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Serum thymic factor in subacute sclerosing panencephalitis patients
Serum P. J. G.
Thymic Factor in Subacute Sclerosing Panencephalitis Patients A.
WIJERMANS,*
KAPSENBERG,
t M. AND
ASTALDI,
*
GROENEWOUD,
P.
TH.
A.
G. C. B. ASTAL.DI, * M. Roes,* C.
* LUCAS,*
SCHELLEKENS*
“Centrul Laborutop of the Netherlunds Red Cross Blood Trunsfkion Service und Laborutory of Clinical und Experimental Immunology. University of Amsterdam. Amsterdum. and tNutionu/ Institute of Public Health. Biltholvn, The Netherlunds Received
June
28. 1978
The activity of thymus-dependent serum factor (SF) was measured in 11 patients with subacute sclerosing panencephalitis (SSPE). A significantly decreased activity was found in 6 out of 11 patients, whereas the level of cyclic AMP in lymphocytes of the patients, a parameter for the maturation of peripheral blood lymphocytes, showed low values in 9 out of 11 patients. These findings were compared with the clinical stage of the disease and with some lymphocyte functions in vitro. No correlation was found between the SF levels and the amount of cyclic AMP in the lymphocytes or between these two parameters and the lymphocyte functions in vitro. It is concluded that a thymic humoral dysfunction is unlikely to play a role in the pathogenesis of SSPE: it appears that such a dysfunction is rather a secondary event in this disease.
INTRODUCTION
Subacute sclerosing panencephalitis (SSPE) is a chronic progressive inflammatory disease of the central nervous system in children and young adults (1). Although it is likely that the chronic encephalitis is the result of a persistent measles or measles-related virus infection (2), it remains unclear which factors could be responsible for the persistence of this viral infection. One hypothesis was proposed by Burnet (3): The persistence of the virus could be due to specific nonresponsiveness of thymus-dependent immunity, whereas normal antibody response to this virus occurs. Alternatively, thymus-dependent. immunodeficiency could be one of the causes of SSPE (4). It is known that thymus-derived (T) lymphocytes play a role both in the humoral and in the cell-mediated immune responses against viruses. A depression of the thymic function might, therefore, be expected in children with SSPE. Studies on the overall cell-mediated immune competence in SSPE patients have yielded conflicting results. Generally, only a slight decrease, if any, of cellular immunity has been observed in most of the SSPE patients (4, 5). Specific unresponsiveness to measles virus has been described (6, 7), however, ability of lymphocytes from SSPE patients to kill measles virus infected targets has been reported (29,30) and attributed to K cells (31). Recently, it was proposed (8) that depression of the cell-mediated immune competence detected in some SSPE patients could be due to immaturity of a part of the T-cell system. Because T-cell maturation takes place, at least partially, under the influence of thymic humoral factors (9), Varsano et. (8) administered one such factor to SSPE patients and reported some favorable effects. 105 0090-1229/79/010105-06$01.00/O Copyright All
rights
ICI of
1979 reproductxm
by
Academic in any
Press.
Inc.
form
reserved
106
WIJERMANS
ET AL
It has been reported earlier (10) that human serum contains a thymus-dependent factor (SF) that can raise the intracellular cyclic AMP concentration in thymocytes. SF exerts its action exclusively on hydrocortisone-sensitive thymocytes: this induction of hydrocortisone resistance in such immature cells is associated with an increase of intracellular cyclic AMP (11). The aim of the present study was to investigate whether a decreased humoral function of the thymus plays a role in the pathogenesis of SSPE. Therefore. we determined the SF activity in the serum of 11 SSPE patients and measured the cyclic AMP level in the patients’ lymphocytes as a parameter for the maturation of peripheral blood lymphocytes (PBL). as suggested by Varsano rt rrl. (8). The clinical stage of the disease was compared with these parameters and with the lymphocyte reactivity to mitogens and alloantigens in \itr.o. PATIENTS
The age, sex. and clinical stage of I1 children with SSPE are shown in Table I. The diagnosis was based on the characteristic clinical and serological findings ( 12). Clinical stages of the disease were defined according to Jabbour et ul. ( 13). In brief, Stage 1 patients show mental and behavioral cerebral signs, Stage 2 show motor signs and convulsions, Stage 3 are comatose, and Stage 4 show loss of function of the cerebral cortex. Anti-measles antibody titers in serum and cerebrospinal fluid (CSF) were measured by a sensitive-complement fixation test. All patients had measles complement fixing antibodies in CSF (Table 1). The CSF serum ratio of measles virus antibodies confirmed the diagnosis of SSPE. Other than thymosin, administered to patient No. 6, the patients were not receiving any medication which could influence the reported immunological findings. METHODS
SF was isolated and assayed as previously described ( IO). In brief, serum separated from defibrinated blood was filtered through Amicon CF 50 filters (molecular weight cut off, 50,000) to remove high-molecular weight inhibitors. The ultrafiltrate TABLE
I Antibody
Age Patient
(years)
Sex
Stage of disease
1 ?
7 5
3 4 5 6 7 8 9 10 I1
7 II 5 6 IO 6 12 9
M F M M M F F M F F M
3 2-3 3 1 2 Z-3 2 2 3 2 I-2
cl Reciprocal
titer.
Serum 7 7 10 7 8 IO 7 8 6 9 9
CSF
titer (log,)‘< Ratio of ’ SerumKSF 4 4 I6 16 Ih 64
4 I6 8 64 72
SERUM
THYMIC
FACTOR
IN
SSPE
PATIENTS
107
was added to an equal volume of mouse thymocyte suspensions (final concentration, 2 x lo6 thymocytes/ml). After 5 min of incubation at 37°C the cells were washed and lysed by freezing in liquid nitrogen and the intracellular cyclic AMP was extracted. Proteins coagulated by boiling for 2 min were spun down in the cold, and the amount of intracellular cyclic AMP in the supernatant was measured in duplicate with the Gilmans competition binding procedure (14) with a cyclic AMP assay kit. Patients’ lymphocytes, isolated by conventional density gradient centrifugation, were tested for their amount of intracellular cyclic AMP. To this end, 2 x lo6 lymphocytes were washed and lysed. Cyclic AMP in the supernatant was determined as described above. Peripheral blood lymphocytes were classified as T cells by rosette formation with sheep erythrocytes. Reaction to mitogens and the response to alloantigen in the mixed lymphocyte reaction (MLR) were tested as described by duBois et al. (15). Results of each patient are expressed as the percentage of stimulation of two normal healthy donors. The partially purified calf thymic extract thymosin fraction V was prepared by Hoffman-La Roche Inc. according to Hooper et al. (16). RESULTS
SF activity in the serum of 11 children with SSPE was measured (Table 2). The mean activity we found was 23 pmol of cyclic AMP/lo’ thymocytes (SE 6.0). This is significantly lower (P < 0.05) than the activity found in sera of a group of 16 healthy controls matched for age, 39 pmol cyclic AMP/lo’ thymocytes (SE 3.4). On the basis of the one-sided 95% confidence interval ( 15.7 pmol) we divided the patients into two groups. Group A (5 of 11 patients) had normal SF activity and group B (6 patients) had significantly decreased activity.
SF Acrtvrrv
Patient Group
(pmol
of cyclic
ASD CYCLIC SF activity AMP/lo’
TABLE AMP
thymocytesY’
2 LEVEI.S
IN
(pmol
LYMPHOCYrES”
Intracellular cyclic AMP of cyclic AMP/lo’ lymphocytes)’
A 1 2 3 4 5
Group 6 7 8 9 10 11 0 Results h Normal r Normal
48 52 31 40 46
-t 2 k ” +
5.7 6.2 3.7 4.8 5.5
I8 48 9 12 22
2 r + 2 2
2.2 5.8 I.1 1.4 2.6
1 2 8 13 13 6
+ k L k k 2
0.1 0.2 0.9 1.5 1.5 0.7
51 24 24 13 15 12
+ 2 k -+ k t
6.1 2.9 2.8 1.6 1.8 I.5
B
are expressed in mean picomoles + SE. value for this age group: 39 k 3.4 pmol. II = 16. value: 41 t 4.9 pmol. n = 16.
108
U’l
JERMANS
ET Al.
We also measured the level of intracellular cyclic AMP in the lymphocytes of the patients and found a mean level of 23 pmol of cyclic AMP/lo’ lymphocytes (SE 4.3), whereas a control group of 16 normal healthy donors had a mean level of 41 pmol of cyclic AMP/lo’ lymphocytes (SE 4.9). which is significantly higher (P < 0.01). No correlation was found between the SF activity. the cyclic AMP content of the lymphocytes (correlation coefficient. 0.0016. II = 111, and the stage of the disease (see Tables I and 2). The results of the tests for cellular immunocompetence irz w’tro are given in Table 3. At variance with others (8). we found a normal percentage of E rosettes in all patients as previously reported by Valdimarsson rt cl/. (7). Most of the lymphocyte reactivity tests were also normal. None of the results of these tests was significantly correlated with either the SF activity or the level of cyclic AMP in lymphocytes (correlation coefficient < 0.5961, II = 10). This confirms the findings of other investigators who found completely normal cell-mediated immunity in rather large groups of SSPE patients (17, 18). Patient No. 6 was treated with the thymic extract thymosin fraction V as part of the ongoing clincial trials (19). Shortly after the start of thymosin treament. the SF level increased to normal, as reported elsewhere (20). and the level of cellular cyclic AMP in this patient’s PBL rose from 51 to 92 pmol/ 10’ cells. However, no clear clinical benefit was observed. DISCUSSION In these studies, 6 out of 11 patients had impaired thymic humoral function as reflected by low SF levels. It could be argued that this might be due to a direct action of the measles virus on the thymus. especially on thymic epithelial cells. Indeed, White and Boyd (21) have reported the involvement of the thymus in acute measles infection. They found that the virus can infect thymic lymphoid cells. The aggregation of large numbers of thymocytes, which is seen in such thymuses, seems to lead to a depletion of the thymic cortex.
Patient
8 9 I0 II
PHA”
ALS
Con
A
too 105 87
42 5s 52
87 84 62
nd II6 151 IS5 I02 94 I64 87
nd XI 375 I.50 64 85 174 74
nd I?5 I34 144 67 63 IO7 71
PWM
MLR
E rosettes
74 74
3-l XI
hi 7x
47 nd 47 97 Ii? x3 49 51
hi nd IO7 100 I31 63 IO1 Ii7
‘I nd 74 69 x.‘; 76 55 77
147
122
hh
” Values for the lymphocyte reaction irl Gtw are expressed as percentage controls. Normal values for the E rosettes: 50 to 80%. b Abbreviations used: PHA, phytohemagglutinin: ALS. antilymphocytic lin A: PWM. pokeweed mitogen: MLR, mixed lymphocyte reaction.
of the mean serum:
Con
of two
(C;)
normal
A. concana\a-
SERUM
THYMIC
FACTOR
IN
SSPE
PATIENTS
109
It is likely that the virus not only can attack thymic lymphoid cells, but can affect the epithelial cells of the thymus too, probably independently from the stage of the disease. This could then lead to a decreased humoral function of the thymus, since it is believed that thymic humoral factors are secreted by the epithelial cells of the thymus (22-24). We also found a decreased cyclic AMP level in the lymphocytes of five of six SSPE patients with low SF levels. Low cyclic AMP, as suggested by Varsano et al. (8), might reflect the presence of an immature subpopulation among peripheral blood lymphocytes, since the intracellular cyclic AMP level in human thymocytes is lower than in mature lymphocytes (25). However, we also observed low cyclic AMP levels in lymphocytes of four patients with normal thymic humoral function, as reflected by normal SF levels. This indicates that the immaturity of some peripheral blood lymphocytes might be independent of thymic humoral function. It is also possible that, in SSPE, the measles virus might attack non-T cells as well as immature T cells, mature T cells, and thymic epithelial cells. An alternative is that an impaired thymic humoral function in these patients cannot be detected by the SF determination. The normal percentages of T cells found in all patients by formation of E rosettes does not rule out the presence of immature cells since it is well known that immature T cells, such as thymocytes, also form E rosettes (26). In accordance with other authors (4, 5), we found only slight abnormalities in the functional tests for cell-mediated immune competence. Therefore, it is unlikely that such a population of immature T cells is responsible for immunodepression, as observed by others (8) in some SSPE patients. Measles virus as such has a marked immunosuppressive effect, especially on cell-mediated immune competence, both in vivo (27) and irr vitro (28). Thus it cannot be excluded that the functional abnormalities in these parameters, reported in SSPE by the other authors mentioned, may well be related to such a mechanism. In our opinion, the low levels of circulating thymic-dependent factor found in some of the SSPE patients are a result of the disease and it is unlikely that a humoral dysfunction of the thymus plays a role in the pathogenesis of SSPE, since we found no correlation between the stage of the disease, the SF levels, the presence of immature lymphocytes, and the lymphocyte function in vitro. The present results and those obtained from other authors do not warrant a clinical trial with thymic factors in SSPE patients. REFERENCES I. Katz. M., In Slow Virus Infection (M. Katz. and V. ter Meulen, Eds.), pp. 106-I 14, SpringerVerlag, Berlin/New York. 1977. 2. Horta-Barbosa. L.. Fuccillo, D. A., London. W. T.. Jabbour. J. T.. Zeman, W.. and Sever. J. L.. Proc. Sot. Exp. Biol. Med. 432, 272, 1969. 3. Burnet. F. M., Lancer 2, 610. 1968. 4. Gerson, K. L., and Haslam. R. H. A., N. Eng. J. Med. 285, 78, 1971. 5. Lischner, H. W.. Sharma. M. K., and Groover, W. D., N. Engl. J. Med. 286, 786, 1972. 6. Jabbour, J. T.. Roane, J. A., and Sever, J. L., Neuro1og.v 19, 929, 1969. 7. Valdimarsson, H., Agnarsdottir, G., and Lachmann. P. J., Proc. Roy. Sot. Med. 67, 1125, 1974. 8. Varsano, I., Danon, Y.. Jaber, L., Livni. E.. Shohat, B., Yakir, Y.. Shneyour, A., and Trainin, N , Iv. J. Med. Sci. 12, I 168. 1976. 9. Bach, J. F.. and Carnaud, C.. Progr. Allergy 21, 342, 1976. 10. Astaldi, A.. Astaldi. G. C. B., Schellekens. P. Th. A., and Eijsvoogel, V. P., Nafure (London) 260, 713, 1976.
110
WIJE.RM”“S
tl
AL
I I. Astaldi. G. C. B.. Astaldi. A.. Groenewoud. M.. WQermans. P.. Schellekens. P. Th. A.. and Eijsvoogel, V. P., Eltr. J. In~inunol. 7, 836. 1977. 12. Sever. J. L.. Krebs, H.. Ley. A.. Barbosa. I.. H.. and Rubinstein. D.. ./. A?hrr. Mrc/. A.\.\. 228, 604. 1974. 13. Jabbour. J. T.. Garcia. J. H., Lemmi. H., Ragland, J.. Duenas. D. A.. and Sever, J. L.. J. .4nlrr. Med. Ass. 207, 2248, 1969. 14. Gilman. A. G., Pro<,. Ndr. Ac,trd. SC;. (USA) 67, 305. 1970. IS. duBois. R., Meinesz, A., Bierhorst, A.. Groenewoud. M.. Schelleken\. P. Th. A.. and Wvoogel. V. P.. Tissr~ ~wi,g~t~.~ 4, 458. 1974. 16. Hooper. J. A.. McDaniel. M. C., Thurman. G. B.. Cohen. G. H.. Schulof, R. S.. and Goldstein. A. L.. Anti. ,Y I’. Ac,ud. SC;. 249, 125, 1975. 17. Kreth. H. W., Kackel. Y. M.. and Meulen. V. ter, Med. Mkrohiol. Inrnntn~~l. 160. 191. 1974. IX. Blaese. R. M.. and Hofstrand. H.. Arch. Neural. 32, 494. 1975. 19. Goldstein. A. L.. Thurman. G. B.. Rossio. J. L., and Costanzi. J. J.. 7’rrr/1sp/~rrr1. Pn,c,. 9. 1141. 1977. 20. Astaldi. A.. Astaldi. G. (.‘. B.. Wijermans. P., Groenewoud. M.. Schellekens. P. Th. A.. and Eijsvoogel. V. P.. .I. Irrtr?rrr,~o/. 119, 1106. 1977. 2 I. White. R. G., and Boyd. J. F., Clint. Eup. Itnn~rtnol. 13, 343, 1973. 22. Stutman. 0.. Yunis. E. J.. and Good, R. A.. J. Erp. Med. 130, 809, 1969. 23. Dardenne. M.. Papiernik, M., Bach. J. F.. and Stutman. G., Immurrolog~ 27, 299. 1974. 74. Kruisbeek. A. M.. Astaldi. G. C. B., Blankwater. J. J.. Zijlstra, J. J.. Levert. I.. A.. and Astaldi. A., Cell. I~nmlrnol. 3.5, 134. 1978. 25. Niaudet. P.. Beaurain. G.. and Bach, M. A.. E~rr. J. Innurrnrjl. 6, 834. 1976. 26. Lay. H. L.. Mendes. N. F.. Bianco. C.. and Nussenzweig. V.. Ncrtrrre (Londorri 230, 531, 1971. 27. Pirquet. C. E.. Duetschc .&fed. Wochensc~hv. 34, 1297, 1908. 28. Lucas, C. J.. Galama, J. M. D.. and Ubels-Postma. J.. Cell. fmmrct~dl. 32, 70. 1977. 29. Perrin. L. H.. Tishor, A. and Oldstone. M. B. A.. .I. Immunol. 118, 282, 1977. 30. Kreth, H. W.. and Meulen. V. ter. .I. fn~nlrrn~>l. 118, 291, 1977. 31. Kreth. H. W. and Wiegand. G.. J. Inrmrrnol. 118, 296, 1977.
Thymic Factor in Subacute Sclerosing Panencephalitis Patients A.
WIJERMANS,*
KAPSENBERG,
t M. AND
ASTALDI,
*
GROENEWOUD,
P.
TH.
A.
G. C. B. ASTAL.DI, * M. Roes,* C.
* LUCAS,*
SCHELLEKENS*
“Centrul Laborutop of the Netherlunds Red Cross Blood Trunsfkion Service und Laborutory of Clinical und Experimental Immunology. University of Amsterdam. Amsterdum. and tNutionu/ Institute of Public Health. Biltholvn, The Netherlunds Received
June
28. 1978
The activity of thymus-dependent serum factor (SF) was measured in 11 patients with subacute sclerosing panencephalitis (SSPE). A significantly decreased activity was found in 6 out of 11 patients, whereas the level of cyclic AMP in lymphocytes of the patients, a parameter for the maturation of peripheral blood lymphocytes, showed low values in 9 out of 11 patients. These findings were compared with the clinical stage of the disease and with some lymphocyte functions in vitro. No correlation was found between the SF levels and the amount of cyclic AMP in the lymphocytes or between these two parameters and the lymphocyte functions in vitro. It is concluded that a thymic humoral dysfunction is unlikely to play a role in the pathogenesis of SSPE: it appears that such a dysfunction is rather a secondary event in this disease.
INTRODUCTION
Subacute sclerosing panencephalitis (SSPE) is a chronic progressive inflammatory disease of the central nervous system in children and young adults (1). Although it is likely that the chronic encephalitis is the result of a persistent measles or measles-related virus infection (2), it remains unclear which factors could be responsible for the persistence of this viral infection. One hypothesis was proposed by Burnet (3): The persistence of the virus could be due to specific nonresponsiveness of thymus-dependent immunity, whereas normal antibody response to this virus occurs. Alternatively, thymus-dependent. immunodeficiency could be one of the causes of SSPE (4). It is known that thymus-derived (T) lymphocytes play a role both in the humoral and in the cell-mediated immune responses against viruses. A depression of the thymic function might, therefore, be expected in children with SSPE. Studies on the overall cell-mediated immune competence in SSPE patients have yielded conflicting results. Generally, only a slight decrease, if any, of cellular immunity has been observed in most of the SSPE patients (4, 5). Specific unresponsiveness to measles virus has been described (6, 7), however, ability of lymphocytes from SSPE patients to kill measles virus infected targets has been reported (29,30) and attributed to K cells (31). Recently, it was proposed (8) that depression of the cell-mediated immune competence detected in some SSPE patients could be due to immaturity of a part of the T-cell system. Because T-cell maturation takes place, at least partially, under the influence of thymic humoral factors (9), Varsano et. (8) administered one such factor to SSPE patients and reported some favorable effects. 105 0090-1229/79/010105-06$01.00/O Copyright All
rights
ICI of
1979 reproductxm
by
Academic in any
Press.
Inc.
form
reserved
106
WIJERMANS
ET AL
It has been reported earlier (10) that human serum contains a thymus-dependent factor (SF) that can raise the intracellular cyclic AMP concentration in thymocytes. SF exerts its action exclusively on hydrocortisone-sensitive thymocytes: this induction of hydrocortisone resistance in such immature cells is associated with an increase of intracellular cyclic AMP (11). The aim of the present study was to investigate whether a decreased humoral function of the thymus plays a role in the pathogenesis of SSPE. Therefore. we determined the SF activity in the serum of 11 SSPE patients and measured the cyclic AMP level in the patients’ lymphocytes as a parameter for the maturation of peripheral blood lymphocytes (PBL). as suggested by Varsano rt rrl. (8). The clinical stage of the disease was compared with these parameters and with the lymphocyte reactivity to mitogens and alloantigens in \itr.o. PATIENTS
The age, sex. and clinical stage of I1 children with SSPE are shown in Table I. The diagnosis was based on the characteristic clinical and serological findings ( 12). Clinical stages of the disease were defined according to Jabbour et ul. ( 13). In brief, Stage 1 patients show mental and behavioral cerebral signs, Stage 2 show motor signs and convulsions, Stage 3 are comatose, and Stage 4 show loss of function of the cerebral cortex. Anti-measles antibody titers in serum and cerebrospinal fluid (CSF) were measured by a sensitive-complement fixation test. All patients had measles complement fixing antibodies in CSF (Table 1). The CSF serum ratio of measles virus antibodies confirmed the diagnosis of SSPE. Other than thymosin, administered to patient No. 6, the patients were not receiving any medication which could influence the reported immunological findings. METHODS
SF was isolated and assayed as previously described ( IO). In brief, serum separated from defibrinated blood was filtered through Amicon CF 50 filters (molecular weight cut off, 50,000) to remove high-molecular weight inhibitors. The ultrafiltrate TABLE
I Antibody
Age Patient
(years)
Sex
Stage of disease
1 ?
7 5
3 4 5 6 7 8 9 10 I1
7 II 5 6 IO 6 12 9
M F M M M F F M F F M
3 2-3 3 1 2 Z-3 2 2 3 2 I-2
cl Reciprocal
titer.
Serum 7 7 10 7 8 IO 7 8 6 9 9
CSF
titer (log,)‘< Ratio of ’ SerumKSF 4 4 I6 16 Ih 64
4 I6 8 64 72
SERUM
THYMIC
FACTOR
IN
SSPE
PATIENTS
107
was added to an equal volume of mouse thymocyte suspensions (final concentration, 2 x lo6 thymocytes/ml). After 5 min of incubation at 37°C the cells were washed and lysed by freezing in liquid nitrogen and the intracellular cyclic AMP was extracted. Proteins coagulated by boiling for 2 min were spun down in the cold, and the amount of intracellular cyclic AMP in the supernatant was measured in duplicate with the Gilmans competition binding procedure (14) with a cyclic AMP assay kit. Patients’ lymphocytes, isolated by conventional density gradient centrifugation, were tested for their amount of intracellular cyclic AMP. To this end, 2 x lo6 lymphocytes were washed and lysed. Cyclic AMP in the supernatant was determined as described above. Peripheral blood lymphocytes were classified as T cells by rosette formation with sheep erythrocytes. Reaction to mitogens and the response to alloantigen in the mixed lymphocyte reaction (MLR) were tested as described by duBois et al. (15). Results of each patient are expressed as the percentage of stimulation of two normal healthy donors. The partially purified calf thymic extract thymosin fraction V was prepared by Hoffman-La Roche Inc. according to Hooper et al. (16). RESULTS
SF activity in the serum of 11 children with SSPE was measured (Table 2). The mean activity we found was 23 pmol of cyclic AMP/lo’ thymocytes (SE 6.0). This is significantly lower (P < 0.05) than the activity found in sera of a group of 16 healthy controls matched for age, 39 pmol cyclic AMP/lo’ thymocytes (SE 3.4). On the basis of the one-sided 95% confidence interval ( 15.7 pmol) we divided the patients into two groups. Group A (5 of 11 patients) had normal SF activity and group B (6 patients) had significantly decreased activity.
SF Acrtvrrv
Patient Group
(pmol
of cyclic
ASD CYCLIC SF activity AMP/lo’
TABLE AMP
thymocytesY’
2 LEVEI.S
IN
(pmol
LYMPHOCYrES”
Intracellular cyclic AMP of cyclic AMP/lo’ lymphocytes)’
A 1 2 3 4 5
Group 6 7 8 9 10 11 0 Results h Normal r Normal
48 52 31 40 46
-t 2 k ” +
5.7 6.2 3.7 4.8 5.5
I8 48 9 12 22
2 r + 2 2
2.2 5.8 I.1 1.4 2.6
1 2 8 13 13 6
+ k L k k 2
0.1 0.2 0.9 1.5 1.5 0.7
51 24 24 13 15 12
+ 2 k -+ k t
6.1 2.9 2.8 1.6 1.8 I.5
B
are expressed in mean picomoles + SE. value for this age group: 39 k 3.4 pmol. II = 16. value: 41 t 4.9 pmol. n = 16.
108
U’l
JERMANS
ET Al.
We also measured the level of intracellular cyclic AMP in the lymphocytes of the patients and found a mean level of 23 pmol of cyclic AMP/lo’ lymphocytes (SE 4.3), whereas a control group of 16 normal healthy donors had a mean level of 41 pmol of cyclic AMP/lo’ lymphocytes (SE 4.9). which is significantly higher (P < 0.01). No correlation was found between the SF activity. the cyclic AMP content of the lymphocytes (correlation coefficient. 0.0016. II = 111, and the stage of the disease (see Tables I and 2). The results of the tests for cellular immunocompetence irz w’tro are given in Table 3. At variance with others (8). we found a normal percentage of E rosettes in all patients as previously reported by Valdimarsson rt cl/. (7). Most of the lymphocyte reactivity tests were also normal. None of the results of these tests was significantly correlated with either the SF activity or the level of cyclic AMP in lymphocytes (correlation coefficient < 0.5961, II = 10). This confirms the findings of other investigators who found completely normal cell-mediated immunity in rather large groups of SSPE patients (17, 18). Patient No. 6 was treated with the thymic extract thymosin fraction V as part of the ongoing clincial trials (19). Shortly after the start of thymosin treament. the SF level increased to normal, as reported elsewhere (20). and the level of cellular cyclic AMP in this patient’s PBL rose from 51 to 92 pmol/ 10’ cells. However, no clear clinical benefit was observed. DISCUSSION In these studies, 6 out of 11 patients had impaired thymic humoral function as reflected by low SF levels. It could be argued that this might be due to a direct action of the measles virus on the thymus. especially on thymic epithelial cells. Indeed, White and Boyd (21) have reported the involvement of the thymus in acute measles infection. They found that the virus can infect thymic lymphoid cells. The aggregation of large numbers of thymocytes, which is seen in such thymuses, seems to lead to a depletion of the thymic cortex.
Patient
8 9 I0 II
PHA”
ALS
Con
A
too 105 87
42 5s 52
87 84 62
nd II6 151 IS5 I02 94 I64 87
nd XI 375 I.50 64 85 174 74
nd I?5 I34 144 67 63 IO7 71
PWM
MLR
E rosettes
74 74
3-l XI
hi 7x
47 nd 47 97 Ii? x3 49 51
hi nd IO7 100 I31 63 IO1 Ii7
‘I nd 74 69 x.‘; 76 55 77
147
122
hh
” Values for the lymphocyte reaction irl Gtw are expressed as percentage controls. Normal values for the E rosettes: 50 to 80%. b Abbreviations used: PHA, phytohemagglutinin: ALS. antilymphocytic lin A: PWM. pokeweed mitogen: MLR, mixed lymphocyte reaction.
of the mean serum:
Con
of two
(C;)
normal
A. concana\a-
SERUM
THYMIC
FACTOR
IN
SSPE
PATIENTS
109
It is likely that the virus not only can attack thymic lymphoid cells, but can affect the epithelial cells of the thymus too, probably independently from the stage of the disease. This could then lead to a decreased humoral function of the thymus, since it is believed that thymic humoral factors are secreted by the epithelial cells of the thymus (22-24). We also found a decreased cyclic AMP level in the lymphocytes of five of six SSPE patients with low SF levels. Low cyclic AMP, as suggested by Varsano et al. (8), might reflect the presence of an immature subpopulation among peripheral blood lymphocytes, since the intracellular cyclic AMP level in human thymocytes is lower than in mature lymphocytes (25). However, we also observed low cyclic AMP levels in lymphocytes of four patients with normal thymic humoral function, as reflected by normal SF levels. This indicates that the immaturity of some peripheral blood lymphocytes might be independent of thymic humoral function. It is also possible that, in SSPE, the measles virus might attack non-T cells as well as immature T cells, mature T cells, and thymic epithelial cells. An alternative is that an impaired thymic humoral function in these patients cannot be detected by the SF determination. The normal percentages of T cells found in all patients by formation of E rosettes does not rule out the presence of immature cells since it is well known that immature T cells, such as thymocytes, also form E rosettes (26). In accordance with other authors (4, 5), we found only slight abnormalities in the functional tests for cell-mediated immune competence. Therefore, it is unlikely that such a population of immature T cells is responsible for immunodepression, as observed by others (8) in some SSPE patients. Measles virus as such has a marked immunosuppressive effect, especially on cell-mediated immune competence, both in vivo (27) and irr vitro (28). Thus it cannot be excluded that the functional abnormalities in these parameters, reported in SSPE by the other authors mentioned, may well be related to such a mechanism. In our opinion, the low levels of circulating thymic-dependent factor found in some of the SSPE patients are a result of the disease and it is unlikely that a humoral dysfunction of the thymus plays a role in the pathogenesis of SSPE, since we found no correlation between the stage of the disease, the SF levels, the presence of immature lymphocytes, and the lymphocyte function in vitro. The present results and those obtained from other authors do not warrant a clinical trial with thymic factors in SSPE patients. REFERENCES I. Katz. M., In Slow Virus Infection (M. Katz. and V. ter Meulen, Eds.), pp. 106-I 14, SpringerVerlag, Berlin/New York. 1977. 2. Horta-Barbosa. L.. Fuccillo, D. A., London. W. T.. Jabbour. J. T.. Zeman, W.. and Sever. J. L.. Proc. Sot. Exp. Biol. Med. 432, 272, 1969. 3. Burnet. F. M., Lancer 2, 610. 1968. 4. Gerson, K. L., and Haslam. R. H. A., N. Eng. J. Med. 285, 78, 1971. 5. Lischner, H. W.. Sharma. M. K., and Groover, W. D., N. Engl. J. Med. 286, 786, 1972. 6. Jabbour, J. T.. Roane, J. A., and Sever, J. L., Neuro1og.v 19, 929, 1969. 7. Valdimarsson, H., Agnarsdottir, G., and Lachmann. P. J., Proc. Roy. Sot. Med. 67, 1125, 1974. 8. Varsano, I., Danon, Y.. Jaber, L., Livni. E.. Shohat, B., Yakir, Y.. Shneyour, A., and Trainin, N , Iv. J. Med. Sci. 12, I 168. 1976. 9. Bach, J. F.. and Carnaud, C.. Progr. Allergy 21, 342, 1976. 10. Astaldi, A.. Astaldi. G. C. B., Schellekens. P. Th. A., and Eijsvoogel, V. P., Nafure (London) 260, 713, 1976.
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