Beneficial and detrimental impact of transplanted canine adipose-derived stem cells in a virus-induced demyelinating mouse model.

In recent years stem cell therapies have been broadly applied in various disease models specifically immune mediated and degenerative diseases. Whether adipose-derived stem cells might represent a useful therapeutic option in virus-triggered central nervous system diseases has not been investigated so far. Theiler's murine encephalomyelitis (TME) and canine distemper encephalitis are established, virus-mediated animal models sharing many similarities with multiple sclerosis (MS). Canine adipose-derived stem cells (ASC) were selected since dogs might serve as an important translational model for further therapeutic applications. The aim of the present study was to investigate whether canine ASC influence clinical signs, axonal damage, demyelination and inflammation during TME. ASC were transplanted intravenously (iv) or intra-cerebroventricularly (icv) at 7 (early) or 42 (late) days post infection (dpi) in TME virus (TMEV) infected mice. TMEV/ASC iv animals transplanted at 7dpi displayed a transient clinical deterioration in rotarod performance compared to TMEV/control animals. Worsening of clinical signs was associated with significantly increased numbers of microglia/macrophages and demyelination in the spinal cord. In contrast, late transplantation had no influence on clinical findings of TMEV-infected animals. However, late TMEV/ASC iv transplanted animals showed reduced axonal damage compared to TMEV/control animals. Screening of spinal cord and peripheral organs for transplanted ASC revealed no positive cells. Surprisingly, iv transplanted animals showed pulmonary follicular aggregates consisting of T- and B-lymphocytes. Thus, our data suggest that following intravenous application, the lung as priming organ for lymphocytes seems to play a pivotal role in the pathogenesis of TME. Consequences of T-lymphocyte priming in the lung depend on the disease phase and may be responsible for disease modifying effects of ASC.

Protective efficacy of adenovirus-mediated small interfering RNAs against encephalomyocarditis virus challenge in mice.

Encephalomyocarditis virus (EMCV) infection can cause myocarditis and sudden death in pre-weaned piglets and severe reproductive failure in sows. There are no specific antiviral drugs for the treatment of the virus infection. In this study, four recombinant adenoviruses expressing small interfering RNAs (siRNAs) targeting to 1D or 3AB protein genes of EMCV were constructed and their inhibition efficiency on the replication of EMCV was evaluated in both Marc-145 cells and mice. The results showed that the rAd5 expressing siRNAs (rAd5) could inhibit EMCV replication in Marc-145 cells in protein and mRNA levels, as well as the virus yield by approximately 100-1000 times. And the inhibition of viral replication was sustained for 72h and dose-dependent. Animal experiment results showed that EMCV VP1 mRNA level in the brain of mice in the rAd5 groups were obviously lower than those in rAd-G1 and challenge control groups. The virus yields in rAd-1D-2 and rAd-3AB-1 groups were markedly decreased by more than 90.0%. The survival rates of mice in rAd-1D-2 group were significantly higher than those in challenge control groups. Furthermore, the survival mice only showed minor microscopic lesions in brain and minor edema of nerve cell, which was obviously slighter than those in challenge control groups. These results indicated that siRNAs mediated by the adenovirus could provide protective efficacy against EMCV challenge in mice. It might provide a potential strategy for combating EMCV.

Method of identifying natural antibodies for remyelination.

INTRODUCTION: Natural autoantibodies are part of the normal human immunoglobulin repertoire. These antibodies react to self-antigens, are usually polyreactive with relatively low affinity, and typically are of the IgM isotype. Natural IgMs in mice that stimulated remyelination in central nervous system (CNS) demyelinating disease all shared the characteristics of binding to the surface of live oligodendrocytes and myelinated tracts in living slices of CNS tissue. METHODS: A screen for human IgMs with similar character resulted in two human natural antibodies, which when injected peripherally into animal models of demyelination induced remyelination. A recombinant human IgM (rHIgM22) that also promoted remyelination in vivo was constructed. RESULTS: Very small doses of this IgM are required for the promotion of remyelination (EC50 is 460 ng per 20-g mouse). It is clear that after peripheral delivery, rHIgM22 enters the CNS and accumulates in CNS lesions. rHIgM22 was tracked in living mice using ferritin-labeled antihuman mu chain antibodies visualized by magnetic resonance imaging and traditional immunocytochemistry. Although the exact antigen recognized by rHIgM22 is not known, all mouse IgMs that promote remyelination bind to myelin membrane lipids, suggesting the antigen for rHIgM22 is similar. CONCLUSIONS: We propose that the IgMs bind to CNS cells and reorganize the membrane, initiating a signal that results in oligodendrocyte proliferation and/or protection with an end result of increased myelin. Recombinant natural human antibodies are potentially important therapeutic molecules that may modulate a wide spectrum of human disease.

Contrasting roles for Valpha14+ natural killer T cells in a viral model for multiple sclerosis.

Most natural killer (NK) T cells express an invariant Valpha14 T-cell receptor. To explore the contribution of NKT cells in an animal model for multiple sclerosis, Theiler's murine encephalomyelitis virus (TMEV) infection, TMEV-infected mice were treated with Valpha14 antibody. Treatment during the early stage of infection delayed the onset of demyelinating disease with higher interleukin-4 production, whereas administration during the late stage or weekly resulted in more severe demyelination with enhanced virus persistence. The effect of in vivo depletion of NKT cells differed depending on the stage of infection, suggesting contrasting roles for NKT cells over the disease course.

Anti-CCL2 treatment inhibits Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus induces a demyelinating disease (TMEV-IDD) of the central nervous system (CNS) in susceptible mouse strains with accompanying histopathology characterized by mononuclear cell infiltrates. In susceptible strains of mice such as SJL, virus establishes a persistent infection in macrophages, induces a CNS infiltration by macrophages, T cells, and B cells, which results in chronic-progressive paralysis. In the present report the authors have investigated the functional role of CCL2 (monocyte chemotactic protein-1) in the induction and progression of demyelinating disease. Treatment of infected mice at day 0, 14, or 28 with anti-CCL2 resulted in a significant decrease in the clinical disease progression. Further analysis of anti-CCL2-treated mice revealed decreased CNS inflammation and mononuclear cell infiltration with an accompanying change in inflammatory cytokine responses. There was an overall decrease in the absolute numbers of CNS-infiltrating CD4+ T cells, macrophages, and B cells. Finally, anti-CCL2 treatment resulted in decreased viral load in the CNS. These data directly demonstrate a role for CCL2 in the pathogenesis of TMEV-IDD.

Biozzi mice: of mice and human neurological diseases.

In 1972 Guido Biozzi selectively bred mice to study the immunopathological mechanisms underlying polygenic diseases. One line, the Biozzi antibody high (AB/H) mouse (now designated the ABH strain) was later found to be highly susceptible to many experimentally induced diseases such as autoimmune encephalomyelitis, autoimmune neuritis, autoimmune uveitis, as well as virus-induced demyelination and has thus been a key mouse strain to study human inflammatory neurological diseases. In this paper we discuss the background of the Biozzi ABH mouse and review how studies with these mice have shed light on the pathogenic mechanisms operating in chronic neurological disease.

CD154 blockade results in transient reduction in Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Transient CD154 blockade at the onset of Theiler's murine encephalomyelitis virus-induced demyelinating disease ameliorated disease progression for 80 days, reduced immune cell infiltration, and transiently increased viral loads in the central nervous system. Peripheral antiviral and autoimmune T-cell responses were normal, and disease severity returned to control levels by day 120.

Cellular sources and targets of IFN-gamma-mediated protection against viral demyelination and neurological deficits.

IFN-gamma is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-gamma, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler's virus model of multiple sclerosis. During viral infections, IFN-gamma is produced by natural killer (NK) cells, CD4(+) and CD8(+) T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-gamma-mediated protection. To determine the lymphocyte subsets that produce IFN-gamma to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-gamma-production. We demonstrate that IFN-gamma production by both CD4(+) and CD8(+) T cell subsets is critical for resistance to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4(+) T cells make a greater contribution to IFN-gamma-mediated protection. To determine the cellular targets of IFN-gamma-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-gamma receptor. We demonstrate that IFN-gamma receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination.

Embryonic stem cells attenuate viral myocarditis in murine model.

We used mice to test our hypothesis that in response to viral invasion, stem cells may migrate into the heart and attenuate the effect of viral myocarditis. Male BALB/c mice were divided into three groups: mouse embryonic stem (ES) cell control, encephalomyocarditis virus (EMCV), and EMCV + ES cells. After administration of ES cells via tail vein, mice were immediately inoculated with EMCV. Mice were sacrificed at different days after EMCV inoculation. Mortality was recorded. Inflammatory cell infiltration and necrosis (major pathological changes of viral myocarditis) were evaluated by hematoxylin-eosin staining. ES cell migration and differentiation were identified by immunofluorescence. The survival rate in the EMCV + ES cell group (80%) was significantly increased (p < 0.05) over the EMCV-alone group (64%). Also, the incidence of inflammatory cell infiltration and myocardial lesions was lower in the EMCV + ES cell mice. Furthermore, the result of green fluorescent protein (GFP) and alpha-actinin analysis indicated that ES cells migrated into the heart and differentiated into myocytes after virus inoculation. In conclusion, ES cells significantly increased the survival of viral myocarditis mice and also decreased the necrosis and infiltration of inflammatory cells. These results demonstrated the ability of stem cells to mitigate the effects of viral infection on the heart and illustrated their potential therapeutic application to other mammalian species, including humans.

Ovine IFN-tau modulates the expression of MHC antigens on murine cerebrovascular endothelial cells and inhibits replication of Theiler's virus.

Interferon-beta (IFN-beta) has been used successfully to treat patients with relapsing-remitting multiple sclerosis (MS). IFN-tau is a new class of type I IFN that is secreted by the trophoblast and is the signal for maternal recognition of pregnancy in sheep. IFN-tau has potent immunosuppressive and antiviral activities similar to other type I IFN but is less cytotoxic than IFN-alpha/beta. The current investigation concerns the effect of recombinant ovine IFN-tau (rOvIFN-tau) on the modulation of MHC class I and II expression on cloned mouse cerebrovascular endothelial (CVE) cells. IFN-tau induced tyrosine phosphorylation of Stat1 and upregulated the expression of MHC class I on CVE. One proposed action by which type I IFN reduce the relapse rate in MS is via interference with IFN-gamma-induced MHC class II expression. IFN-tau was shown to downregulate IFN-gamma-induced MHC class II expression on CVE and, hence, may be of potential therapeutic value in downregulating inflammation in the central nervous system (CNS). IFN-tau did not upregulate the expression of MHC class II on CVE. IFN-tau also inhibited the replication of Theiler's virus in CVE. These in vitro results suggest that IFN-tau may be of therapeutic value in the treatment of virus-induced demyelinating disease.

Immunoglobulin treatment prevents congestive heart failure in murine encephalomyocarditis viral myocarditis associated with reduction of inflammatory cytokines.

We have previously shown that immunoglobulin therapy suppressed murine coxsackievirus B3 myocarditis. In the present study, we examined the effects of immunoglobulin upon murine myocarditis induced by encephalomyocarditis virus, which is not pathogenic to humans. Antiviral activity of immunoglobulin (Venilon) against encephalomyocarditis virus could not be detected in vitro. The production of cytokines was decreased in virus-infected macrophages by the treatment of immunoglobulin in vitro. Immunoglobulin (1 g/kg/day) was administered intraperitoneally to the virus-infected C3H/He mice daily for 2 weeks, beginning simultaneously with virus inoculation in experiment I and on day 14 after virus inoculation in experiment II. In experiment I, survival rate did not differ significantly between immunoglobulin-treated and untreated groups. In experiment II, survival rate was higher in immunoglobulin compared with control groups. Immunoglobulin administration suppressed the development of myocardial necrosis with T-lymphocyte infiltrates in mice not only in the acute viremic but in the chronic aviremic stages concomitantly associated with the reduction of inflammatory cytokines, i.e., tumor necrosis factor-alpha, interferon-gamma, macrophage inflammatory protein-2, and interleukin-6. Taken together, immunoglobulin therapy could have the potential to prevent congestive heart failure.

Enhanced production of macrophage inflammatory protein 2 (MIP-2) by in vitro and in vivo infections with encephalomyocarditis virus and modulation of myocarditis with an antibody against MIP-2.

Interleukin-8 (IL-8) is a chemotactic cytokine for neutrophils and lymphocytes. Macrophage inflammatory protein 2 (MIP-2) is a murine counterpart of IL-8. The present study was performed to determine whether MIP-2 aggravates murine myocarditis. We examined (i) the MIP-2-producing activity of encephalomyocarditis (EMC) virus-infected cultured macrophages, (ii) serial plasma MIP-2 levels in EMC virus-induced mice by enzyme-linked immunosorbent assay, and (iii) the effects of antimouse MIP-2 monoclonal antibody (MAb) in vivo upon myocarditis. The production of MIP-2 increased in an infection dose- and time-dependent manner in virus-infected RAW 264. 7 macrophages. Five-week-old C(3)H/He mice were inoculated with EMC virus. Plasma MIP-2 levels were significantly elevated in mice on days 7 and 14 postinfection. Mice were injected subcutaneously with anti-MIP-2 MAb at 10 microg/day (group 2) or 100 microg/day (group 3) on days 0 to 5 and were observed until day 21. Uninfected control mice (group 1) were prepared. The survival rate was higher in the anti-MIP-2-treated group (group 3), but not in group 2, than in the control group. Histopathological analysis revealed that cellular infiltration and myocardial necrosis with macrophage and T-cell accumulation were less prominent in the anti-MIP-2 MAb-treated group, but not in group 2, compared to the level in the controls. MIP-2 is an important naturally occurring inflammatory cytokine in myocarditis, and anti-MIP-2 MAb treatment may prevent the inflammatory response.

High-dose mouse immunoglobulin G administration suppresses Theiler's murine encephalomyelitis virus-induced demyelinating disease.

We studied the effect of high-dose mouse IgG on TMEV-induced demyelinating disease (TMEV-IDD). We injected TMEV intracerebrally into susceptible SJL/J mice and induced TMEV-IDD. Mouse IgG were injected intraperitonealy, and clinical course and various immunological indicators were studied. The results show that TMEV-IDD was significantly suppressed both clinically and histologically (P<0.01) when IgG were administered in the effector phase. The delayed type hypersensitivity and T cell proliferative response specific for TMEV were decreased by this treatment. In an ELISPOT assay, the number of TNF-alpha producing lymphocytes in the spinal cords was low in high-dose IgG treated mice compared with PBS treated control mice. These data suggest that administration of IgG suppresses TMEV-IDD and may be promising treatment to prevent exacerbation of human multiple sclerosis.

Viral hyperinfection of the central nervous system and high mortality after hematopoietic stem cell transplantation for treatment of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus (TMEV) establishes a persistent infection in the central nervous system (CNS) leading to an inflammatory demyelinating disease of the CNS in which the histology and clinical course is similar to multiple sclerosis (MS). Disease pathogenesis is primarily due to T-cell-mediated destruction of myelin, which has been attributed to cytopathic effects of the virus, but immune-mediated destruction of myelin mediated via both virus-specific and myelin-specific T cells appear to play the major role. To determine if bone marrow transplantation would be an effective therapy for a virus-initiated autoimmune disease and to better separate viral cytopathic effects from immune-mediated demyelination, we ablated the immune system of TMEV-infected animals with 1,100 cGy total body irradiation, and then the animal's immunity was reconstituted by transplantation of disease-susceptible SJL/J mice with syngeneic marrow or disease-susceptible DBA/2J with marrow from disease-resistant (C57Bl/6 x DBA/2)F1 (B6D2) donors. Hematopoietic transplant performed after onset of disease resulted in 42% mortality in SJL/J syngeneic transplants, 47% mortality in diseased DBA2 recipients restored with marrow from naive B6D2 donors, and 12% in diseased DBA2 recipients receiving marrow from B6D2 donors previously infected with TMEV. Delayed type hypersensitivity (DTH) to both virion and myelin proteins was decreased in surviving mice that underwent transplantation; however, CNS viral titers were significantly elevated compared with nontransplanted controls. We conclude that a functional immune system with appropriate T-cell responses are important in prevention of lethal cytopathic CNS effects from TMEV. Relevant to the clinical use of bone marrow transplantation, attempts to ablate the immune system in viral-mediated immune diseases or virus-initiated autoimmune disease may have acute and lethal consequences. Our results raise concern about the attempted use of autologous hematopoietic transplantation in patients with MS, an autoimmune disease with a suspected virus etiology, particularly if the graft is aggressively depleted of lymphocytes.

Contrasting effects of anti-adhesion molecule therapy in experimental allergic encephalomyelitis and Theiler's murine encephalomyelitis.

An augmentation of experimental allergic encephalomyelitis (EAE) was observed when monoclonal antibody (mAb) to intercellular adhesion molecule 1 (ICAM-1) was administered after adoptive transfer. Clinical disease was more severe in the ICAM-1 specific mAb-treated EAE mice and included prominent ataxia compared to the PBS-treated controls or Theiler's murine encephalomyelitis virus (TMEV) infected mice treated with ICAM-1 specific mAb. Neuropathologic evaluation demonstrated a distinctly different distribution of lesions in the anti-ICAM-1-treated EAE mice which featured prominent demyelination and inflammation in the cerebellum, brainstem and cerebrum. These structures were minimally involved in the control mice and mAb treatment did not alter the neuropathology in TMEV-infected mice. These results indicate that anti-ICAM-1 can alter trafficking of lymphocytes and mononuclear cells in EAE but not TMEV-induced demyelinating disease.

Enhancement by muramyl peptides of the protective response of interferon-alpha/beta against encephalomyocarditis virus infection.

The use of interferon-alpha (IFN-alpha) in the treatment of infectious diseases has shown limited efficacy and dose-limiting toxicity. We have selected safe immunomodulators of the muramyl peptide family with the potential of enhancing the efficacy of IFN-alpha without resulting in increased toxicity. One of these synthetic muramyl dipeptide (MDP) derivatives, namely murabutide which is in a clinical stage of development, has been recently found to synergize with IFN-alpha 2a in the selective induction of anti-inflammatory mediators and to enhance the biological activities of the therapeutic cytokine. The present study was performed to assess the antiviral activity of such muramyl peptides and a possible potentiation of the antiviral activity of IFN-alpha/beta by associated therapy using the classical assay of Encephalomyocarditis virus (EMCV) infection. In vitro, pretreatment of Moloney Sarcoma virus (MSV)-transformed cell line with MDP derivatives followed by treatment with IFN-alpha/beta showed a synergistic protection against the cytopathogenic effect of a subsequent EMCV infection. None of the MSV cultures could be protected by stimulation with muramyl peptides alone. In vivo, all of the muramyl peptide derivatives tested were found to be more potent than the parent molecule MDP in inducing protection against death or in the prolongation of the mean survival time of infected mice. Sequential administration of suboptimal doses of exogenous IFN-alpha/beta and muramyl peptides established a strong antiviral state and considerably improved the protective effect of the cytokine, frequently leading to an abortive infection. Our findings suggest that combination therapy with safe muramyl peptides and IFN-alpha/beta could constitute a highly effective and new regimen for the treatment of viral infections in humans.

Immunomodulating agents for the management of heart failure with myocarditis and cardiomyopathy--lessons from animal experiments.

We have developed murine models of viral myocarditis induced by the encephalomyocarditis (EMC) virus in which there is a high incidence of severe myocarditis, congestive heart failure and dilated cardiomyopathy. From these models, we have learned of their natural history and pathogenesis and assessed new diagnostic methods and therapeutic and preventive interventions. Our recent studies showed that increased circulating cytokines have been detected in patients with acute myocarditis and cardiomyopathy and suggest that cytokines may play some role in the pathogenesis of myocardial injury in these diseases. In our animal model of EMC virus myocarditis, plasma tumour necrosis factor-(TNF)-alpha was elevated in the acute stage and exogenously administered anti-TNF-alpha antibody improved survival and reduced the myocardial lesion, suggesting the importance of TNF-alpha in the pathogenesis. A recently developed positive inotropic agent, vesnarinone, was effective in the treatment of EMC virus myocarditis by its immunomodulating effects such as inhibition of production of TNF-alpha The plasma angiotensin II level was increased in EMC virus myocarditis, and a new angiotensin II type I antagonist, TCV-116, prevented development of myocarditis.

The in vivo antiviral activity of interleukin-12 is mediated by gamma interferon.

The injection of 20 ng of mouse interleukin-12 (IL-12) protects mice from a lethal infection with encephalomyocarditis virus. In vitro, an anti-gamma interferon (anti-IFN-gamma) monoclonal antibody but not an anti-IL-12 monoclonal antibody neutralizes the antiviral activity present in the supernatants of splenocytes stimulated with IL-12. Finally, IL-12 fails to protect 129 Sv/Ev IFN-gamma R0/0 mice against encephalomyocarditis virus infection. These results demonstrate that IL-12 exerts its antiviral activity through the induction of endogenous IFN-gamma.

Effect of combination therapy with OK432 and recombinant human interferon-alpha A/D on atrial natriuretic peptide gene expression in mice with viral myocarditis.

The effects of combination therapy with the immunomodulators OK432 (derived from the Su strain of Streptococcus pyogenes A3; 1 unit corresponds to 0.1 mg of dried streptococci dissolved in 0.1 ml of saline) and human recombinant interferon-alpha A/D (IFN) on cardiac atrial natriuretic peptide (ANP) gene expression and myocardial hypertrophy were examined in a murine model of viral myocarditis with congestive heart failure. Therapy was started 24 h after inoculation with encephalomyocarditis virus and was continued for 14 days. The plasma ANP concentration in untreated infected mice was significantly (P < .01) increased on day 10 (115 +/- 48 pg/ml) and day 30 (43 +/- 22 pg/ml) after inoculation relative to that in uninfected controls (5 +/- 4 pg/ml), whereas plasma ANP levels in treated mice were significantly (P < .01) reduced on day 10 (14 +/- 13 pg/ml) and day 30 (11 +/- 9 pg/ml) in comparison with untreated infected mice. The atrial and ventricular ANP messenger RNA (mRNA) concentrations in untreated mice showed increases of approximately 1.4- and 29.3-fold, respectively, on day 10 and increases of 1.8- and 34-fold, respectively, on day 30 compared with the concentration in uninfected controls. Combined OK432 and IFN significantly (P < .01) reduced the increase in ANP mRNA concentration in ventricles to 6.0- and 6.7-fold on days 10 and 30, respectively. Neither OK432 nor IFN monotherapy reduced the ANP mRNA concentrations in atria and ventricles compared with those in untreated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of virus-induced myocardial injury with a novel immunomodulating agent, vesnarinone. Suppression of natural killer cell activity and tumor necrosis factor-alpha production.

Controversy still exists concerning the therapy for viral myocarditis which manifests a wide variety of clinical symptoms. Vesnarinone, a quinolinone derivative that was developed as a positive inotropic agent with complex actions, including phosphodiesterase inhibition and cation channel modification, has recently been confirmed to improve the prognosis of patients with chronic heart failure. However, the precise mechanism of this beneficial effect is not yet clearly understood. In this study, using a murine model of acute viral myocarditis resulting from encephalomyocarditis virus infection, survival and myocardial damage were markedly improved by treatment with vesnarinone. In contrast, survival was not improved by treatment with amrinone, a phosphodiesterase inhibitor. Although vesnarinone did not inhibit viral replication or protect myocytes from viral direct cell injury, it did inhibit the increase in natural killer cell activity after viral infection. On the other hand, amrinone failed to inhibit natural killer cell activity. Both vesnarinone and amrinone suppressed the production of tumor necrosis factor-alpha. Therefore, we postulate that vesnarinone exerted its beneficial effects through an inhibition of natural killer cell activity, and that it serves as an immunomodulator providing new therapeutic possibilities for the treatment of viral myocarditis and/or immunological disorders.