Therapy With Immunoglobulin Suppresses Myocarditis in a Murine Coxsackievirus B3 Model
Antiviral and Anti-inflammatory Effects
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 cell infiltration and the severity of necrosis and calcification 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.
Infiltration of the myocardium with inflammatory cells occurs during infection with a variety of viruses.1 The infiltrate usually consists of mononuclear cells that are either focal or diffusely scattered in the myocardium. Myofiber necrosis is an important feature of this lesion. Myocyte hypertrophy has also been documented after myocardial necrosis.1 2 3 4
Enteroviruses, particularly coxsackievirus B (CB3), have been established as the predominant cause of viral myocarditis in humans,1 2 5 and viral myocarditis is considered to be a cause of dilated cardiomyopathy.1 2 5 Immune or autoimmune mechanisms may be involved in the pathogenesis of viral myocarditis and the subsequent cardiomyopathy.1 2 3 4 5 To address unresolved questions regarding the mechanisms operating in viral myocarditis, an experimental model of CB3 myocarditis in mice may be of great value.6
The therapeutic efficacy of high-dose immunoglobulin in inflammatory diseases, eg, Kawasaki disease,7 8 9 systemic vasculitis,10 and idiopathic thrombocytopenic purpura (ITP),11 has been reported. It recently was shown that immunoglobulin treatment in children with myocarditis has therapeutic value.12 Although the precise mechanisms responsible for the efficacy of this treatment are still unknown, immunoglobulin therapy is considered to have an immunosuppressive or anti-inflammatory effect. Because it has been established that the immune system is involved in myocarditis, studies have been undertaken in which immunosuppressive and anti-inflammatory agents have been used in both clinical and experimental settings. Accordingly, to clarify the precise immunologic mechanisms of such treatment, we investigated the effects of immunoglobulin on murine CB3 myocarditis and analyzed splenic lymphocyte subset populations.
In Vitro Study
Immunoglobulin (Venilon) was kindly supplied by Fujisawa Co, Ltd. Venilon is a sulfonated human immunoglobulin (classes G, A, and M) with Fc activity and is purified by Cohn’s cold ethanol method.10 11 Antiviral activity was assayed by a plaque-reduction method. A sterile solution of immunoglobulin was prepared. Serially diluted immunoglobulin was incubated with 100 plaque-forming units (PFU) of CB3 at 37°C for 1 hour. The reaction was stopped at 4°C for 30 minutes. The sample was added to confluent monolayers of VERO (African green monkey kidney) cells in six-well plastic plates.
After 2 days of incubation at 37°C, the cells were fixed with acetic acid and methanol and stained with crystal violet; then the plaques were counted. Plaque formation was expressed as a percentage of the number of control plaques. The drug concentration required to reduce the number of plaques by 50% from the number in the control well (median inhibitory dose) was calculated from the graph relating plaque number and drug concentration on a semilog plot (linear regression).
In Vivo Study
The virus stock of CB3 was prepared in cultures of VERO cells in Eagle’s minimum essential medium (EMEM). Virus suspensions were centrifuged after the cytopathic effect had developed, and the viral stock had a titer of more than 109 PFU/mL determined in tissue cultures.
Two-week-old male, inbred, certified virus-free C3H/He mice (Shizuoka Laboratory Animal Center) were used. They were supplied, together with their dams; when they were 4 weeks old, the dams were removed. The animals were inoculated intraperitoneally with 0.1 mL virus suspension containing 103 PFU.
The studies were approved by the institution’s Animal Care and Use Committee.
Immunoglobulin (Venilon, human immunoglobulin) was administered intraperitoneally daily; the actual dose in each experiment was calculated from the mouse weight at the beginning of the experiment. From previous studies, the dose of immunoglobulin used was 1 g · kg−1 · d−1.7 8 9 13 14 15 16 Immunoglobulin antigenicity between different species does not seem to be a problem.15 17 Fig 1⇓ shows the treatment protocol.
Experiment 1: early protocol. Mice (n=56) were randomized to two groups in which they received either no treatment (n=28) or treatment with immunoglobulin (n=28). Mice in the untreated group were injected intraperitoneally with 0.1 mL saline during the treatment period. Beginning simultaneously with the virus inoculation, treatment was given for 14 days. The mice were observed daily, and necropsy was performed immediately on those mice found dead. Seven mice in each group were killed on day 7 for virological study and for an age-matched study of cardiac pathology. Accordingly, the survival study covered 21 mice in each of the two groups. Mice surviving until the end of treatment period were killed. The thymus, spleen, and heart were weighed, and the organ-to-body-weight ratios were calculated. The organs were processed for pathological study.
Additional control groups were uninfected mice treated for 14 days with saline (n=5) and with immunoglobulin (n=5).
Experiment 2: late protocol. Mice surviving until 14 days after virus inoculation (n=50) were randomized to either of two groups: no treatment (n=25) or treatment with immunoglobulin (n=25). Treatment was given for 14 days, ie, until 28 days after virus inoculation. The mice were observed daily, and necropsy was carried out on those mice that died during the course of the experiment. At the end of the treatment period, the same procedure as that for experiment 1 was performed.
Additional control groups were age-matched uninfected mice treated for 14 days with saline (n=5) and with immunoglobulin (n=5), in parallel with late protocol.
Tissues (heart, lung, liver, thymus, spleen, pancreas, and muscle) were processed by standard methods, embedded in paraffin, cut into 5-μm-thick sections, and stained with hematoxylin and eosin. Myocardial lesions were graded by two of the authors (H.T. and C.K.) blinded to the respective treatment groups to determine the severity of cellular infiltration, necrosis, and calcification of the ventricles. The mean value was cited.
The pathological criteria for grading the severity of myocardial infiltration, necrosis, and calcification were as follows: grade 1 (mild), one or two small foci; grade 2 (slight), several small foci; grade 3 (moderate), multiple small foci or several large foci; and grade 4 (severe), multiple large foci or diffuse infiltration, necrosis, or calcification.
To avoid postmortem changes and to match the time course, pathological studies were performed only in mice killed on days 7, 14, and 28. The other organs were evaluated for evidence of viral or other pathological lesions.
For the infectivity assay, portions of the heart were weighed and homogenized aseptically in 2 mL PBS. After a 15-minute centrifugation at 1500g, virus titers in the supernatants were determined by a plaque assay method.4 In brief, VERO cells suspended (1×106/mL) in EMEM with 5% FCS plus 100 μg/mL each penicillin and streptomycin were placed in six-well plates and allowed to grow for 2 or 3 days at 37°C in 5% CO2. After adsorption, the cells were overlaid with 3 mL EMEM containing 5% FCS and 1% methylcellulose. After a 2-day incubation at 37°C in a humidified atmosphere containing 5% CO2, the cells were fixed with acetic acid and methanol (1:3) and stained with 1% crystal violet; plaques were then counted with an inverted microscope.
Serum-Neutralizing Antibody Titers
Blood was obtained under sterile conditions from the retro-orbital plexus, and the serum was inactivated at 56°C for 30 minutes. Each sample was titrated serially by determining the fourfold dilution in 5 mL EMEM supplemented with 3% FCS that protected the VERO cell monolayer against a challenge of 100 PFU of CB3. The neutralizing antibody titer was expressed as the reciprocal (log 4) of the highest serum dilution showing 50% plaque reduction.
Experiment 3: lymphocyte subset protocol. This experiment was conducted to clarify the effects of treatment of immunoglobulin on the lymphoid organ in the host. Virus inoculation and immunoglobulin treatment of mice were the same as in experiments 1 and 2. For the analysis of splenic lymphocyte subsets by flow cytometry and an immunoperoxidase method, further control and treated groups of mice were killed on days 7 and 21. The mice killed in this study were not included in the pathological or virological study.
The lymphocyte immunostaining methods used were similar to those described previously.3 4 For flow cytometry, the spleen was mechanically dissociated in RPMI-1640 medium with a 23-gauge needle. After the erythrocytes were lysed with 0.83% NH4Cl in Tris buffer (pH 7.2), the lymphocyte fractions were obtained by Ficoll gradient centrifugation.
The B cells were determined by staining with fluorescein isothiocyanate (FITC)–labeled rabbit anti-mouse immunoglobulin. T cells were stained with monoclonal rat anti-Thy 1.2 (pan T, clone 30-H12,18 Becton-Dickinson) as the first layer of antibody and with FITC-labeled rabbit anti-mouse immunoglobulin as the second antibody. The antibodies were diluted 10-fold. The cells were centrifuged at 1500g for 3 minutes. After centrifugation, the cell pellet was suspended in 100 μL RPMI-1640 medium with 2.5% FCS or the first layer of antibody. After a 30-minute incubation at 4°C, the cells were washed three times and suspended in 100 μL of the second antibody. They were incubated at 4°C for 30 minutes; then they were washed twice and suspended in 0.5 mL RPMI-1640 medium with 2.5% FCS.
The percentage of positive fluorescent cells was determined by laser flow cytometry (FACScan, Becton-Dickinson). The percentage of T cells was obtained by subtracting the percentage of B cells from the values obtained after staining with monoclonal rat antibody and FITC-labeled rabbit anti-mouse immunoglobulin.
An indirect horseradish immunoperoxidase technique was used for in situ analysis of the distribution of splenic lymphocyte subsets, as previously described.3 4 Spleens were quickly frozen in OCT compound. Sections 6 μm thick were cut from the frozen blocks. Endogenous peroxidase activity was blocked with cold methanol. Horseradish peroxidase activity was visualized with diamino benzidine as chromogen. The monoclonal antibodies used are Thy 1.2 for pan T cells18 and Bet-119 for B cells. Bet-1 reacts specifically to the allotypic marker of mouse IgM.19 Three to four spleens were examined in each group. Semiquantitative analysis of the distribution of the areas of positive-stained cells in the T- or B-cell zone was performed; − indicates nonreactive (no staining); 1+, ≦10% stained; 2+, ≦25% stained; 3+, ≦50% stained; and 4+, ≦90% stained.
Age-matched uninfected mice with and without immunoglobulin treatment were also prepared, and lymphocyte subsets in these groups were also examined.
Survival was analyzed by the Kaplan-Meier20 method. Statistical analysis of the data for body weight, organ-to-body-weight ratios, histological scores, and the percentage of lymphocyte subsets was determined by ANOVA. When significant differences were found, the two-tailed t test was used as a post-ANOVA test for establishing differences. A value of P<.05 was considered statistically significant.
Antiviral Activity In Vitro
The percent plaque formation was 118±32% at an immunoglobulin concentration of 10−3.0 mg/mL, 64±18% at 10−2.5 mg/mL, 36±20% at 10−2.0 mg/mL, 12±6% at 10−1.5 mg/mL, and 0±0% at 10−1.0 mg/mL (each n=4). Linear regression analysis showed a good negative correlation of Y=−68.9−57.4 log10X (r=−.90, P<.01; Fig 2⇓) between PFU and the logarithms of immunoglobulin concentrations. Thus, the dose of immunoglobulin inhibiting 50% of plaques was 0.01 mg/mL.
In Vivo Study
Infection with CB3 produced a similar pathological picture to that reported previously.3 4 6 21 22 In brief, 3 days after virus inoculation, the mice appeared ill; some showed coat ruffling, weakness, and irritability. Grossly, the myocardium had pale yellow patches that correlated with the inflammation; necrosis and calcification were seen microscopically.
Mortality. Eleven mice in the control group and no mice in the treated group had died by day 14; the survival rate on day 14 was 47.6% (10 of 21) in the control group and 100% (21 of 21) in the treated group. The difference between treated and control groups was significant (P<.01, Fig 3⇓).
Cardiac pathology. In the mice killed on days 7 and 14, the scores for cellular infiltration and myocardial necrosis were lower in the immunoglobulin-treated group than in the control group (Fig 4⇓, Table 1⇓). Notably, there was no myocarditis in any of the immunoglobulin-treated mice; ie, immunoglobulin administration completely suppressed the development of myocarditis.
Pathology of other organs. Pancreatitis, probably virus-induced, was noted in several mice in the untreated group but in no mice in the treated group (Fig 5⇓). No viral lesions were noted in the lung, liver, muscle, spleen, or thymus in either group. Notably, marked atrophy of lymphoid follicles (B-cell areas) was noted in the spleens of immunoglobulin-treated mice.
Serum-neutralizing antibody titers. Antibody titers on days 7 and 14 were significantly higher in the immunoglobulin-treated group than in the untreated group (Table 2⇓).
From in vitro analysis of origin of antibodies (fluorescent antibody technique), it was demonstrated that the circulating serum antibodies in immunoglobulin-treated mice on days 7 and 14 were primarily of human origin, ie, exogenous (data not shown).
Myocardial virus titers. No viruses were recovered from the hearts of immunoglobulin-treated mice; ie, an exogenous antibody had neutralized CB3. On day 7, there was a significant difference (P<.05) in myocardial virus titers between treated and untreated groups. On day 14, the virus was not detected in the myocardium in either group (Table 2⇑).
Organ weights. On day 7, body weight was greater, the ratio of heart to body weight was smaller, the ratio of thymus to body weight was higher, and the ratio of spleen to body weight was smaller in the treated mice than in the untreated mice (Table 3⇓). On day 14, body weight was greater, and the ratios of heart and spleen to body weight were smaller in the immunoglobulin-treated group than in the untreated group. Although no evidence was available, it would seem that the reciprocal changes in thymus and spleen weights seen in the immunoglobulin-treated group on day 7 are related to the host response induced by high-dose immunoglobulin.
Uninfected groups. None of the uninfected mice in the two groups died throughout the entire period. Lymphoid follicle atrophy was observed in the spleens of the immunoglobulin-treated mice. No abnormal pathological changes were evident in other organs examined in either of these groups.
Mortality. Fifteen mice in the control group and 10 in the immunoglobulin-treated group had died by day 28; the difference in the survival rates was not significant (Fig 6⇓).
Cardiac pathology. Cellular infiltration and myocardial necrosis in the treated group were less severe than in the control group (Table 1⇑).
Pathology of other organs. The results were similar to those in experiment 1. Pancreatitis was noted in several mice in both groups. Lymphoid follicle atrophy was also noted in the spleens of immunoglobulin-treated mice. No viral lesions were noted in other organs in either of the two groups.
Serum-neutralizing antibody titers. There was no significant difference in the serum-neutralizing antibody titers on day 28 between the two groups (Table 2⇑). The circulating antibodies in immunoglobulin-treated mice were both endogenous and exogenous (data not shown).
Organ weights. Body weight was greater, and the ratio of spleen to body weight was smaller in immunoglobulin-treated than in untreated mice (Table 3⇑).
Uninfected groups. No uninfected mice in the two groups died throughout the entire period. Lymphoid follicle atrophy was noted in the spleens of the immunoglobulin-treated mice. No abnormal pathological changes were evident in other organs examined in either of these groups.
Before analysis of lymphocyte subsets on day 7, 4 of 15 mice in the control untreated group and 0 of 15 in the treated group had died; 4 of 12 mice in the untreated group and 3 of 12 in the treated group had died on days 14 through 21 (Fig 7⇓, Table 4⇓).
In the flow cytometric study, the percentages of B cells in treated mice were markedly decreased on days 7 and 21 compared with those in untreated mice. Differences in the percentage of pan T cells between control and treated groups on days 7 and 21 were not significant.
Mice treated with immunoglobulin showed a positive reaction with a great majority of the T-cell zone (marginal zone, paracortical area and periarterial sheath) in the Thy 1.2 (pan T) staining and with a great minority of the B-cell zone (lymphoid follicle) in the Bet-1 staining in the spleen. Untreated mice, however, showed a positive reaction with a great majority of the T-cell zone in the Thy 1.2 staining and with a great majority of the B-cell zone in the Bet-1 staining in the spleen.
Table 4⇑ also shows the results on day 7 for age-matched uninfected mice with and without immunoglobulin treatment. The B-cell population in immunoglobulin-treated mice also decreased significantly.
In summary, immunoglobulin administration produced a marked decrease of the splenic B lymphocyte subset population in treated mice during the course of CB3 infection.
This study showed that immunoglobulin therapy completely suppressed CB3 myocarditis through an anti-CB3 effect in the acute viremic stage and partially suppressed the condition by an anti-inflammatory effect in the subsequent aviremic stage. The associated reduction of the splenic B cell subset may have been caused by a negative feedback of the high-dose immunoglobulin administration; splenic pathology revealed that immunoglobulin treatment produced marked atrophy of the lymphoid follicle (B-cell area) in CB3-infected mice. This finding may be of great value in aiding our understanding of the pathogenesis of CB3 myocarditis.3 4 6 21 22
As noted previously, immunoglobulin therapy is of value in the treatment of autoimmune and inflammatory diseases.7 8 9 12 13 14 15 16 The mechanisms responsible for its efficacy, however, are unknown. The prophylactic administration of immunoglobulin was reported to be of clinical value against respiratory syncytial virus in high-risk infants,23 and this effect was due to the capacity of immunoglobulin to neutralize this virus. The successful treatment of ITP with immunoglobulin appears to result from the blockade of Fc receptors.9 24 25 Intravenous immunoglobulin also was shown to act as a sump for activated complement components in Forssman shock syndrome.15 The rapidity of the effect of immunoglobulin in children with Kawasaki disease makes it doubtful that immunoglobulin is neutralizing a microorganism.7 13 14 This rapid effect in such children may be due to the neutralization of a microbial toxin by immunoglobulin, which acts as a superantigen that binds nonspecifically to major histocompatibility class II molecules or to certain viable regions of the T-cell–antigen receptor. Alternatively, this may result in the downregulation of the secretion of cytokines that increase inflammation or in the secretion of cytokines that downregulate inflammatory responses. More recently, the therapeutic efficacy of immunoglobulin in patients with dermatomyositis,16 in which a distinct myopathy is characterized by rash and a complement-mediated microangiopathy, was reported: immunoglobulin treatment improved the neuromuscular symptoms, resolved complement deposition on capillaries, and reduced the expression of intercellular adhesion molecule-1 and major histocompatibility class I antigens.16 Most recently, Drucker et al12 reported that, in a pediatric population, the use of high-dose intravenous immunoglobulin for the treatment of acute myocarditis, which complicated most cases of Kawasaki disease, was associated with improved recovery of left ventricular function and a tendency for better survival. Accordingly, clarification of the mechanisms underlying this treatment in myocarditis is warranted.
The present study points to two probable major mechanisms. First, polyclonal immunoglobulin could directly affect an infectious agent by the transfer of anti-viral antibody. This appears to be the mechanism that would explain the results in experiment 1; in vitro demonstration of the dose-dependent anti-CB3 activity of immunoglobulin, the lack of myocardial lesions in immunoglobulin-treated mice, and the higher serum levels of neutralizing antibody titers in the treated mice support this interpretation. We consider that an all-or-nothing effect of immunoglobulin in mortality and tissue pathology in experiment 1 was due to the exogenous antibodies present simultaneously with the beginning of the treatment. Second, regarding the mechanism whereby the anti-inflammatory effects of immunoglobulin are exerted, it is possible that immunoglobulin administration may alter immune responses, thus leading to a decrease in cardiac inflammation.26 27 28 29 The accepted mechanisms of action are reticuloendothelial system blockade and induction of idiotype–anti-idiotype networks. Reticuloendothelial system blockade implies that high concentrations of immunoglobulin could prevent antigen presentation and stimulation of immune responses by several mechanisms. Polyclonal immunoglobulin may bind to Fc receptors on macrophages and prevent internalization of the antigens. Exogenous proteins may occupy phagocytic vesicles to the exclusion of autoantigens, thus inhibiting autoimmune processes. An overabundance of immunoglobulin peptides may competitively prevent autoantigen peptides from binding to relevant major histocompatibility class I or II molecules. Polyclonal immunoglobulin-treated macrophages may show deficiencies in inflammatory cytokine secretion. Anti-idiotypic antibodies are antibodies to an antibody. Pooled normal serum should contain a wide variety of naturally occurring anti-idiotypes to self-antibodies. Some of these may be directed against the autoimmune effectors in myocarditis. We consider that the reduction of the splenic B-cell population in experiment 2 may have been caused by a negative feedback of the high-close immunoglobulin and was closely associated with an anti-inflammatory effect; this phenomenon may at least reflect changes in the immunologic environment in the immunoglobulin-treated host. Indeed, the decrease of the peripheral B-cell population in immunoglobulin-treated children with Kawasaki disease was documented.14
It is generally accepted that a biphasic disease process results when mice are infected with CB3.1 2 3 4 5 6 18 19 During the acute phase, viral replication in the myocardium results in myocardial necrosis and inflammation in the first week. After the virus has been eliminated from the myocardium, chronic inflammation results in progressive myocyte damage. It was suggested that the chronic phase results from a cell-mediated immune response to a neoantigen developed during the acute phase of the illness.1 2 3 4 5 6 21 22 Several investigators reported that, during the acute stage of myocarditis, sensitized T cells migrate toward the target organ, ie, the heart, where they may play a role in the development of myocarditis.1 2 3 4 5 6 21 22 Furthermore, it has been shown that both the expression of intercellular adhesion molecule-126 and the behavior of inflammatory cytokines27 28 29 play roles in the development of myocarditis. Autoantibodies against cardiac tissue components also were suggested to play a role in the development of this disease30 31 ; eg, in a clinical setting, the significant role played by autoantibodies against ventricular myosin was demonstrated in the sera of patients with myocarditis.30 31 Recently, Weller and Huber17 proposed the induction of idiotype–anti-idiotype networks by immunoglobulin as the mechanism whereby immunoglobulin protects against murine CB3 myocarditis. Most recently, it was implicated that a superantigen that selectively interacts with a T-cell receptor Vβ component may be associated with the pathogenesis of CB3 myocarditis.32 Seko et al32 suggested the possibility of using antibodies specific for the T-cell receptor Vβ gene products to prevent T-cell–mediated myocardial damage in the late stage of CB3 myocarditis. On the other hand, it was shown that normal pooled immunoglobulin contains antibodies against a major group of the superantigens, suggesting a possible immunoregulatory role for the exogenous antibodies in vivo.33 34 Therefore, changes in the immunologic environment induced by high-dose immunoglobulin, ie, the possible induction of antibodies against the superantigens,32 33 34 reduction of autoantibodies against the heart,17 30 31 suppression of both inflammatory cytokines27 28 29 and the expression of intercellular adhesion molecule-1,16 26 and suppression of major histocompatibility complexes,16 29 may lead to amelioration of myocarditis and improve survival.
In conclusion, immunoglobulin treatment suppressed CB3 murine myocarditis through the transfer of an anti-viral antibody and by exerting an anti-inflammatory effect. There is as yet no general agreement on effective treatment for viral myocarditis. Trials with steroids,35 nonsteroidal anti-inflammatory drugs,36 immunosuppressive agents,21 22 37 β-blockers,38 angiotensin-converting enzyme inhibitors,39 and other therapeutic modalities have been attempted. Immunoglobulin is safer and better tolerated than corticosteroids or other immunosuppressive agents. Accordingly, more widespread clinical use of this treatment in patients with myocarditis of viral and unknown origins appears warranted.
This work was supported in part by research grants from the Japanese Heart Foundation, an education fellowship from Toyama Medical and Pharmaceutical University, Iwaki fellowships from the Japanese Education of Science and Welfare (Nos. 01570478, 03670445, and 06670702), and the Uehara Memorial Foundation.
Presented in part at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 11-14, 1993.
- Received November 16, 1994.
- Revision received February 19, 1995.
- Accepted February 28, 1995.
- Copyright © 1995 by American Heart Association
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