Clin. exp. Immunol. (1976) 26, 86-90.
Anti-heart autoantibodies in ischaemic heart disease patients D. T. GOLAN * & A. KURS BAUM Clinical Immunology Service, Rothschild Hospital, Department of Medicine B, Rambam Hospital, Aba Khoushy School of Medicine, Haifa, Israel
(Received 27 February 1976)
SUMMARY
One hundred and ninety-nine ischaemic heart disease (IHD) patients were studied with regard to the prevalence of anti-heart autoantibodies (AHA). The incidence of AHA in IHD patients was 1%: one out of 102 patients who suffered acute myocardial infarction (AMI), one out of seventy-two patients who suffered from acute coronary insufficiency (ACI), and none out of twenty-five patients with other signs and symptoms of IHD, had AHA in their sera. An additional 2% of patients who suffered from AMI developed detectable antibody levels during a follow-up period of 15 days. In comparison, 53%o of patients (eight out of fifteen) who underwent heart surgery and who had no AHA prior to operation, developed these antibodies in their sera during 1-2 weeks following operation. INTRODUCTION The relation of cardiac manifestations to autoimmune responses i.e. appearance of AHA, has remained uncertain or even controversial (Kaplan & Frengley, 1969) although many years have elapsed since it was first described (Cavelti, 1945). Serum autoantibodies reactive with heart tissue constituents (either homologous or heterologous) may occur in a number of cardiac disorders (Ehrenfeld, Gery & Davies, 1961; Hess et al., 1964; Engle et al., 1974) presumably as a result of stimulation by antigens that have been released or altered by myocardial injury due to infection, infarction, cardiac surgery or trauma to heart or pericardium. However, circulatory autoantibodies may also appear after streptococcal infection in response to an antigenically cross-reactive constituent of the group A Streptococcus. (Zabriskie & Freimer, 1966). The relation of such autoantibodies to heart tissue to underlying cardiac disease remains an active field of investigation. In order to further elucidate this issue an attempt was made to find out the prevalence of AHA in a relatively large group of IHD patients and correlate this finding with their clinical state. MATERIALS AND METHODS Serum specimen. Freshly separated sera, harvested with sterile technique were stored at - 20'C until examination. Blood was collected on the first day of admission from all patients. A second sample was withdrawn from patients of groups I on 14-15th day of hospitalization, whereas from group no. II blood was collected before discharge (on day 7). Blood collection from group no. IV was performed preoperatively and 2-3 weeks following operation. Patients. Group no. I. Patients who were admitted to the hospital because of AMI (diagnosed on the basis of clinical history, typical ECG changes and appropriate elevated enzyme levels). Group no. II. Patients who were admitted to the hospital because of ACI (diagnosed on the basis of clinical history, ischaemic ECG changes without a pattern of AMI and without elevation of appropriate enzyme levels). Group no. III. Patients with IHD (a documented clinical history of a previous MI, or anginal pains) who were admitted to the hospital because of an unrelated acute disease. Group no. IV. Patients who were admitted to the hospital for heart surgery (congenital or rheumatic heart diseases). Immunofluorescence (IF). Rat heart sections were cut at 4 pm thickness in a cryostat at - 220C and thawed on a slide. The sections were then air-dried and given a final rinse in phosphate-buffered saline (PBS) pH 7-2, 0-01 M. Diluted test serum
Correspondence: Dr Dov T. Golan, Clinical Immunology Service, Rothschild Hospital, Aba Khoushy School of Medicine, P.O.Box 4940, Haifa, Israel.
86
Anti-heart autoantibodies
87
(1:10) was applied to the sections for 30 min in a moist chamber at room temperature (RT); the slides were then washed for 30 min in three changes of PBS at 370C and incubated with fluorescein isothiocyanate conjugated goat anti-human polyvalent immunoglobulins (Hyland, Travenol Laboratory) diluted 1:30 for 30 min at RT. The slides were again washed and the sections mounted in buffered glycerol. A 'Zeiss' standard fluorescence microscope (with a HBO-200W/4 mercury lamp, exciter filter UG-I, red-excluding filter BG-38, barrier filters 47,65), was used for viewing the sections. Specificity of the reaction was demonstrated by negative results for other tissue specific antigens such as anti-thyroid, anti-parietal cells, or anti-mitochondrial antibodies. Readings. The slides were read in duplicates independently by two investigators (D.G., A.K.). The degree of fluorescence was graded on a scale of 0-3+, 1 + fluorescence being required for a positive reading. This standard was adopted when experience showed that a + degree of fluorescence was difficult to interpret. Disagreement occurred on some of the total of 730 slides only where the distinction between 0 and + had to be made. Throughout the study many sera (both positive and negative ones) were repeatedly retested with consistent results.
RESULTS The age and sex distribution of patients of groups I-III is shown in Table 1. In these groups that represent various stages of IHD the male/female (M: F) ratio ranges between 3: 1 to 1 5: 1, with a reversal of that trend at the higher age groups as is well known in this disease. Table 2 summarizes the results of the various subgroups of patients with IHD. Only one out of 102 patients (1%) with AMI (group no. I) had a positive result on day of admission and remained so until discharge. Two other TABLE 1. Age and sex distribution of IHD patients
Group No. I Group No. II Group No. III Age
M
F
Anti-heart autoantibodies in ischaemic heart disease patients D. T. GOLAN * & A. KURS BAUM Clinical Immunology Service, Rothschild Hospital, Department of Medicine B, Rambam Hospital, Aba Khoushy School of Medicine, Haifa, Israel
(Received 27 February 1976)
SUMMARY
One hundred and ninety-nine ischaemic heart disease (IHD) patients were studied with regard to the prevalence of anti-heart autoantibodies (AHA). The incidence of AHA in IHD patients was 1%: one out of 102 patients who suffered acute myocardial infarction (AMI), one out of seventy-two patients who suffered from acute coronary insufficiency (ACI), and none out of twenty-five patients with other signs and symptoms of IHD, had AHA in their sera. An additional 2% of patients who suffered from AMI developed detectable antibody levels during a follow-up period of 15 days. In comparison, 53%o of patients (eight out of fifteen) who underwent heart surgery and who had no AHA prior to operation, developed these antibodies in their sera during 1-2 weeks following operation. INTRODUCTION The relation of cardiac manifestations to autoimmune responses i.e. appearance of AHA, has remained uncertain or even controversial (Kaplan & Frengley, 1969) although many years have elapsed since it was first described (Cavelti, 1945). Serum autoantibodies reactive with heart tissue constituents (either homologous or heterologous) may occur in a number of cardiac disorders (Ehrenfeld, Gery & Davies, 1961; Hess et al., 1964; Engle et al., 1974) presumably as a result of stimulation by antigens that have been released or altered by myocardial injury due to infection, infarction, cardiac surgery or trauma to heart or pericardium. However, circulatory autoantibodies may also appear after streptococcal infection in response to an antigenically cross-reactive constituent of the group A Streptococcus. (Zabriskie & Freimer, 1966). The relation of such autoantibodies to heart tissue to underlying cardiac disease remains an active field of investigation. In order to further elucidate this issue an attempt was made to find out the prevalence of AHA in a relatively large group of IHD patients and correlate this finding with their clinical state. MATERIALS AND METHODS Serum specimen. Freshly separated sera, harvested with sterile technique were stored at - 20'C until examination. Blood was collected on the first day of admission from all patients. A second sample was withdrawn from patients of groups I on 14-15th day of hospitalization, whereas from group no. II blood was collected before discharge (on day 7). Blood collection from group no. IV was performed preoperatively and 2-3 weeks following operation. Patients. Group no. I. Patients who were admitted to the hospital because of AMI (diagnosed on the basis of clinical history, typical ECG changes and appropriate elevated enzyme levels). Group no. II. Patients who were admitted to the hospital because of ACI (diagnosed on the basis of clinical history, ischaemic ECG changes without a pattern of AMI and without elevation of appropriate enzyme levels). Group no. III. Patients with IHD (a documented clinical history of a previous MI, or anginal pains) who were admitted to the hospital because of an unrelated acute disease. Group no. IV. Patients who were admitted to the hospital for heart surgery (congenital or rheumatic heart diseases). Immunofluorescence (IF). Rat heart sections were cut at 4 pm thickness in a cryostat at - 220C and thawed on a slide. The sections were then air-dried and given a final rinse in phosphate-buffered saline (PBS) pH 7-2, 0-01 M. Diluted test serum
Correspondence: Dr Dov T. Golan, Clinical Immunology Service, Rothschild Hospital, Aba Khoushy School of Medicine, P.O.Box 4940, Haifa, Israel.
86
Anti-heart autoantibodies
87
(1:10) was applied to the sections for 30 min in a moist chamber at room temperature (RT); the slides were then washed for 30 min in three changes of PBS at 370C and incubated with fluorescein isothiocyanate conjugated goat anti-human polyvalent immunoglobulins (Hyland, Travenol Laboratory) diluted 1:30 for 30 min at RT. The slides were again washed and the sections mounted in buffered glycerol. A 'Zeiss' standard fluorescence microscope (with a HBO-200W/4 mercury lamp, exciter filter UG-I, red-excluding filter BG-38, barrier filters 47,65), was used for viewing the sections. Specificity of the reaction was demonstrated by negative results for other tissue specific antigens such as anti-thyroid, anti-parietal cells, or anti-mitochondrial antibodies. Readings. The slides were read in duplicates independently by two investigators (D.G., A.K.). The degree of fluorescence was graded on a scale of 0-3+, 1 + fluorescence being required for a positive reading. This standard was adopted when experience showed that a + degree of fluorescence was difficult to interpret. Disagreement occurred on some of the total of 730 slides only where the distinction between 0 and + had to be made. Throughout the study many sera (both positive and negative ones) were repeatedly retested with consistent results.
RESULTS The age and sex distribution of patients of groups I-III is shown in Table 1. In these groups that represent various stages of IHD the male/female (M: F) ratio ranges between 3: 1 to 1 5: 1, with a reversal of that trend at the higher age groups as is well known in this disease. Table 2 summarizes the results of the various subgroups of patients with IHD. Only one out of 102 patients (1%) with AMI (group no. I) had a positive result on day of admission and remained so until discharge. Two other TABLE 1. Age and sex distribution of IHD patients
Group No. I Group No. II Group No. III Age
M
F
