Effect of immunostimulant therapy on acute viral myocarditis in an animal model The effect of immunostimulant therapy on acute viral myocarditis, which was induced with encephalomyocarditis virus, was investigated in 4-week-old male BALBlc mice. In vitro, peritoneal exudate cells and spleen cells from mice that were pretreated with a synthetic immunoactivating peptide, FK565 (heptanoyl-~-D-glutamyl-(L~meso-diaminopimelyl-(D)-alanine), significantly inhibited the multiplication of encephalomyocarditis virus in BALBlc 3T3 cells compared with peritoneal exudate cells and spleen cells from untreated mice (6.14 + 0.21 log10 plaque-forming units (pfu) per milliliter, control: 6.59 f. 0.03, p < 0.05; 3.55 + 0.23, control: 5.64 _t 0.09, p < 0.01, respectively), although FK565 did not inhibit viral replication directly. The mice were inoculated intraperitoneally with 100 pfu of encephalomyocarditis virus. FK565 (1 mglkglday), which was administered 1 day before or simultaneously with virus inoculation, effectively inhibited myocardial viral replication (2.77 _+ 0.17 log10 pfulmg, 2.46 * 0.35, log10 pfulmg, respectively, control: 3.33 s 0.26, p < 0.025) and increased survival (70% and 60%, respectively, control: 20%, p < 0.01). Histopathologic findings were scored on a scale of 0 to 4. Treatment with 1 mglkglday of FK565 that was started 1 day before virus inoculation was most effective in reducing the inflammatory response (1.2 + 0.63, control: 2.0 + 0.61, p < 0.05) and myocardial necrosis (1.2 i- 0.42, control: 2.0 t 1.00, p < 0.025). The present study suggests that immunostimulant therapy improves the course of viral myocarditis during the virus-mediated phase. The inhibitory activity of FK565 seems to be due to the activation of host defense mechanisms such as macrophages or lymphocytes. (AM HEART J 1992;124:426.)

Yukihito Sato, MD, Shingo Maruyama, MD, Chuichi Kawai, MD, and Akira Matsumori, MD Kyoto, Japan

With the increasing use of endomyocardial biopsy, evidence has suggested that dilated cardiomyopathy may result from prior viral myocarditis.‘, 2 With the use of gene amplification by polymerase chain reaction, enteroviral signals have been obtained from cardiac biopsy specimens that were taken from some patients with myocarditis and dilated cardiomyopathy.3 Encephalomyocarditis (EMC) virus is a picornavirus that is biologically similar to coxsackievirus. It can cause severe myocarditis in experimental animals and may also infect human beings. The disease in an animal model is characterized by myocardial necrosis and inflammation in the acute stage; necro-

From The Third Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto, Japan. Supported in part by a research grant from the Ministry of Health and Welfare and Grant-in-Aid for General Scientific Research from the Ministry of Education, Science and Culture, Japan and Kanazawa Research Fund. Received for publication Oct. 31,199l; accepted Jan. 20, 1992. Reprint requests: Akira Matsumori, MD, The Third Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Kawaracho Shogoin, Sakyo-ku, Kyoto 606, Japan. 4/l/38103

426

sis and inflammation are followed by myocardial fibrosis, dilatation, and hypertrophy (which resemble dilated cardiomyopathy) in the chronic stage.4,5 It is clear that myocardial damage, especially in the acute stage, occurs because of the direct cytopathic effect of the virus on myocardial cells; therefore, appropriate immunostimulant therapy may have a positive effect on the degree of myocardial injury. It is well known that host factors against viral infection involve several different mechanisms: (1) the antibody response, (2) cell-mediated immunity including cytotoxic T cells or macrophages, (3) antibody-dependent cell cytotoxity, and (4) interferons. Attempts to enhance the host defense mechanism against infection have been reported.” FK565 is a synthetic immunoactive peptide, isolated from culture filtrates of Streptomyces olivaceogriseus. Although it has no direct effect, it induces resistance against somemicrobial, fungal, and viral infections in vivo by activating host defense mechanisms.7-‘0 In the present study, we investigated the effect of immunostimulant therapy with FK565 on an animal model of acute viral myocarditis that was induced by EMC virus.

Volume Number

124 2

METHODS

All experiments were performed in accordancewith the Guidelines for Animal Experiments of Kyoto University. Cells. BALB/c 3T3 cells and human amniotic cellswere grown in Eagle’sminimum essentialmedium (EMEM) that was supplementedwith 10% newborn calf serum. Virus. The myocardial variant of EMC virus, which results predominantly in myocardial disease,wasused.Virus stock wasprepared in cultures of human amniotic cells in EMEM and stored at -70° C until it was diluted for use. Virus titers were determined by plaque formation on human amniotic cell monolayers. Human amniotic cellswere suspendedat a concentration of 1 X lo6 cells per milliliter in EMEM with lO@O fetal calf serumin 6-well plastic plates and allowedto grow for 4 days at 37OC in 5% CO2.Volumes (0.1 ml) of decimal dilutions of virus suspendedin EMEM were adsorbed to human amniotic cell monolayers for 60 minutes at 37’ C in 5% CO?. After adsorption, the cells were overlaid with 3 ml of medium contained 2 % fetal calf serumand 1Y methylcellulose. After 2 days of incubation at 37’ C in a humidified atmosphere that contained 5’0 COz, cells were fixed in an acetic acid and methanol mixture (at a ratio of 1:3) and stained with gentian violet (1‘T;,). Plaques were counted with t,he useof an inverted microscope. Agent. FK565 (heptanoyl-r-D-glutamyl-(L)-meso-diaminopimelyl-(D)-alanine) (Fig. 1) wassynthesized at the Product Development Laboratories of Fujisawa Pharmaceutical Co., Ltd. in Osaka,Japan. FK565 powder wasdissolved in phosphate-buffered saline solution (PBS) and stored at 4’ C. Antiviral activity of FK565. The antiviral activity of FK,565wasassayedby a plaque reduction method. Confluent monolayers of human amniotic cells in plastic plates were treated with medium that contained various concentrations (0, 1, 10,or 1000pg/ml) of FK565 (six wellsfor each concentration). Twenty-four hours later, the cells were washedwith PBS and then inoculated with 100 plaqueforming units (pfu) of EMC virus in 0.1 ml of PBS. After 60 minutes of virus absorption at 37” C in 5 $6COz, EMEM that contained 2“; fetal calf serum, 15: methylcellulose, and various concentrations of FK565 (0, 1, 10, or 1000wg/ ml) was overlaid. After 48 hours of incubation, the cells were fixed and stained, and plaques were counted as describedabove. Plaque formation wasexpressedasa percentage of the control. Preparation of peritoneal exudate cells. Four-week-old male BALB/c mice were given FK565 intraperitoneally at a doseof 0.01 mg/kg per day. Three days after the first injection, peritoneal lavage was performed with 3 ml of Hanks balanced salt solution. Peritoneal exudate cells (PECs) were centrifugated and washed.PECs that were obtained from untreated mice were usedascontrols. In order to study the effect of FK565 on type and yield of PECs, 4.week-old male ICR mice were treated with 0.01 mg/kg/ day of FK565 for 4 days. The control mice were injected with PBS. After peritoneal lavage was performed, cells were stained with Giemsastain. The number of PECs from treated mice was 4.67 t 0.52 x lo6 per cavity (macro-

Immunostimulant

therapy

and viral myocarditis

429

CD) cH,(cH,),CO-HNCHCOOH CL) I ~LLcOHN~HCO-HNAHCOOH

CH, (0)

H,NCHCOOH (D) Fig. 1. Chemicalstructure of FK565 (heptanoyl-y-D-glu-

tamyl-(L)-meso-diaminopimelyl-(D)-alanine).

phages,91.4%; polymorphonuclear neutrophil leukocytes; 1.20,; lymphocytes; 7.44’;; n = 5) and that of control mice was 3.76 t 0.32 X lo6 per cavity (macrophages;93.8!‘;,; polymorphonuclear neutrophil leukocytes; 0.2CiJ,lymphocytes, 6.OY’;);n = 5). Preparation of spleen cells. Four-week-old male BALB/c micewere given FK565 intraperitoneally at a dose of 0.01 mg/kg per day. Spleenswere removed, and spleen cellsweredissociatedmechanically by squeezingthe spleen through a mesh screen. These cells were suspendedin RPMI-1640 medium (Gibco, Grand Island, N.Y .). Spleen cellsthat were obtained from untreated mice were usedas controls. Inhibition of viral multiplication by PECs and spleen cells. Inhibition of viral multiplication wasdetermined by

assayaccording to the method of Morahan et al.‘l ConAuent BALB/c 3T3 cells were infected with EMC virus at multiplicity of infection 1. After virus adsorption for 1 hour at 37” C, PECs or spleencells were added to the BALB/c 3T3 cell cultures (at an effector to target ratio 10 and 100, respectively) and incubated for 16hoursat 37’ C in EMEM in a humidified atmospherethat contained 5ci COz. The culture supernatantswerecollected after the cellshad been frozen and thawed, and virus titration wasperformed. Effect of FK565 in vivo. Four-week-old BALB/c mice were inoculated intraperitoneally with 100pfu of EMC virus suspendedin 0.1 ml of PBS. Experiment 1. To evaluate the virus titer and the histologic condition of the heart, FK565 (1 mg/kg once a day) wasadministeredintraperitoneally, beginning 1 day before (group 1, n = 10) or on the day of virus inoculation (group 2, n = 10). The control mice were given intraperitoneal injections of 0.1 ml of PBS (group 3, n = 9). FK565 was administered for 4 consecutive days, and the animals were killed on day 8. Experiment 2. To evaluate the effect of FK565 on survival, five groups of animals were studied. FK565 (0.01 mg/kg once a day) was administered intraperitoneally, starting 1 day before virus inoculation (group 4, n = 10). Other groupsof mice received FK565 (1 mg/kg oncea day) beginning 1 day before (group 5, n = lo), on the sameday of (group 6, n = lo), on 1 day after (group 7, n = 10) virus

430

August

Sat0 et al.

Amencan

Table I. Jnhibition spleen cells ____--__

FK565

(pg/ml.

each

of viral

multiplication

Heart

1992 Journal

by PECs anti

n=6)

Fig. 2. Effect of FK565 on EMC virus infection in human amniotic cells. Treatment of cells with FK565 did not reduce plaque formation.

E6

I

n=7

n=lO

n=lO *P

Effect of immunostimulant therapy on acute viral myocarditis in an animal model.

The effect of immunostimulant therapy on acute viral myocarditis, which was induced with encephalomyocarditis virus, was investigated in 4-week-old ma...
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