Heart 1996;75:596-601

Long-term follow up of patients with dilated heartmuscle disease treated with human leucocyticinterferon alpha or thymic hormonesInitial results

Milutin Miric, Jovan Vasiljevic, Milovan Bojik Popovic, Keserovic,Milan Pe§ic

CardiologyDepartment,"Dedinje"CardiovascularInstitute, Beograd,YugoslaviaM MiricJ VasiljevicM BojicZ PopovicInstitute forImmunobiology andVirusology "Torlak",Beograd, YugoslaviaN KeserovicInstitute forImmunology andThymus Research,Bad Harzburg,GermanyM PedikCorrespondence to:Dr M Miric, DedinjeCardiovascular Clinic, 27.mart 41, 11000 Beograd,Yugoslavia.Accepted for publication13 December 1995

AbstractObjective-To determine whether givinginterferon-a or thymomodulin in additionto conventional treatment improves car-

diac function in patients with idiopathicmyocarditis and idiopathic dilated car-

diomyopathy.Design-Single-centre, randomised, open

label, parallel group comparison of con-

ventional treatment plus interferon-a,conventional treatment plus thymomod-ulin, and conventional treatment alone.Patients-38 patients aged 19-54 years (23men) with biopsy-proven myocarditis or

dilated cardiomyopathy. 12 were treatedwith conventional treatment alone, 13were treated with interferon-a and con-ventional treatment, and 13 with thymo-modulin and conventional treatment.Setting-Tertiary cardiac referral centre.Main outcome measures-Clinical evalua-tion, echocardiography, and Holter moni-toring at baseline, 6 months, and 1 and 2years. Radionuclide ventriculography atrest and during exercise after 2 years.

Endomyocardial biopsy at baseline andafter a year if the initial diagnosis was

myocarditis.Results-Left ventricular ejection frac-tion was improved in 21 (81%) of 26patients after interferon-a or thymomod-ulin administration and in 8 (66%) of 12conventionally treated patients (P < 0.05)at 2 year follow up. The maximum exer-cise time was significantly longer at 2-year follow up in patients treated withimmunomodulators (mean (SEM) 5.1(0.6) minutes for interferon-a and 5*0(0.4) minutes for thymomodulin) than in

conventionally treated patients (3.3 (0.4)minutes). Left ventricular ejection frac-tion during exercise (assessed by radionu-clide ventriculography) improved in 9 of12 patients treated with interferon-a, 10of 12 patients treated with thymomodulin,and 3 of 9 conventionally treated patientsat 2 year follow up. The electrocardio-gram was normal in 21 (88%) of 24patients after interferon-a or thymomod-ulin treatment and 2 (22%) of 9 conven-

tionally treated patients. At 2 year followup, 19 (73%) of 26 patients treated withimmunomodulators and 4 (25%) of 12conventionally treated patients hadimproved their fimctional class.Conclusions-The results suggest that

treatment of idiopathic myocarditisand/or idiopathic dilated cardiomyopathywith interferon-a or thymomodulininduces an earlier and significantly supe-rior clinical improvement than conven-tional treatment alone.

(Heart 1996;75:596-601)

Keywords: idiopathic myocarditis; idiopathic dilatedcardiomyopathy; interferon-o, thymic hormones

Enteroviruses and other viruses or viralsequences have been found in the cardiac tis-sues of patients with idiopathic myocarditis andidiopathic dilated cardiomyopathy.'-3 Possiblycardiotropic viruses cause not only a sympto-matic or asymptomatic initial infection but alsopersist in the myocardium of some patients.s6During persistent infections there is frequentlya reduction in the virally encoded surface glyco-proteins expressed on the outer membrane ofinfected cells.78 This reduction in viral proteinson infected cells provides an advantage for thepersisting virus, because such cells are less vul-nerable to immunological attack by humoral orcell-mediated effector mechanisms. At thesame time, such infected cells fail to activatelymphocytes with natural killer cell activity.9 Adeficiency in host defenses, such as the inter-feron system, might contribute to the develop-ment of persistent infections. Ifviruses do causeidiopathic myocarditis and some cases of idio-pathic dilated cardiomyopathy, treatment withinterferon or its inducers may be appropriate,either because of its direct action on the targetcell or indirect effects (activation of cycotoxic-ity, modulation of immune responses, andinteraction with other mediators of immuneresponse). This was the rationale for our clini-cal trial of interferon-a or thymic hormones inpatients with dilated heart muscle disease. Wechose interferon-a because it is highly diffusibleand easily reaches distant organs after subcuta-neous injection and thymic hormones becausethey induce a rise in the concentration ofendogenous interferon and stimulate naturalkiller and T cell activities.'0 We performed aserial evaluation of clinical data over a two yearperiod to document the possible influence ofimmunomodulatory treatment on the naturalcourse of idiopathic myocarditis and idiopathicdilated cardiomyopathy, because these diag-noses could represent two forms of the samedisease."I

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Long-termn follow up ofpatients with dilated heart muscle disease treated with human leucocytic interferon alpha or thymic hormones

Patients and methodsWe studied 38 patients (15 women and 23men) in whom the diagnosis of idiopathicmyocarditis or idiopathic dilated cardiomy-opathy had been confirmed by biopsy and whohad a left ventricular ejection fraction (LVEF)of < 45% at angiography. Those with postpar-tum and familial cardiomyopathy, giant-cellmyocarditis, and all forms of secondarymyocarditis were excluded, as were patientswith ischaemia, valvar or congenital heart dis-ease, severe hypertension, alcoholism, preg-nancy or postpartum, and autoimmune orother life-threatening disease. Initial evalua-tion included clinical symptoms, immunologi-cal tests, chest x rays, electrocardiogram, crosssectional echocardiographic examination, rightand left heart haemodynamic studies, andright ventricular endomyocardial biopsy.Three or four myocardial tissue samples weretaken and studied by light microscopy toestablish the histological diagnosis, accordingto the Dallas criteria'2 (femoral approach,King's bioptome). Molecular biological tech-niques including in situ hybridisation andpolymerase chain amplification were not avail-able at the time of the study.

IMMUNOLOGICAL INVESTIGATIONSBlood was taken for measurement of virusneutralising antibody titres, humoral, and cel-lular studies. Humoral studies included: (a) asearch for circulating antibodies against car-diac muscle, skeletal muscle, smooth muscle,and parietal cells as well as those against com-ponents of the nuclei and the mitochondria(immunofluorescence assay) 13; (b) analysis ofsera for IgM, IgG, IgA, IgD, and IgE, (lasernephelometry) 14; and (c) detection of circulat-ing immune complexes (laser nephelometrywith LN Latex CIC Reagent, Behring).15Among cellular studies, phenotyping ofperipheral blood mononuclear cells and nat-ural killer cell assay (5"Cr release assay)'6 wereperformed to define the patient's lymphocytesubpopulations and non-specific cell-killingability. For phenotyping of peripheral bloodmononuclear cells we used monoclonal anti-bodies (OKT3, OKT4, OKT8 (OrthoDiagnostics, Raritan, NJ) and Leu-7 (Becton-Dickinson, Mountain View, CA)), fluoresceinisothiocyanate staining, and flow cytometricenumeration.'7-'9 Immunological studies wererepeated weekly and monthly during the firstsix months of follow up.The study design was a prospective, ran-

domised, open label comparison of conven-tional treatment with conventional treatmentplus immunomodulation with interferon-a orthymic hormones. After approval by theInstitution Review Board and after consentforms were signed, the patients were randomlyassigned to three treatment groups: (1) 12patients received conventional treatment forcongestive heart failure, (2) 13 patientsreceived conventional treatment plus inter-feron-a; and (3) 13 patients received conven-tional treatment plus thymic hormone.Treatment lasted for three months. For thenext three months all patients received con-

ventional treatment alone. The criteria foradministration of conventional treatment wereset at the beginning of the study. A specificstepwise conventional regimen, with identicaldoses in all treatment groups, was given by thesame physician in the order listed below:* reduced salt intake (less than 4 g a day).* Digoxin (serum concentration of 1-2

mg/ml).* Frusemide (40-160 mg/day).* Captopril (25 mg three times a day).* Metolazone (2-5-5 mg/day).* Warfarin (ifLVEF was < 35%) to maintain

the international normalised ratio for pro-thrombin time in the range 2 5-4 0.These steps were followed in order as neces-

sary.Purified leucocytic interferon-a (Torlak,

Beograd) was given at 3 million U/M2 of bodysurface area subcutaneously three times aweek. Thymus-TFX Thymomodulin (10 mg)(Thymoorgan, Pharmazie, Vieneburg,Germany) was given subcutaneously threetimes a week. Thymus-TFX thymomodulin is abiologically active extract of the thymus glandof calves consisting of polypeptides between4000 and 6000 daltons.

At six month, one year, and two year followup we performed clinical evaluation, echocar-diographic studies (biplane method for LVEFmeasurements), chest x rays, and Holter mon-itoring. At one year, we repeated the right ven-tricular endomyocardial biopsy if the initialdiagnosis was myocarditis. At two yearsradionuclide ventriculography gated blood-pool technique was performed at rest and dur-ing exercise.

STATISTICAL ANALYSISMeasurements were mean (SEM). Statisticalanalysis was performed using analysis of vari-ance for repeated measures (Newman-Keulsmultiple comparisons) and analysis of varianceby the modified t test (Bonferroni) as appro-priate. Results were regarded as significant if Pwas less than 0 05.

ResultsFour patients had histological diagnosis ofactive and 11 patients of borderline myocardi-tis. Their average age at presentation was 29-3years (range 19-40 years). Twenty threepatients had a histological diagnosis of idio-pathic dilated cardiomyopathy (mean age atpresentation of 31-7 years (range 19-54years)).We found raised virus neutralising antibody

titres, usually to Coxsackie B viruses, in 14patients.

At the start of the study two patients were inNew York Heart Association class II, 26patients were in class III, and 10 in class IV.There was no significant baseline medical dif-ference among the three treatment groups.Left ventricular function assessed by angiogra-phy at the beginning of the study was similar(LVEF was 22-7 (1-2)% in patients treatedwith interferon-a, 22 (14)% in patients treatedwith thymomodulin, and 23-9 (1.1)% in con-

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Miri4 Vasiljevi4 Boji4 Popovi4 Keserovi4 Pelic

Figure 1 Natural killercells as a percentage ofperipheral bloodmononuclear cells (PBMC)in 38 patients with dilatedheart muscle disease duringtreatment. IFN, interferon-a; TH, thymomodulin,P < 00001 (ANOVA forrepeated measures,Newman-Keuls multiplecomparisons, error bars =SEM).

Figure 2 Effects of threetreatments on natural killercell activity andneutralising antibody titresduring six month follow upin 38 patients with dilatedheart muscle disease.IFN = Interferon-a.

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Left ventricular ejection fraction (LVEF) at the beginningof the study and after two yearfollow up in patients withdilated heart muscle disease according to diagnosis andtreatment

No Start No 2y

Idiopathic myocarditis:5 23-0 (1-2) 31-0 (2-1)5 23-6 (1-3) 32-8 (1-4)5 23-4 (1-0) 27-6 (1-4)

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ventionally treated patients). Thirty fivepatients had electrocardiographic abnormali-ties. Natural killer cells were absent or signifi-

50 cantly decreased in all patients, while natural40

killer cell activity was low in 35 patients.__40 The relative number of natural killer cells

30 increased significantly after two weeks of treat-_ - 20 S ment in the interferon-a group and returned

_ 10 to the baseline count after six months (fig 1).t0 In contrast to this sharp, short term increase in

24 natural killer cells, we found a progressive,long term increase in the thymomodulingroup. A significant increase in natural killercell activity was seen in the interferon-a and

0thymomodulin treated patients. This started at

40 two weeks and lasted for months (from 30-630 (1-4)% to 48-5 (1'9)% in the interferon-a0group, and from 286 (1-2)% to 44.7 (1.6)%

20 0 in the thymomodulin group). We did not find al t10 change in natural killer cell activity in conven-

24 ° tionally treated patients (P < 0 0001, analysisof variance for multiple comparisons).Increase in the relative number of natural

ent killer cells and natural killer cell activity wasfollowed by significant decrease in neutralising-50 antibody viral titres in patients treated withJ 40 immunomodulators (fig 2).

=4 - 30 . In 10 interferon-a treated and 11 thymo-20 - modulin treated patients, resting LVEF

- 10 assessed by echocardiography improved at six0 months and showed further improvement at

24 two year follow up (fig 3). Resting LVEFimproved in eight conventionally treated

Antibody titre patients at two year follow up. This improve-NK cell activity ment was found in a significantly lower pro-

portion than in patients treated withimmunomodulators (P < 0 05, analysis of

Figure 3 Effects of threetreatments on leftventricular ejection fraction(LVEF) in 38 patientswith dilated heart muscledisease treated with IFN-aand conventionaltreatment; thymomodulinand conventionaltreatment, andconventional therapy alone(mean (SEM), P <0 0001) (ANOVA forrepeated measures,Newman-Keuls multiplecomparisons).

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Long-term follow up ofpatients with dilated heart muscle disease treated with human leucocytic interferon alpha or thymic hormones

Figure 4 Left ventricularejection fraction (LVEF)during exercise in 33patients at two yearfollowup during treatment withIFN-a and conventionaltreatment, thymomodulinand conventionaltreatment, andconventional treatmentalone (mean (SEM), P <0 0001) (ANOVA forrepeated measures,Newman-Keuls multiplecomparisons).

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variance for multiple comparisons). The tableshows changes in resting LVEF during twoyear follow up in the treatment groups accord-ing to the diagnosis. There were too fewpatients in each group for these changes to beanalysed statistically. At the two year follow upleft ventricular ejection fraction was higher atpeak exercise than at rest in nine patientstreated with interferon-a and 10 with thymo-modulin (fig 4). In seven conventionallytreated patients, the ejection fractiondecreased or failed to increase on exercise,indicating an abnormal haemodynamicresponse. Maximum exercise time at two yearfollow up was 5-1 (0 6) minutes in the inter-feron-a group, 5 (0 4) minutes in thymomod-ulin group, and 3-3 (0-4) minutes in theconventionally treated group (P < 0-01, analy-sis of variance, modified t test, Bonferroni).

Biopsy was repeated at one year. In fivepatients with initial diagnosis of myocarditiswho were treated with interferon-a (two activeand three borderline) myocarditis hadresolved. Of five patients treated with thymo-modulin (one active and four borderlinemyocarditis), one had resolving myocarditisand four had resolved myocarditis. In the con-ventionally treated group, one patient who ini-tially had active myocarditis had resolvingmyocarditis, two who initially had borderlinemyocarditis had resolved myocarditis, and twowho initially had borderline myocarditis had ahistological diagnosis of dilated cardiomyopa-thy.

NYHA Interferon-a0 1 yr 2yr

Thymomodulin0 1 yr 2 yr

Conventional treatment0 1 yr 2 yr

1

51~~~~~~~~~~~~~~~~~~~~

1~~~~~~~~~~~~~

Figure 5 NYHA classification at two yearfollow up in 38 patients with dilated heartmuscle disease.

In nine patients treated with interferon-aand six treated with thymomodulin NYHAclass had improved at one year (fig 5). At twoyear follow these patients maintained theirimprovement. In five conventionally treatedpatients the functional class had improved atone year, and in one of them it had worsenedagain at two year follow up.One interferon-a treated patient with

biopsy confirmed idiopathic dilated cardiomy-opathy died five months after presentation. Itwas a sudden death outside hospital. One thy-momodulin treated patient died at ninemonths of an embolic cerebrovascular acci-dent. Three patients in the conventionallytreated group died during two year followup-one suddenly and two from end stage car-diac failure.

DiscussionDilated heart muscle disease can be idiopathic(IDC) or secondary to other causes, such asviral myocarditis or viral perimyocarditis.'0The criteria for IDC in our study were angio-graphic evidence of cardiomegaly and reducedejection fraction (< 45%); exclusion of coro-nary artery disease, valvar heart disease, hyper-tension, and other forms of secondary heartmuscle diseases; and histopathological find-ings of hypertrophy and branching of themyocytes, with diffuse or focal fibrosis. Thecriteria for idiopathic myocarditis were biopsyconfirmed inflammation of the myocardiumwith infiltrate, focal necrosis, and interstitialoedema with or without fibrosis. Fourteenpatients also had significant changes (> three-fold increase) in virus neutralising antibodytitres against cardiotropic viruses. RNA andDNA probes for cardiotropic viruses were notavailable in our study.Though there are many causes of dilated

cardiomyopathy and the aetiology is frequentlydifficult to determine, it is becoming increas-ingly apparent that after an acute or subacuteepisode of viral myocarditis the virus can persistin the cardiac tissues and cause dilated car-diomyopathy.621 In human diseases thought tobe caused by putative persistent infections,

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Miric Vasiljevi ; Bojuc, Popovic Keserovic Pelic

there is evidence of diminished interferon pro-duction and natural killer activity by lympho-cytes.22 23

Left ventricular ejection fractions improvedin 21 (81%) of 26 patients after interferon-aor thymomodulin administration and to alesser extent in eight (66%) of 12 convention-ally treated patients (P < 001) at six monthfollow up. Maximum exercise time was signifi-cantly longer at two year follow up in patientstreated with immunomodulators. Left ventric-ular reserve, assessed by rest and exerciseradionuclide ventriculography was signifi-cantly higher in patients treated withinterferon-a or thymomodulin. The electro-cardiogram was normal in 21(88%) of 24patients after interferon-a or thymomodulintreatment and in two (22%) of nine conven-tionally treated patients. At two year followup, 19 (73%) of 26 patients treated withimmunomodulators and four (25%) of 12conventionally treated patients showed animprovement in functional class.We found a significant increase in relative

number of natural killer cells and enhance-ment of natural killer cell activity in patientstreated with interferon-a or thymomodulin.Both the increase in natural killer cell numberand activation occurred quickly with inter-feron-a and disappeared after four weeks oftreatment. Activation was slower with thymo-modulin but lasted significantly longer.Patients treated conventionally had nochanges in relative number of natural killercells and natural killer cell activity.

Increased natural killer cell activity is associ-ated with a more effective inflammatoryresponse and less severe initial myocarditis.'4The natural killer cell response is mainlyresponsible for elimination of virus in the earlyphase of the disease, but when viral infectionpersists it could also be important in eliminat-ing virus in late phase of the disease. Our find-ings accord with the suggestion of Mason etaP4 that a strong spontaneous immuneresponse could be a beneficial rather than aninitiating factor in myocarditis24 and also withthe concept that stimulation of the immuneresponse by immunomodulators could be ofbenefit even in idiopathic dilated cardiomy-opathy.Our data strongly suggest treatment of

myocarditis and idiopathic dilated cardiomy-opathy with interferon-a or thymomodulin,and in our opinion they accord with the con-clusions of Mason et al, who do not recom-mend the routine use of immunosuppressivedrugs. Nonetheless, immunohistological find-ings in some groups of patients indicate thatimmunosuppressive drugs may be beneficial.

There were minor complications of endo-myocardial biopsy in two of the 38 patientsbefore treatment was started and in one (6%)of the 15 repeat biopsies. Eleven of the 13patients treated with interferon-a had a 'flu-like symptom dominated by fever, a few hoursafter the start of treatment. The peak tempera-ture tended to decrease with repeated injec-tions and usually disappeared after 10 days oftreatment. In two patients it was associated

with lassitude and malaise. Cardiovascularand neurological symptoms were not reported.Reversible mild leucothrombocytopenia wasseen in six patients. Treatment did not haveany significant effect on serum transaminases.Reversible heart dilatation25-27 was not seeneither.

Four of the 13 patients treated with thymo-modulin had a slight, but transient increase inweight. In two women menstrual bleeding wasprolonged.

Patients with a left ventricular ejection frac-tion < 30% have a poor prognosis and manysuch patients are on a progressively downhillcourse and are likely to die within a few years.The poor prognosis of idiopathic heart muscledisease justifies the continued pursuit of newtreatments. We believe that the improvementin cardiac function that occurred during thetwo year follow up in patients with idiopathicmyocarditis or idiopathic dilated cardiomy-opathy treated with interferon-a or thymo-modulin may have been the result ofenhancement of immune function in patientswho had a chronic myocardial viral infection.Our results suggest that a combination of

interferon-a and thymomodulin couldimprove function more than either drug givenon its own-because of the early effects ofinterferon and the sustained effects of thymo-modulin on immune function.

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2 Kandolf R, Ameis D, Kirschner P, Canu A, HofschneiderPH. In situ detection of enteroviral genomes in myocar-dial cells by nucleic acid hybridization: an approach tothe diagnosis of viral heart disease. Proc Nail Acad SciUSA 1987;84:6272-6.

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