Acute rheumatic fever cellular immunity rheumatic carditis

Pediat. Res. 13: 16-20 (1979)

Cellular Immunity in Children with Acute Rheumatic Fever and Rheumatic Carditis NURTEN MERIC'~" Dr. Sami Ulus Children's Hospital, Ankara, Turkey

Immunology Laboratory, Hacettepe Children's Hospital, Institute of Child Health, Hacettepe University, Ankara, Turkey

Summary Cell-mediated immunity (CMI) was examined in 74 children with acute rheumatic fever (ARF) grouped as with or without carditis and inactive disease with valvular lesions and 26 healthy controls of the same age. Absolute lymphocyte count, skin tests with PPD, Candida, and streptokinase streptodornase (SK-SD) antigens were employed. The lymphocyte transformation test was performed by using phytohemagglutinin (PHA), SK-SD, Candida, and cardiac antigens, and incorporation of tritiated thymidine into DNA was measured. Absolute lymphocyte counts in all groups were not significantly different than those of controls. Skin test responses to PPD, Candida, and SK-SD antigens were significantly decreased in patients with rheumatic activity. In contrast, inactive rheumatic patients with valvular lesions showed higher percentages of positive reactions which were not significantly different than those of the healthy controls. The in vitro response to PHA was decreased in active rheumatic fever (RF) patients, whereas patients with inactive R F with valvular lesions showed a hyperactivity to this mitogen. Patients with inactive R F with valvular lesions also exhibited an exaggerated cellular reactivity to SK-SD antigen, but the patients in active groups showed more of a decreased reactivity than did the controls. Patients with evidence of rheumatic carditis exhibited the same degree of cellular reactivity to these antigens and mitogen as did patients without clinical evidence of rheumatic heart disease. Candida did not cause significant stimulation of blastic transformation in any of the study groups. The blastic transformation response to heart homogenates was present in 25% of the cases with active rheumatic carditis, in 15%without carditis, and in 46% of the patients with inactive valvular lesions. Plasma from the patients with active R F caused inhibition of the in vitro response of the normal lymphocytes to PHA. In the last few years, it has become increasingly apparent that, in addition to the well documented alterations in humoral immunity, ARF is also accompanied by abnormalities in cell-mediated immunity (CMI) where the precise significance in pathogenesis of rheumatic carditis remains controversial. Speculation Patients with active rheumatic fever may have depressed cellular immune responses both in vivo and in vitro to PHA and specific antigens. Observations on the responses to SK-SD and heart homogenate in inactive rheumatic fever patients suggest that patients with rheumatic carditis build up a cellular immune response to the heart. Our findings also suggest that the possibility of lymphocyte sensitization by heart tissue antigens might play a part in the

pathogenesis of rheumatic carditis. But the reason for hyporeactivity and the exact link if any between lymphocyte abnormalities and tissue lesions of rheumatic carditis remain to be elucidated. The concept of immunologic cross-reactivity is suggested by the presence of organ-specific antigens in mammalian heart, sharing of common antigens by group A Streptococci and the mammalian heart, and antibody reactive with the heart in some patients with rheumatic carditis, and diffuse deposits of y-globulin in the hearts of patients with active carditis (4, 14, 16, 28-30). A histologic inspection of the microscopic cardiac lesions in ARF, such as the area of acute myocarditis or Aschoff bodies themselves, often shows prominent cellular infiltrations with lymphocytes. There is indeed a voluminous literature dealing with various humoral antibody responses to streptococcal products or antecedent streptococcal infections in such patients. But the number of publications on abnormalities or anormalities of behavior among peripheral lymphocytes in patients with ARF is rather limited (5-7, 11, 20, 27). Little attention has been given to the role of cell-mediated immune responses in this disease (20,23), despite clear evidence that this type of immunity is important in experimental autoimmune diseases (12, 18, 19). In this study various parameters of cellular immunity were investigated with the hope of clarifying the status of CMI in ARF. MATERIALS AND METHODS

Seventy-four patients with ARF between the ages of 4 and 16 seen at Ankara, Dr. Sami Ulus Children's Hospital were included this study. All active patients classified as A R F satisfied the Jones criteria (1). Acute cases were studied within 1 week after the onset of the acute rheumatic fever episode. No patient was receiving steroids at the time of collection of peripheral blood leukocytes and the highest serum salicylate level of the patients included in this study was less than 3.2 mg/dl, which was shown not to affect in vitro lymphocyte response (25). The hemoglobin values were above 10 g/dl in all patients and controls. Patients and controls were divided into 4 groups: Group I consisted of 10 females and 16 males, between the ages of 6 and 16, with A R F and active carditis. Group I1 had 8 females and 15 males, from 4 to 13 years old, with A R F without active carditis. The diagnosis of active carditis rested on the presence of significant murmurs plus two or more of the following: muffled S1, S3 gallop, pericardial friction rub, radiologically evident cardiomegaly, conduction abnormalities or left ventricular hypertrophy o n electrocardiogram, and clinical signs of frank congestive failure. Group I11 consisted of 12 females and 13 males with inactive R F with valvular lesions. Acute phases of their illness had been previously

17

CELLULAR IMMUNlTY IN CHILDREN

observed by one of us (N. M.). The intervals between acute disease and testing in patients of this group ranged from 5 months to 4 years. Their ages ranged from 8 to 16 years. No patient in this group had positive acute phase reactants during this study. Group IV consisted of 26 control children in good health with no history of rheumatic fever. Their economic and racial background, as well as their geographic location, were essentially the same as that of the study patients. The ages of 14 females and 12 males ranged from 5 to 16 years. SKIN TESTS

All groups received intradermal injections of the following antigens: PPD, 3 TU in 0.1 m1 (3 1); Candida albicans, 50 PNU in 0.1 mi (32); and streptokinase-streptodornase (SK-SD), 50 U SK in ml and 12.5 U SD in ml(33). A positive response was considered to be 5 rnrn or more induration at 48 hr. LYMPHOCYTE CULTURES

Blastogenic transformation studies were performed according to the modification of the method of Moorhead et al. (24), as described previously (2). Venous blood was drawn into plastic syringes containing preservative-free heparin (Pan heparin) (34) and was allowed to sediment at 37' for 45 to 60 min. Then the plasma and buffy coat was harvested and centrifuged at 900 rpm for 10 min. The resulting supernatant was centrifuged at 1000 x g for 10 min to obtain cell-free plasma. Leukocytes were washed two or three times in Medium 199 (35) containing 10,000 U penicillin, 10,000 pg streptomycin (36) and 1% pooled heat-inactivated AB serum. Then the pellet was resuspended in Medium 199 containing 15% AB serum and the same antibiotic mixture. The final cell concentration was adjusted to 0.5 x lO%ononuclear cells/ml of culture in the 16 x 100-mm glass tubes with screw caps. The preservative-free antigens were added into the respective culture tubes in duplicate in the following doses which were found to be optimal after establishing dose-response curves in previous experiments performed in this laboratory: phytohemagglutinin (35a), 0.05 ml of 1/25 dilution; C. albicans (32); 0.15 ml. of 1/100 dilution in 50% glycerine, SK-SD (33), 25 pg in 0.05 ml. The cardiac antigen was prepared from the heart valves, papillary

muscles, and appendices obtained from the rheumatic fever patients at surgery (36) according to the method of Kaplan (15). In each experiment the following doses were used for each duplicate tube: 4000, 2000, 200, and 20 pg of cardiac antigen in a 0.1-ml volume. The tubes were incubated at 37O in a humidified 5% COs atmosphere for 55 hr for PHA and 144 hr for the antigens. Then 1 pCi of tritiated thymidine (specific activity, 17.0 Ci/mol) (37) was added to each tube. Control lymphocyte cultures without the antigen or mitogen were also pulsed with tritiated thymidine at the same time. Sixteen hours later, all of the cultures were harvested according to the modified filter paper disc method of Mans and Novelli (22). Then the discs were placed with 5 ml scintillation fluid in glass counting vials and placed in a beta scintillation counter (38) at 4'. Each sample was counted for 10 min. The results were expressed as cpm/106 lymphocytes. A stimulation index (SI) (cpm of stimulated/cpm unstimulated) of greater than 20 for PHA and greater than 2 for Candida, SK-SD, and cardiac antigens was considered a positive response (8, 13). The experiments on both the patients and controls were performed simultaneously using the same batches of the PHA and the antigens. STATISTICAL ANALYSIS

Student's t-test and variance analysis method were employed for comparing the results of the patients and the controls (17). Statistical analysis of the blastogenic transformation studies was based on the cpm, the SI, and the number of cases (%) responding to mitogen and antigens. RESULTS TOTAL LYMPHOCYTE COUNT

The mean values of the total lymphocyte counts of each group are listed in Table 1. There were no significant differences among the groups with active RF, inactive RF with valvular lesions, and the controls (P > 0.05). SKIN TESTS

The results are tabulated in Table 1. The patients with active RF and rheumatic carditis (group I) had less positive reactions to

Table I. Absolute [ymphocyte count and skin test response Skin Tests

Groups I. Active RF with carditis

PPD' Candida SK-SD Absolute lymphocyte No. of cases count/pl NO. positive % positive No. positive % positive No. positive % positive 26

2,873 f 344' (800-7,800)3 10 (or 38.470)~

1

3.8

5

19.2

8

30.8

17

65.4

18

69.2

3,517 + 4372 (1,100-10,000)~ 7 (or 30.4%)4 3,176 f 276' (800-10,000)3 1 1 (or 34.4%)'

11. Active RF without carditis

Active RF total

111. Inactive RF with valvular lesions

25

2,572 f 222' (1,000-5,100)~ 7 (or 28%)4

IV. Controls

26

3,119 f 2602 (1,10&5,900)3 6 (or 26%)4

' All were immunized with BCG previously. 'Mean % SE. Range. Number and percent with low count.

19

73

18

MERIC AND BERKEL

PPD, candida, and SK-SD antigens in comparison to groups 111 and 1V. This low response was also observed in patients with active R F without carditis (group 11). Skin test responses of the patients with inactive R F with valvular lesions to these antigens was not significantly different from those of the control group ( P > 0.05). The difference between the active R F patients (with or without carditis) and the control group (group IV) or the inactive R F patients with valvular lesions (group 111) was statistically significant ( P < 0.05-0.001). There was no significant difference between groups 111 and IV. I N VITRO

LYMPHOCYTE (BLASTOGENIC TRANSFORMATION) RESPONSE

Lymphocyte blastogenic transformation response to PHA was depressed in active R F patients (mean cpm, 42,575 ; S1, 30) when compared with the control subjects (mean cpm, 53,021; SI, 42.7) (Table 2). The patients with inactive R F and valvular lesions had approximately 2-fold higher reactivity than control subjects (mean cpm, 101,611; SI, 65). Although the degree of stimulation with PHA in active R F patients appears to be lower than that of the controls, statistical analysis of the data failed to reveal any significant difference when the results were expressed as total thymidin uptake (cpm) and as SI. But the number of cases responding to this mitogen in the active R F group was significantly lower (61.2%) than that of the controls (100%) (P < 0.001). The number of cases responding to PHA in the inactive R F patients was also fewer than that of the control group ( P < 0.05) (Table 2). The in vitro lymphocyte response to Candida antigen was also depressed in active R F patients (mean cpm, 1,778; SI, 1.36) when

compared with the inactive R F group with valvular lesions (mean CPM, 10,587; SI, 5.4) or to the healthy control group (cpm, 8,022; SI, 3.35). However, statistical comparison of the groups for the numbers of responding lymphocytes showed no significant differences (Table 3). The blastogenic response to SK-SD in patients with active ARF was significantly lower (mean cpm, 2,677; SI, 1.57) than that of controls (mean cpm, 7,848; SI, 4.6) ( P < 0.05) The patients with inactive A R F and valvular lesions exhibited an exaggerated cellular reactivity (mean cpm, 17, 019; SI, 9.1) to this antigen ( P < 0.05). The results in patients with carditis (mean cpm. 2.969; S1, 1.7) did not differ from patients without carditis (mean cpm, 2,330: SI, 1.4) (Table 4). The in vitro response to cardiac antigen (heart homogenate) was observed in 25% of the patients with active R F with carditis, in 15% of the patients with active R F without carditis. and in 46% of the patients with inactive R F with valvular lesions. The lymphocytes of the control group did not show any response to the stimulation with this antigen. Statistical analysis showed that the blastic transformation response to the cardiac antigen was significantly higher in patients with inactive R F with valvular lesions (group 111) than in patients with active R F (groups I and 11) ( P < 0.05). The comparison of the groups showed the following significant differences: the patients with active R F (groups I and 11) and the controls (group IV) ( P < 0.01), the patients with inactive R F with valvular lesions (group 111). and the controls group (IV) ( P < 0.001). The difference between groups I and 11 was not significant ( P > 0.05) (Table 4). The in vitro response to the cardiac antigen was usually ob-

Table 2. In vitro Iymphocyte (blastogenic transformation) response to PHA No. responding/no. % responding SI' cpm'. ' studied

Groups

Unstimulated cpm', "

I. Active RF with carditis 11. Active RF without carditis Active RF total 111. Inactive RF with valvular lesions

20/24

83.3

65 + 13.3 (0.9-2 17)

IV. Controls -

' Values given as means + SE; range given in parentheses. cpm: counts per minute after PHA stimulation. Table 3. In vitro lymphocyte (blasto~enictransformation) response to Candida anti~en No. responding/no. cum'. % res~onding S1' studied

Unstirnulated cum

I. Active RF with carditis 11. Active RF without carditis

Active RF total 111. Inactive RF with valvular lesions

9/24

IV. Controls I

Values given as means + SE; range given in parentheses. cpm: counts per minute after stimulation with Candida.

37.5

*

5.4 2.10 (0.1-32.9)

*

10,587 4,802 (1 89-76,4 19)

1,829 + 381 (378-6,958)

19

CELLULAR IMMUNITY IN CHILDREN Table 4. In vitro lymphocyte (blastogenic transformation) response to SK-SD and cardiac antigens1 SK-SD Cardiac antigen

Grou~s

No. responding/no. % restudied s~ondine.

-

S12

c~m"

No. responding/no. % restudied sponding

S12

cpm2.

I. Active RF with carditis

5/25

20

1.7 + 0.4 (0.3-9.3)

2,969 + 1,643 (273-41,8 11)

6/24

25

2.07 + 0.69 (0.7- 17)

1,723 -t 308 (336-5,229)

11. Active RF without carditis Active RF total

4/21

19

15

19.6

2,330 + 642 (84-9,954) 2,677 + 932 (84-41,811)

3/20

9/46

1.4 + 0.35 (0.3-7) 1.57 zk 0.29 (0.3-9.3)

9/44

20

1.48 + 0.25 (0.5-3.75) 1.79 + 0.38 (0.5-17.0)

1,699 + 187 (609-3,234) 1,693 -1- 186 (336-5,229)

111. Inactive RF with valvular lesions

8/24

33.3

9.1 + 3.58 (0.4-32.9)

17,019 + 6,033 (42677,301)

11/24

46

11.38 + 4.37 (0.3-46.5)

23,675 + 13,070 (399-103,030)

IV. Controls

8/22

36.3

4.6 + 2.48 (0.2-55.6)

7,848 f 3,421 (420-63,483)

0/22

0

1.07 -t 0.1 (0.7-1.9)

2,933 -+ 741 (12613,629)

Unstimulated cpm and SE are the same as in Table 3. Values given as means f SE; range given in parentheses. cpm: counts per minute after stimulation with Candida. Table 5. Effect of plasma from patients with active RF on in vitro lymphocyte response to PHA from normal controls PHA response (cpm) Healthy control % case no. Before plasma After ~lasma inhibition 65 77 None 98.6 98 93.5 98.5 98 93 84 14.5 None 69.4

' P: plasma sample. served in between dose ranges of 200 and 2000 pg. One patient responded to 4000 pg and the other four responded to a dose of 20 Pg. The effect of plasma from patients with active R F on the in vitro lymphocyte response to PHA from normal controls was checked in 10 experiments by using plasma from 13 such patients (Table 5). Ten of 13 plasma samples from patients with active R F caused 50% or more inhibition in the in vitro response of lymphocytes of the healthy controls. DISCUSSION An autoimmune mechanism is claimed in the pathogenesis of acute rheumatic fever and rheumatic carditis on the basis of studies performed by several investigators (7, 11, 14, 16, 20, 23). Depression of CMI plays an important role in the development of autoimmune diseases. Apparent defects in the lymphocyte response in the rheumatic states have been previously reported by a group of investigators. A diminished reactivity to streptolysin S among patients with R F was noted by Hirshhorn et al. (1 I). Francis and Oppenheim (5) reported hyporesponsiveness in peripheral lymphocyte cultures stimulated by pathogenic strains of group A streptococci when patients with rheumatic heart disease were compared with healthy controls. However, the in vitro response to PHA was normal in those patients (5). Decreased reactivity of lymphocytes in mixed

leukocyte culture from patients with R F was observed by Lueker and Williams (20). In this particular study, lymphocytes from A R F patients were found to be incapable of stimulating or responding to the lymphocytes of other acute R F patients. Although A R F cells were able to respond to the stimulation by normal cells in 50% of the experiments performed, they were usually incapable of stimulating normal cells (20). A study by Read et al. (26), using the migration inhibition system and testing streptococcal wall and membrane antigens, has shown that in ARF patients there is a significant cellular reactivity to streptococcal membrane antigens which contain the cross-reacting antigen with the heart. This abnormal response to streptococcal membrane antigens appeared to persist in rheumatic subjects for at least 5 years after the initial attack of RF. McLaughlin et al. (23) studied the in vitro response of peripheral blood lymphocytes to PHA, streptococcal cell wall antigens, and heart homogenates in children with rheumatic carditis and in healthy controls by using autologous plasma in the culture system. Neither the rheumatic children nor the control subjects showed statistically significant lymphocyte responses to heart homogenates. In contrast, both groups exhibited significant responses to PHA and streptococcal cell walls (23). The results of our study appear to indicate some sort of cellular deficiency or anomalous behavior among lymphocytes from patients with A R F when studied by different in vivo and in vitro tests. Although we did not observe any significant difference among the absolute lymphocyte counts of the patients with ARF, the patients with inactive R F and valvular lesions and the controls, the delayed hypersensitivity response assessed by skin testing with PPD, Candida, and SK-SD was markedly decreased in patients with active R F with or without carditis. In patients with inactive R F with valvular lesions, skin test responses were not significantly different than those of the normal controls. The in vitro response of the lymphocytes (checked by blastogenic transformation) to PHA was found to be decreased in a significant number of patients with active RF. The SK-SD response of active R F lymphocytes was significantly lower than that of controls. However, patients with inactive R F exhibited an exaggerated cellular reactivity to SK-SD and PHA. We observed a specific in vitro response of the peripheral blood lymphocytes from patients with rheumatic fever to the cardiac antigen. We chose the use of heart homogenate in our experiments rather than heart fractions, so that all antigenic determinants could be represented in the in vitro culture system. Because the optimal doses'of cardiac antigens were not known by us, we decided to use different concentrations of heart homogenate (between 20 and 4000 pg/ml culture) in

20

MERIC AND BERKEL

order to avoid some false-negative results which might have been due to insufficient or excessive amounts of antigen. The in vitro response to cardiac antigen was more prominent in the group of patients with inactive R F and valvular lesions (1 1 of 24 patients or 46% responded) than the active RF with carditis (25% of the patients responded), or the active R F without carditis (only 15% responded). The lymphocytes of the control children showed no blastic transformation response to the heart homogenate. We were unable to explain the discrepancy between our findings about cardiac antigens and those of McLaughlin et al. (23). The answer possibly lies in the antigen doses employed by their study, which was 10 and 100 pg. It is known that optimal antigen doses are necessary for the stimulation of lymphocytes. Decreased response of lymphocytes of our patients with the active RF to SK-SD antigen confirms the reports of Hirshhorn et al. ( I I ) and Francis and Oppenheim (5); but is not in agreement with that of Read et al. (26), who applied a different in vitro test, namely, the migration inhibition system to measure the response to streptococcal antigens. In our study the lymphocytes from patients with inactive RF with valvular lesions showed significant increases in DNA synthesis in the presence of SK-SD or PHA. Recently, Greenberg et al. (9) have shown that lymphocytes from ~ndividualscarrying HLA-B5 antigen are hyperreactive when incubated in vitro with varying concentrations of SK-SD antigen. Because HLA typing was not performed in our patients during this study, we cannot draw any conclusions in this respect. Studies of Malakian and Schwab (21) have shown that an immunosupressant factor is derived from group A streptococci. Recently, Ceppellini et al. (3) reported the occurrence of inhibition in the presence of serum with anti-HLA activity which may block the antigens on lymphocytes, and thus abrogate cell stimulation. Hirata and Terasaki (10) observed a cross-reactivity between M streptococcal protein and several HLA antigens. If, during the course of ARF, patients developed antibodies to M protein or other streptococcal antigens that cross-react with human cell surface antigens, particularly materials present on the surface of lymphocytes, such antibodies could conceivably block or interfere with cross stimulation. However, Lueker and Williams (20) reported that no evidence for such blocking antibodies was obtained in their studies. In our study, a 50% or more inhibition of the blastic transformation response of the normal lymphocytes to PHA by the plasma from active RF patients was observed in 10 of 13 cases. This suggests the presence of an inhibitory factor in the plasma of patients with active RF causing some degree of depression in the lymphocyte functions or cell-mediated immune response in these patients. However, the significance of enhancement of the PHA response by the plasma from two active ARF patients is not known at present. - In summary the present study serves to point out that, by the criterion of delayed hypersensitivity responses (skin tests and in vitro lymphoblastic transformation), CMI in acute rheumatic fever is abnormal. Further investigations are needed to find the exact relation between the lymphocyte abnormalities and the tissue lesions of carditis. REFERENCES AND NOTES I. American Heart Association, Council on Rheumatic Fever and Congenital Heart Disease: Jones Criteria (revised) for guidance in the diagnosis of rheumatic fever. Circulation. 32: 664 (1965). 2. August. C. S.. Merler. E., Lucas, D. 0 . and Janeway, C. A,: The response in vltro of human lymphocytes to phytohemagglutinin and to antigens after fraction on discontinuous density gradients of albumin. Cell. Immunol.. 1: 603 (1970). 3. Ceppellini. R.. Bonnard. G. D., Coppo, F.. and Miggiano. V. C.: Mixed leukocyte cultures and HL-A antigens: 11. Inhibition by anti-HL-A sera. Transplant. Proc.. 3: 63 (1971). 4. Espinosa, E.. Kushner. I., and Kaplan, M. H.: Antigenic composition of heart tissue. Am. J. Cardiol.. 24: 508 (1969). 5. Francis. T. C., and Oppenheim J. J.: Impaired lymphocyte stimulation by some Copyright O 1979 International Pediatric Research Foundation, Inc. 003 1-3998/79/1301-0016 $02.00/0

streptococcal antigens In patients with recurrent aphthous stomatit~s and rheumatic heart disease. Clin. Exp. Immunol.. 6: 573 (1970). 6. Francis. T. C.. Oppenheim. J. J.. and Barile. M. F.: Lymphocyte transformation by streptococcal antigens in guinea pigs and man. In: W. 0 . Rieke: Proceedings of the Third Annual Leukocyte Conference. p. 501 (Appleton Century-Crofts. New York, 1969). 7. Friedman, I., Laufer, A,, Ron, N., and Davies, A. M.: Experimental myocarditis: In vitro and in vivo studies of lymphocytes sensitized to heart extracts and groups A Streptococci. Immunology, 20: 225 (1971). 8. Gelfand, E. W., Berkel, A. I., Godwin, H. A., Rocklin, R. E., David, J. R., and Rosen, F. S.: Pernicious anemia, hypogammaglobulinaemia and altered lymphocyte reactivity. Clin. Exp. Immunol., 11: 187 (1972). 9. Greenberg, L. J., Gray, E. D., and Yunis, E. J.: Association of HLAs and immune responsiveness in vitro to streptococcal antigens. J. Exp. Med., 141: 933 (1975). 10. Hirata. A. A.. and Terasaki. P. I.: Cross-reactions between streptococcal M proteins and human transplantation antigens. Science (Wash. D.C.). 168: 1095 ( 1970). I I. Hirschhorn, K., Schreibman. R. R.. Verbo. S.. and Gruskin R. H.: The action of streptolysin S on peripheral lymphocytes of normal subjects and patients with acute rheumatic fever. Proc. Natl. Acad. Sci. USA. 52: 1151 (1964). 12. Jankovic. B. D., Mitrovic, K., Popeskovic. L.. and Milosevic. D.: Passive transfer of experimental thyroiditis, delayed hypersensitivity to thyroglobulin and anti thyroglobulin antibody formation in inbred Lewis rat. Clin. Exp. Immunol.. 5: 29 (1969). 13. Jehn, U. W., Nathanson, L.,and Schwarz, R.: In vitro lymphocyte stimulation by soluble antigen from malignant melanoma. N. Engl. J. Med., 283: 329 (1970). 14. Kaplan, M. H.: Autoimmunity to heart and its relation to heart disease. A review. Prog. Allergy. 13: 408 (1969). 15. Kaplan. M. H.. and Dallenbach. F. D. Immunologic studies of the heart tissue. J. Exp. Med., 113: 1 (1961). 16. Kaplan. M. H.. and Frengley. J. D.: Autoimmunity to the heart In cardiacdisease. Current concepts of the relation of autoimmunity to rheumatic fever, post cardiotomy and post infarction syndromes and cardiomyopathies. Am. J. Cardiol., 24: 459 (1969). 17. Kutsal, A., and Muluk Z.: Uygulamali Temel Istatistik. Hacettepe Universitesi yayinlari, Ankara, 1972. 18. Lennon, V. A., and Byrd, W. J.: Role of T lymphocytes in the pathogenesis of experimental autoimmune encephalomyelitis. Eur. J. Immunol., 3: 243 (1973). t9. Levin, S.. and Wenk. E.: The production and passive transfer of allergic adrenalitis. Am. J. Pathol., 52: 41 (1968). 20. Lueker. R. D., and Williams. R. C.: Decreased reactivity of lymphocytes in mixed-leukocyte culture from patients with rheumatic fever. Circulation. 46: 655 (1972). 21. Malakian, A. H.. and Schwab. J. H.: Biological characterization of an immunosuppressant from group A streptococci. J. Exp. Med.. 134: 1253 (1971). 22. Mans, R. J., and Novelli, G. D.: Measurement of the incorporation of radioactive aminoacids into protein by a filter paper disc method. Arch. Biochem. Biophys.. 94: 48 (1961). 23. McLaughlin. J. F.. Paterson. P. Y.. Hartz. R. S.. and Embury. S. H.: Rheumatic carditis: in vitro responses of peripheral blood leukocytes to heart and streptococcal antigens. Arthritis Rheum., 15: 600 (1972). 24. Moorhead, P. S., Nowell, P. C.. Mellman. W. J.. Battips. D. M.. and Hungerford, D. A.: Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell. Res., 20: 613 (1960). 25. Opelz, G.. Terasaki. P. I., and Hirata, A. A.: Supress~onof lymphocyte transformation by aspirin. Lancet. 2: 478 (1973). 26. Read. S. E.. Fisehetti. V. A., el al: Cellular reactivity studies to streptococcal antigens. J. Clin. Invest.. 54: 439 (1974). 27. Taranta, A,. Cuppari. G.. and Quagliata. F.: Dissociation of hemolytic and lymphocyte-transforming activities of streptolysis S preparations. J. Exp. Med., 129: 605 (1969). 28. Zabriskie. J. B.: Mimetic relationships between group A streptococci and mammalian tissues. Adv. Immunol.. 7: 147 (1967). 29. Zabriskie. J. B.: The relat~onshipof streptococcal cross-reactive antigens to rheumatic fever. Trans. Proc.. I: 968 (1969). 30. Zabriskie. J. B.. Hsu. K. C.. and Seegal B. C.: Heart reactive antibody associated with rheumatic fever: Characterization and diagnostic significance. Clin. Exp. Immunol.. 7: 147 (1970). 31. Refik Saydam Central Hygiene Institute. Ankara. Turkey. 32. Hollister Stier Laboratory. Yeadon. PA. 33. Lederle Laboratories. Pearl River. NY. 34. Abbott Laboratories, Detroit, MI. 35. Gibco Laboratory, Grand Island, NY . 35a. Difco Laboratories, Detroit, MI. 36. Kindly provided by Dr. Kemal Bayazid, Center for Cardiovascular Surgery, Ankara, Turkey. 37. Amersham. Buckinghamshire, England. 38. Packard. Tricarb, Model 3224. 39. Requests for reprints should be addressed to: Nurten Meriq. M.D.. Tunus Cad. 70/9 Ankara. Turkey. 40. Received for publication March 23. 1977. 41. Accepted for publication March 21. 1978. Printed in U. S. A .

Cellular immunity in children with acute rheumatic fever and rheumatic carditis.

Acute rheumatic fever cellular immunity rheumatic carditis Pediat. Res. 13: 16-20 (1979) Cellular Immunity in Children with Acute Rheumatic Fever an...
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