality in this infection model. The inflammatory and necrotic lesions in the ventricular myocardium 7 days after the inoculation covered 2.2% of the tissue section area in infected control mice. This damage was increased (n.s.) by 50% (to 3.3% of the tissue section area) in MeHg-treated mice. The response pattern of lymphocyte subsets in situ in myocardial inflammatory lesions was corroborated using an immune histological technique. MeHg treatment tended to increase (2.2-fold n.s.) the number of Mac 2 + cells (macrophages) in the heart muscle in this infection. Plasma levels of both TNF-α and IFN-γ increased on day 3 of the infection in MeHg-treated as well as in non-MeHg-treated mice, but the mean IFN-γ response was more pronounced in the MeHg-treated mice. On day 7 of the infection, when most animals still showed clinical signs of disease, cytokine levels were back to normal. MeHg-exposure in non-infected mice did not affect cytokine levels. In situ hybridization of virus RNA in myocardial tissue showed remaining virus in those mice who had the lowest plasma IFN-γ levels. A 20% increased ( P < 0.05) lymphoproliferative response to the T cell mitogen Con A was observed as a result of the MeHg treatment. Even heart tissue lesions and virus persistence tended to be influenced by MeHg in a direction compatible with the development of chronic disease.

Nitric oxide and murine coxsackievirus B3 myocarditis: Aggravation of myocarditis by inhibition of nitric oxide synthase

Objectives. This study sought to investigate the effects of nitric oxide inhibition in a murine model of coxsackievirus B3 myocarditis.Background. Little is known about the contribution of nitric oxide to the pathophysiology of myocarditis.Methods. Antiviral activity was tested in vitro using nitric oxide inhibition by treatment with activated macrophages of NG-nitro-l-arginine methyl ester and NG-nitro-d-arginine methyl ester (both at 100 μg/ml) were administered to C3H/He mice early (days 0 to 14) and late (days 14 to 35) after infection with coxsackievirus B3.Results. In the in vitro experiments with interferon-gamma-and lipopolysaccharide-induced activated murine macrophages, treatment with the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester, but not its inactive enantiomer NG-nitro-d-arginine methyl ester, restored coxsackievirus B3 titers. In the in vivo experiments in the early treatment group, myocardial virus titers were higher in NG-nitro-l-arginine methyl ester-treated than infected untreated animals, and both inflammatory cell infiltration and necrosis were more severe. In the late treatment group, more severe necrosis and more dense myocardial and perivascular fibrosis were observed in NG-nitro-l-arginine methyl ester-treated than in infected untreated animals. NG-Nitro-d-arginine methyl ester administration was ineffective.Conclusions. Nitric oxide inhibition increases myocardial virus titers, resulting in the aggravation of cardiac pathology in the early stage of coxsackievirus B3 myocarditis. In the late stage, it induces more severe cardiomyopathic lesions. Nitric oxide plays a defensive role in the pathogenesis of coxsackievirus B3 myocarditis.

Antiviral treatment with WIN 54 954 reduces mortality in murine coxsackievirus B3 myocarditis.

Coxsackieviruses B (CBVs) are dominant causative agents in myocarditis and are associated with pathogenesis is some cases of dilated cardiomyopathy, a clinical entity with a poor survival without heart transplantation. In vitro, the antiviral agent WIN 54 954 was shown to inhibit replication of CBV3 at a minimal inhibitory concentration value of 0.02 mg/L. Administration of WIN 54 954, 100 mg/kg BID PO, beginning on the day of infection resulted in complete protection from enteroviral mortality (P < .01). WIN 54 954 treatment did not abrogate the inflammatory reaction in the myocardium. No difference was found in the expression of surface lymphocyte subset markers. At 3 weeks, macrophages seemed to dominate the inflammatory reaction, regardless of treatment. There was no difference in CBV3 antibody titers, indicating that WIN 54 954 does not interfere with the development of protective immunity. Complement factors C3 and B were synthesized at a higher level during infection and correlated well with the degree of inflammatory reaction. The results show that WIN 54 954 is a potent antiviral agent with a highly significant effect on survival in CBV-induced myocarditis in the A/J mouse if treatment is started early. It is suggested that the reduction in mortality seen with WIN 54 954 administration is due to an inhibitory effect on virus replication in affected organs that does not interfere with cellular or humoral immunity.

Viral evolution as driven by host nutritional selective factors: influence of dietary oxidative stress

The endemic juvenile cardiomyopathy known as Keshan disease occurs in regions of China with poor selenium nutrition, but a role for an infectious agent was suggested by seasonal changes in disease incidence. Mice fed a selenium-deficient diet suffered more heart damage than normal mice when infected with a myocarditic coxsackievirus B3 (CVB3/20). Increased heart damage was also observed when CVB3/20 was inoculated into vitamin E-deficient mice. Feeding diets deficient in either vitamin E or selenium allowed an amyocarditic coxsackievirus (CVB3/0) to become myocarditic. When CVB3/0 was harvested from deficient mice, passed through HeLa cells and inoculated into normal (non-deficient) mice, it retained its increased cardiovirulence. Virus obtained from the selenium-deficient mice contained six nucleotide changes in the genome compared with the input strain. This is the first report of a nutritional deficiency driving changes in a viral genome. Host nutritional status could have important public health implications for the spread of influenza, hepatitis, polio and perhaps even AIDS.

Effects of granulocyte colony-stimulating factor upon coxsackievirus B3 myocarditis in mice.

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.

Colony-stimulating factors and coxsackievirus B3 myocarditis in mice: Macrophage colony-stimulating factor suppresses acute myocarditis with increasing interferon-α

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 × 106 U/kg/day M-CSF and 20 μg/kg/day G-CSF were injected subcutaneously every day on day 0 to day 14 starting simultaneously with virus inoculation. Serum interferon-α 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-α 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-α. Thus macrophages may play a defensive role in this model.

Therapy with immunoglobulin suppresses myocarditis in a murine coxsackievirus B3 model. Antiviral and anti-inflammatory effects.

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. 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. 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.

Effects of naloxone, an opiate receptor antagonist, on coxsackievirus B3 myocarditis in the mouse*1

Effects of naloxone, an opiate receptor antagonist, on coxsackievirus B3 myocarditis in the mouse*1

Changed distribution and immune effects of nickel augment viral-induced inflammatory heart lesions in mice

We have used the myocarditic coxsackievirus B3 (CB3) infection in Balb/c mice to investigate immunotoxic effects of a ten-week low-dose (0.002 M) administration of nickel chloride (NiCl 2) prior to infection. This dose did not influence CB3-induced mortality. Whole-body autoradiography of [ 63Ni] during the disease showed the pancreas, lungs and myocardium to be new target organs in this disease. Seven days after the inoculation, impulse counting of these organs showed the infection-induced increase of [ 63Ni] to be 5-fold ( P < 0.01) in the pancreas, 2.2-fold ( P < 0.05) in the lungs and 1.3-fold ( P < 0.05) in the heart. Nickel tended to increase spleen B- and T-cell activities, but thymocyte activity was unaffected. The activity of spleen natural killer (NK) cells decreased by 30% ( P < 0.05), whereas blood-cell activity in fact increased by 51% ( P < 0.05). The inflammatory and necrotic lesions in the ventricular myocardium seven days after the inoculation covered 3.31% of the tissue section area in infected control mice. This damage was increased by 43% (to 4.74% of the tissue section area) in nickel-treated mice. The response pattern of lymphocyte subsets in situ in myocardial inflammatory lesions was elucidated by an immune histochemical staining technique. The number of cytotoxic T-cells, helper T-cells and Mac 2 + cells (macrophages) in these lesions decreased by 46% ( P < 0.05), 41% ( P < 0.05) and 27% (not significant), respectively, with the nickel treatment. The number of helper T-cells was negatively correlated to the size of the inflammatory area ( r = −0.529, P < 0.02). The results indicate that nickel may contribute to the progression of target organ pathology in infection-induced diseases of an autoimmune and/or inflammatory character, such as diabetes and myocarditis.

Coxsackievirus B3 Infection Leads to Cell Death of Cardiac Myocytes

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.

Cytokine and murine coxsackievirus B3 myocarditis. Interleukin-2 suppressed myocarditis in the acute stage but enhanced the condition in the subsequent stage.

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. 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. 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.

Vitamin E Deficiency Intensifies the Myocardial Injury of Coxsackievirus B3 Infection of Mice

AbstractFeeding 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.

Role of oxygen derived free radicals in the pathogenesis of coxsackievirus B3 myocarditis in mice.

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. 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. 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. 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.

The effects of lobenzarit disodium, a novel immunomodulator, upon murine coxsackievirus B3 myocarditis.

The aim of this study is to test the therapeutic efficacy of immunomodulation with lobenzarit disodium (CCA) upon coxsackievirus B3 (CB3) myocarditis. Two-week-old C3H/He mice were inoculated with 10(3) plaque-forming units of CB3. CCA, 2.5 mg/kg per day, was administered 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 (groups 1 and 3). For the analysis of splenic lymphocyte subsets, additional mice in untreated and treated groups were killed on day 7, and the percentages of Thy 1.2 (CD3), L3T4 (CD4) and, Ly 2 (CD8) subsets were analyzed by laser flow cytometry (Experiment III). In Experiment I, the survival rate did not differ significantly between groups 1 and 2. Cellular infiltration in the CCA group was less severe. Myocardial virus titers and serum neutralizing antibody titers did not differ significantly between the two groups. In Experiment II, the survival rates between the two groups did not differ significantly. Myocardial necrosis in the CCA group was less severe compared to the control. In Experiment III, the percentages of Thy 1.2 (CD3) and L3T4 subsets (CD4) of the treated group were significantly higher than those of the control group. Thus, CCA increased splenic T cells and improved cardiac pathology in acute murine CB3 myocarditis.

Enhancement of coxsackievirus B3 myocarditis in mice by lobenzarit disodium through inhibition of splenic pan T cells

The aim was to test the efficacy of the immune system modulator lobenzarit disodium in the treatment of coxsackievirus B3 myocarditis. 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). 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)%. Lobenzarit disodium decreased splenic pan T cells and aggravated both clinical course and cardiac pathology in acute murine coxsackievirus B3 myocarditis.

Effects of polyethylene glycol conjugated superoxide dismutase on coxsackievirus B3 myocarditis in mice

The aim was to examine the role of oxygen derived free radicals in the development of myocarditis by investigating the effects of polyethylene glycol conjugated superoxide dismutase (PEG-SOD), a potent scavenger of oxygen free radicals, upon coxsackievirus B3 (CB3) myocarditis. Two week old male C3H/He mice were inoculated intraperitoneally with 10(3) plaque forming units of CB3. PEG-SOD, 1 x 10(3), 5 x 10(3), 1 x 10(4), and 1 x 10(5) U.kg-1 x d-1, was given subcutaneously daily on days 0 to 14. Treated groups were compared to the infected control. The survival rate of the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group was lower than that of the infected control group (40% v 78%, p < 0.01). The survival rates of the other treated groups did not differ significantly from the infected control. The myocardial calcification score in the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group was higher than in the infected control group on d 7, when myocardial virus titres did not differ significantly among the five groups. The scores for myocardial cellular infiltration and myocardial necrosis in the 1 x 10(3) and the 5 x 10(3) U.kg-1 x d-1 PEG-SOD groups were significantly lower than in the infected control on d 14, when myocardial viruses were not detected in the five groups. However, the myocardial necrosis and myocardial calcification scores in the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group were higher than in the infected control. The improvement of cardiac pathology in the 1 x 10(3) and the 5 x 10(3) U.kg-1 x d-1 PEG-SOD groups seems to have resulted not from the reduction in myocardial virus titres but from inhibition of generation of oxygen free radicals. The mechanism of the impaired survival and aggravation of cardiac pathology in the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group is unknown. The results suggest that oxygen free radicals may be involved in the pathogenesis and development of CB3 myocarditis and that appropriate dosages of PEG-SOD have therapeutic potential for clinical CB3 myocarditis, although caution must be paid to the treatment window.

Treatment of coxsackievirus B3 myocarditis by immunoactive peptide in an animal model

The effect of immunostimulant therapy on acute viral myocarditis, induced with coxsackievirus B3 (CB3), was investigated in C3H/He mice. Peritoneal exudate cells (PEC) or spleen cells (SC) from mice pretreated with a synthetic immunoactivating peptide FK565 significantly inhibited the multiplication of CB3 in C3H/He mouse embryo fibroblast cells compared with PEC or SC from nontreated mice in vitro (6.45 ± 0.20 log 10 PFU/ml; control: 6.85 ± 0.05, P < 0.05; 6.40 ± 0.07, control: 6.78 ± 0.07, P < 0.05, respectively), although FK565 did not inhibit viral replication directly. Mice were inoculated intraperitoneally with 3 × 10 5 plaque-forming units of CB3. FK565, 1 or 10 μg/kg, given intraperitoneally daily started on the same day of viral inoculation. To determine CB3 virus titer, mice were killed on Day 3. To study survival and myocardial histopathology, mice were killed on Day 20. Histopathological findings were scored on a scale of 0 to 4. FK565, 10 μg/kg/day, effectively inhibited myocardial viral replication (3.23 ± 0.42 log 10 PFU/mg, control: 3.71 ± 0.40, P < 0.05), reduced the cellular infiltration (1.3 ± 0.7, control: 2.5 ± 0.5, P < 0.05), myocardial necrosis (1.4 ± 0.9, control: 3.0 ± 0.6, P < 0.01), and calcification of the heart (1.0 ± 0.6, control: 2.5 ± 0.5, P < 0.01) and increased survival (60%, control: 30%, P < 0.05). The present study suggests that immunostimulant therapy improves the course of viral myocarditis during the viral-mediated phase. The inhibitory activity of FK565 seems to be due to the activation of host defense mechanisms.