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Serum migration-inhibitory activity in children with acute infectious mononucleosis
CLINICAL
IMMUNOLOGY
AND
19, 314-318 (1981)
IMMUNOPATHOLOGY
Serum Migration-Inhibitory Infectious
Activity in Children with Acute Mononucleosis
A. BERTOTTO, DANIELA CAPRINO, R. VACCARO, AND FRANCA SONAGLIA Puericultura
Institute,
University
of Perugia.
Perugia.
Italy
Received July 15, 1980 Leukocyte migration-inhibitory activity (LIF) was found in the sera of 11114 (78.5%) children with heterophil-positive infectious mononucleosis as opposed to only 1115 (6.6%) control children (p < 0.001). It is postulated that the cellular unresponsiveness observed during the acute phase of the infection may be partly explained by the production of an inhibitory factor(s) with LIF-like activity, possibly by a subpopulation of suppressor lymphocytes present in excessive concentrations in Epstein-Barr virusinfected patients.
INTRODUCTION
When appropriately stimulated in vitro, lymphocytes release various biologically active factors known as lymphokines. Although some lymphokine activities are undoubtedly biological artifacts with little or no physiological relevance, experimental evidence indicates that the mechanisms controlling cell-mediated immune reactions in vivo involve substances which are identical or similar to those responsible for the in vitro phenomena (1- 5). These observations suggest the possibility that lymphokine-like activity may be detectable in vivo in certain human pathologic states. Indeed, significant serum macrophage or leukocyte migration-inhibitory factor(s) (serum MIF or LIF), or substances with MIF- or LIF-like activity, have been detected in patients with malignant lymphoproliferative disorders (6-9), post-transplantation hepatic dysfunction (lo), infectious diseases which elicit a cellular immune response (11, 12), and in immunologically competent cancer patients (13, 14). Since lymphocytes from patients with acute infectious mononucleosis (IM) spontaneously release LIF in culture (15) and since this disease is associated with alterations in immunologic status, which may be related to impaired lymphokinedependent mechanisms (15-20), it was of interest to investigate LIF activity in acute IM patients’ sera. MATERIALS
AND METHODS
Patients. Fourteen children, male and female between 7 and 14 years of age, with acute IM admitted to the Puericultura Institute at Perugia University were studied. The diagnosis was based on fever, sore throat, lymphadenopathy, splenomegaly, a positive heterophil index, and the presence of atypical lymphocytes in the peripheral blood. Blood samples (5 ml) for serum LIF activity detection were collected within the first 5 days after admission (average 10 days after 314 0090-1229/81/060314-05$01.00/O Copyright All rights
@ 1981 by Academic Press. Inc. of reproduction in any form reserved.
SERUM
LIF
IN
INFECTIOUS
MONONUCLEOSIS
315
onset of symptoms). The blood was centrifuged at SOOg for 10 min and the sera stored at 4°C until used, which was a maximum of 1 week. Blood from 15 children seen for routine well-child care at the same institute during the same time period was also assayed. These children were age and sex matched with the patients. Lculcocyte migration-inhibition assay. Twenty milliliters of venous blood were drawn from a normal donor into a syringe containing sodium citrate as anticoagulant, and allowed to sediment with 6% dextran in saline (Plander-Pierrel) for 30 to 45 min. The white cell-rich plasma was aspirated and spun at 4OOg for 5 min. The cell pellet was incubated for 7 min at 37°C with Tris-buffered ammonium chloride (0.83%) to lyse erythrocytes. The cells were then washed three times in RPM1 1640 culture medium (Eurobio-Paris) supplemented with antibiotics (penicillin, 100 III/ml; streptomycin, 100 pg/ml) and adjusted to a final concentration of 4 x 10’ cells/ml. Several microcapillary tubes (length: 63 mm) (Behringwerke, OTCL 20/21) were tilled with the cell suspension, plugged at one end, and centrifuged at SOOg for 3 min. The tubes were cut at the cell-fluid interface and inserted into hematocrit nonheparinized capillaries (internal diameter: 1, 1- 1, 2 mm) (Drummond Scientific Co., Broomal, Pa.) previously filled with a medium containing 50% decomplemented serum of the children to be tested and 50% RPM1 culture medium. The hematocrit chamber was sealed, fixed on a glass slip with silicone grease, and then incubated for 18 hr at 37°C. Each experiment consisted of four duplicates, which were averaged to obtain the mean length of migration. The migration index (MI) was calculated by dividing the migration length in the serum sample by the migration length in an allogeneic control serum obtained from a healthy individual. Inhibition greater than 20% of the leukocyte control migration (MI< 0.8) was considered significant, in accordance with the findings of Bruley-Rosset et al. (1 l), who developed a similar, but not identical, technique. Statistical analysis. The x2 test was used to determine the significance of differences in results between paired sets of data. RESULTS The accompanying Fig. 1 represents a scattergram of migration indices for all experiments carried out. Sera from healthy children did not show any significant inhibitory activity, with the exception of the one putatively normal subject with a positive reaction (l/15; 6.6%). In contrast, the majority of the patients in the experimental group studied had LIF-like activity in their serum (11/14; 78.5%). The difference between the percentages of positive serum LIF tests was statistically significant (x2: 12.6; p < 0.001). The intensity of reaction (serum LIF production) was not correlated to the degree of lymphocytosis in the blood; in particular, there was no correlation with the concentration or proportion of morphologically activated lymphocytes. Likewise, no correlation was found with age, sex, duration of symptoms, body temperature, liver damage, or heterophil antibody titers.
316
BERTOTTO
ET AL.
FIG. 1. Migration-index (MI) values of children with acute infectious mononucleosis as compared to index values of healthy children. Each dot represents the mean of quadruplicate samples. Inhibition of migration (MI < 0.8) is shown by 11 (78.5%) young patients compared with one (6.6%) control @ -C 0.001).
DISCUSSION The Epstein-Barr virus (EBV), the primary causal agent of IM, is a human herpes group virus with a striking tropism for lymphocytes. It selectively infects B lymphocytes via a specific virus receptor (21). During acute IM, the peripheral blood contains a large number of activated lymphocytes. The majority of these cells are thought to be lymphocytes reacting to EBV-infected cells; they are mainly T lymphocytes but, also, include B lymphocytes. A smaller proportion of the activated cells are EBV genome-carrying B lymphoblasts (22). Lymphokines are released by activated lymphocytes and such a release by mononuclear cells from patients with acute IM has been documented in the absence of antigenic challenge in experiments which used the macrophage or leukocyte migration-inhibition techniques (23, 15). However, these situations all represent in vitro phenomena. Using an unidirectional leukocyte migration-inhibition assay, Bruley-Rosset ef al. (11) showed serum LIF activity in four out of five IM patients sampled during the acute phase of the infection, These findings are at present the only data available on the in vivo production of lymphokine-like activity in IM. Our results, on a larger number of patients, confirm the previously published data and induce us to speculate on the nature and biological significance of the inhibitory factor(s). Although various substances, including specific antibody and circulating immune complexes, are implicated in human serum-induced migration inhibition (24-27), the assay adopted by us does not totally rule out a possible cellular origin for serum LIF. Therefore, the release of this factor(s) by activated B lymphocytes (28) and/or virus-infected lymphoid cells (29, 30) cannot be excluded. However,
SERUM LIF IN INFECTIOUS
MONONUCLEOSIS
317
when considering a possible lymphocytic origin for serum LIF, it would, perhaps, be more relevant to put forward evidence that supports the presence of an intense suppressor T-cell activity in blood from patients with IM. Tosato er al. (31) and Johnsen ef al. (32) demonstrated that during the acute phase of the illness, noncytotoxic suppressor T-lymphocytes are involved in an immune reaction which inhibites EBV target B-cell activation and proliferation. Suppressor cells in rats exert their effect by soluble factors which pass through a dialysis membrane (33) and Rich and Pierce (34) documented the production in supernatant fluids of suppressive factors, which may act on antigen-activated B cells to limit their clonal expansion or terminal differentiation into antibodysynthesizing cells. Since the kinetics of appearance of suppressive activity in culture medium is similar to that of MIF secretion (35), the suppressive factors and MIF may represent different biological functions of the same molecule (36). Viral antigens stimulate suppressor T cells to release these substances, and a feedback suppression by lymphocyte products may explain the transitory anergy observed in certain human infections (37). Experimental evidence that intravenously administered exogenous MIF-rich supernatants can suppress delayed hypersensitivity skin reactions in immunized guinea pigs (5) supports the existence of such a mechanism. It is, therefore, quite possible that circulating endogenous MIF or other lymphokines in patients with infectious diseases exert similar inhibitory effects not only on skin reactivity but also on the various other manifestations of cellular immunity. Thus, it can be postulated that cutaneous anergy (16- 18) and in vitro lymphocyte hyporesponsiveness (15, 19, 20, 38-40) observed in acute IM are partly due to the presence of an inhibitory factor(s) with LIF-like activity, possibly produced by a subpopulation of suppressor lymphocytes present in excessive concentrations in EBV-infected patients. Studies are underway in our laboratory to correlate migration inhibition with material obtained by centrifuging on Amicon 50 membranes acute IM patients’ sera. Preliminary results suggest that serum LIF is a relatively small molecule (molecular weight below 50.000). Whether other physical and chemical properties of serum LIF are similar to those of previously characterized lymphokines remains to be elucidated. REFERENCES 1. Yamamoto, K., and Takahashi, Y., Nature (London), New Biol. 233, 261, 1971. 2. Salvin, S. B., Youngner, .I. S., and Lederer, W. H., Infect. Immun. 7, 68, 1973. 3. Hay, J. B., Lachmann, P. J., and Tmka, Z., “Proceedings of the 7th Leukocyte Culture Conference” (F. Daguillard, Ed.), pp. 341-348. Academic Press, New York, 1973. 4. Yoshida, T., and Cohen, S., “Mechanisms of Cell-Mediated Immunity” (R. T. McCluskey and S. Cohen, Eds.), p. 43. Wiley, New York, 1974. 5. Yoshida, T., and Cohen, S., .I. Immunol. 112, 1540, 1974. 6. Cohen, S., Fisher, B., Yoshida, T., and Bettigole, R. E., N. Engl. .I. Med. 290, 882, 1974. 7. Yoshida, T., Edelson, R., Cohen, S., and Green, I., J. Immunol. 114, 915, 1975. 8. Ambrogi, F., Polidori, R., Azzara, A., Petrini, M., and Grassi, B., Eur. J. Cancer 14, 1107, 1978. 9. Fassas, A., Bruley-Rosset, M.. Guibout, C., and Jasmin, C., C/in. Immunnl. fmmunopathol. 14. 368, 1979. 10. Torisu, M., Yoshida, T., and Cohen, S., C/in. Immunol. Immunopathol. 3, 369, 1975. 11. Bruley-Rosset, M., Botto, H. G., and Goutner, A., Eur. J. Cancer 13, 325, 1977.
318
BERTOTTO
ET AL.
12. Bertotto, A., Stagni, G., Sonaglia, F., Caprino, D., and Velardi, A., J. Pediar., in press. 13. Botto, H. G., Gauthier, H., PouilIart, P., Bruley-Rosset, M., and Huguenin, P., Eur. J. Cancer 13, 329, 1977. 14. Fassas, A., and Bruley-Rosset, M., “Adjuvant Therapies and Markers of Post-surgical Minimal Residual Disease” (G. Bonnadona, G. Mathe, and S. Salmon, Eds.), pp. 88-92. Springer-Verlag, Heidelberg, 1978. 15. Palit, J., Bendtzen, K., and Andersen, V., C/in. Exp. Immunol. 31, 66, 1978. 16. Bentzon, J. W., Tubercle 34, 34, 1953. 17. Haider, S., Coutinho, M. DE L., Emond, R. T. D., and Sutton, R. N. P., Lancer 2, 74, 1973. 18. Mangi, R. J., Niederman, J. C., Kelleher, J. E., Dwyer, J. M., Evans, A. S., and Kantor, F. S., N. Engl. J. Med. 291, 1149, 1974. 19. Lai, P. K., Alpers, M. P., and Mac-Kay-Scollay, E. M., Infect. Immun. 17, 28, 1977. 20. Wainwright, W. H., Veltri, R. W., and Sprinkle, P. M., J. Infect. Dis. 140, 22, 1979. 21. Jondal, M., and Klein, G., 1. Exp. Med. 138, 1365, 1973. 22. Epstein, M. A., and Achong, B. G., Lancer 2, 1270, 1977. 23. Papageorgiou, P. S., Sorokin, C. F., and Glade, P. R., J. Immunol. 112, 675, 1974. 24. Cochran, A. J., Klein, G., Kiessling, R., and Gunven, P., J. Nar. Cancer Inst. 51, 1431, 1973. 25. Ortiz-Ortiz, L., Zamacona, G., Garmilla, C., and Arellanomt, T., J. Immunol. 113, 993, 1974. 26. Kotkes, P., andPick, E., C/in. Exp. Immunol. 19, 105, 1975. 27. Cochran, A. J., Mackie, R. M., Ross, C. E., Ogg, L. J., and Jackson, A. M., Inf. J. Cancer 18, 274, 1976. 28. Chess, L., Rocklin, R. E., Macdermott, R. P.. David, J. R.. and Schlossman, S. F., J. Immunol. 115, 315, 1975. 29. Flanagan, T. D., Yoshida, T., and Cohen, S., Infect. Immun. 8, 145, 1973. 30. Flanagan, T. D., Yoshida, T., and Genco, R. J., Fed. Proc. 32, 841, 1973. 31. Tosato, G., Magrath, I., Koski, I., Dooley, N., and Blaese, M., N. Engl. J. Med. 301, 1133. 1979. 32. Johnsen, H. E., Madsen, M., and Kristensen, T., Stand. J. Immunol. 10, 251, 1979. 33. Tardieu, M., and Daguillard, F., Cell. Zmmunol. 19, 148, 1975. 34. Rich, R. R., and Pierce, C. W., J. Immunol. 112, 1360, 1974. 35. David, J. R., and David, R. R., Progr. Allergy 16, 300, 1972. 36. Jirillo, E., and Fumarola, D., Ann. Sclavo (Siena) 21, 573, 1979. 37. Kantor. F. S., N. Engi. J. Med. 292, 629, 1975. 38. Nikoskelainen, J., Ablashi, D. V., Isenberg, R. A., Neel, E. Il., Miller, R. G.. and Stevens. D. A., J. Immunol. 121, 1239, 1978. 39. Claesson, M. H., and Andersen, V., Clin. Exp. Immunol. 38, 483, 1979. 40. Moody, C. E., Casazza, B. A., Christenson, W. N., and Weksler. M. E.. J. Exp. Med. 150, 1448. 1979.
IMMUNOLOGY
AND
19, 314-318 (1981)
IMMUNOPATHOLOGY
Serum Migration-Inhibitory Infectious
Activity in Children with Acute Mononucleosis
A. BERTOTTO, DANIELA CAPRINO, R. VACCARO, AND FRANCA SONAGLIA Puericultura
Institute,
University
of Perugia.
Perugia.
Italy
Received July 15, 1980 Leukocyte migration-inhibitory activity (LIF) was found in the sera of 11114 (78.5%) children with heterophil-positive infectious mononucleosis as opposed to only 1115 (6.6%) control children (p < 0.001). It is postulated that the cellular unresponsiveness observed during the acute phase of the infection may be partly explained by the production of an inhibitory factor(s) with LIF-like activity, possibly by a subpopulation of suppressor lymphocytes present in excessive concentrations in Epstein-Barr virusinfected patients.
INTRODUCTION
When appropriately stimulated in vitro, lymphocytes release various biologically active factors known as lymphokines. Although some lymphokine activities are undoubtedly biological artifacts with little or no physiological relevance, experimental evidence indicates that the mechanisms controlling cell-mediated immune reactions in vivo involve substances which are identical or similar to those responsible for the in vitro phenomena (1- 5). These observations suggest the possibility that lymphokine-like activity may be detectable in vivo in certain human pathologic states. Indeed, significant serum macrophage or leukocyte migration-inhibitory factor(s) (serum MIF or LIF), or substances with MIF- or LIF-like activity, have been detected in patients with malignant lymphoproliferative disorders (6-9), post-transplantation hepatic dysfunction (lo), infectious diseases which elicit a cellular immune response (11, 12), and in immunologically competent cancer patients (13, 14). Since lymphocytes from patients with acute infectious mononucleosis (IM) spontaneously release LIF in culture (15) and since this disease is associated with alterations in immunologic status, which may be related to impaired lymphokinedependent mechanisms (15-20), it was of interest to investigate LIF activity in acute IM patients’ sera. MATERIALS
AND METHODS
Patients. Fourteen children, male and female between 7 and 14 years of age, with acute IM admitted to the Puericultura Institute at Perugia University were studied. The diagnosis was based on fever, sore throat, lymphadenopathy, splenomegaly, a positive heterophil index, and the presence of atypical lymphocytes in the peripheral blood. Blood samples (5 ml) for serum LIF activity detection were collected within the first 5 days after admission (average 10 days after 314 0090-1229/81/060314-05$01.00/O Copyright All rights
@ 1981 by Academic Press. Inc. of reproduction in any form reserved.
SERUM
LIF
IN
INFECTIOUS
MONONUCLEOSIS
315
onset of symptoms). The blood was centrifuged at SOOg for 10 min and the sera stored at 4°C until used, which was a maximum of 1 week. Blood from 15 children seen for routine well-child care at the same institute during the same time period was also assayed. These children were age and sex matched with the patients. Lculcocyte migration-inhibition assay. Twenty milliliters of venous blood were drawn from a normal donor into a syringe containing sodium citrate as anticoagulant, and allowed to sediment with 6% dextran in saline (Plander-Pierrel) for 30 to 45 min. The white cell-rich plasma was aspirated and spun at 4OOg for 5 min. The cell pellet was incubated for 7 min at 37°C with Tris-buffered ammonium chloride (0.83%) to lyse erythrocytes. The cells were then washed three times in RPM1 1640 culture medium (Eurobio-Paris) supplemented with antibiotics (penicillin, 100 III/ml; streptomycin, 100 pg/ml) and adjusted to a final concentration of 4 x 10’ cells/ml. Several microcapillary tubes (length: 63 mm) (Behringwerke, OTCL 20/21) were tilled with the cell suspension, plugged at one end, and centrifuged at SOOg for 3 min. The tubes were cut at the cell-fluid interface and inserted into hematocrit nonheparinized capillaries (internal diameter: 1, 1- 1, 2 mm) (Drummond Scientific Co., Broomal, Pa.) previously filled with a medium containing 50% decomplemented serum of the children to be tested and 50% RPM1 culture medium. The hematocrit chamber was sealed, fixed on a glass slip with silicone grease, and then incubated for 18 hr at 37°C. Each experiment consisted of four duplicates, which were averaged to obtain the mean length of migration. The migration index (MI) was calculated by dividing the migration length in the serum sample by the migration length in an allogeneic control serum obtained from a healthy individual. Inhibition greater than 20% of the leukocyte control migration (MI< 0.8) was considered significant, in accordance with the findings of Bruley-Rosset et al. (1 l), who developed a similar, but not identical, technique. Statistical analysis. The x2 test was used to determine the significance of differences in results between paired sets of data. RESULTS The accompanying Fig. 1 represents a scattergram of migration indices for all experiments carried out. Sera from healthy children did not show any significant inhibitory activity, with the exception of the one putatively normal subject with a positive reaction (l/15; 6.6%). In contrast, the majority of the patients in the experimental group studied had LIF-like activity in their serum (11/14; 78.5%). The difference between the percentages of positive serum LIF tests was statistically significant (x2: 12.6; p < 0.001). The intensity of reaction (serum LIF production) was not correlated to the degree of lymphocytosis in the blood; in particular, there was no correlation with the concentration or proportion of morphologically activated lymphocytes. Likewise, no correlation was found with age, sex, duration of symptoms, body temperature, liver damage, or heterophil antibody titers.
316
BERTOTTO
ET AL.
FIG. 1. Migration-index (MI) values of children with acute infectious mononucleosis as compared to index values of healthy children. Each dot represents the mean of quadruplicate samples. Inhibition of migration (MI < 0.8) is shown by 11 (78.5%) young patients compared with one (6.6%) control @ -C 0.001).
DISCUSSION The Epstein-Barr virus (EBV), the primary causal agent of IM, is a human herpes group virus with a striking tropism for lymphocytes. It selectively infects B lymphocytes via a specific virus receptor (21). During acute IM, the peripheral blood contains a large number of activated lymphocytes. The majority of these cells are thought to be lymphocytes reacting to EBV-infected cells; they are mainly T lymphocytes but, also, include B lymphocytes. A smaller proportion of the activated cells are EBV genome-carrying B lymphoblasts (22). Lymphokines are released by activated lymphocytes and such a release by mononuclear cells from patients with acute IM has been documented in the absence of antigenic challenge in experiments which used the macrophage or leukocyte migration-inhibition techniques (23, 15). However, these situations all represent in vitro phenomena. Using an unidirectional leukocyte migration-inhibition assay, Bruley-Rosset ef al. (11) showed serum LIF activity in four out of five IM patients sampled during the acute phase of the infection, These findings are at present the only data available on the in vivo production of lymphokine-like activity in IM. Our results, on a larger number of patients, confirm the previously published data and induce us to speculate on the nature and biological significance of the inhibitory factor(s). Although various substances, including specific antibody and circulating immune complexes, are implicated in human serum-induced migration inhibition (24-27), the assay adopted by us does not totally rule out a possible cellular origin for serum LIF. Therefore, the release of this factor(s) by activated B lymphocytes (28) and/or virus-infected lymphoid cells (29, 30) cannot be excluded. However,
SERUM LIF IN INFECTIOUS
MONONUCLEOSIS
317
when considering a possible lymphocytic origin for serum LIF, it would, perhaps, be more relevant to put forward evidence that supports the presence of an intense suppressor T-cell activity in blood from patients with IM. Tosato er al. (31) and Johnsen ef al. (32) demonstrated that during the acute phase of the illness, noncytotoxic suppressor T-lymphocytes are involved in an immune reaction which inhibites EBV target B-cell activation and proliferation. Suppressor cells in rats exert their effect by soluble factors which pass through a dialysis membrane (33) and Rich and Pierce (34) documented the production in supernatant fluids of suppressive factors, which may act on antigen-activated B cells to limit their clonal expansion or terminal differentiation into antibodysynthesizing cells. Since the kinetics of appearance of suppressive activity in culture medium is similar to that of MIF secretion (35), the suppressive factors and MIF may represent different biological functions of the same molecule (36). Viral antigens stimulate suppressor T cells to release these substances, and a feedback suppression by lymphocyte products may explain the transitory anergy observed in certain human infections (37). Experimental evidence that intravenously administered exogenous MIF-rich supernatants can suppress delayed hypersensitivity skin reactions in immunized guinea pigs (5) supports the existence of such a mechanism. It is, therefore, quite possible that circulating endogenous MIF or other lymphokines in patients with infectious diseases exert similar inhibitory effects not only on skin reactivity but also on the various other manifestations of cellular immunity. Thus, it can be postulated that cutaneous anergy (16- 18) and in vitro lymphocyte hyporesponsiveness (15, 19, 20, 38-40) observed in acute IM are partly due to the presence of an inhibitory factor(s) with LIF-like activity, possibly produced by a subpopulation of suppressor lymphocytes present in excessive concentrations in EBV-infected patients. Studies are underway in our laboratory to correlate migration inhibition with material obtained by centrifuging on Amicon 50 membranes acute IM patients’ sera. Preliminary results suggest that serum LIF is a relatively small molecule (molecular weight below 50.000). Whether other physical and chemical properties of serum LIF are similar to those of previously characterized lymphokines remains to be elucidated. REFERENCES 1. Yamamoto, K., and Takahashi, Y., Nature (London), New Biol. 233, 261, 1971. 2. Salvin, S. B., Youngner, .I. S., and Lederer, W. H., Infect. Immun. 7, 68, 1973. 3. Hay, J. B., Lachmann, P. J., and Tmka, Z., “Proceedings of the 7th Leukocyte Culture Conference” (F. Daguillard, Ed.), pp. 341-348. Academic Press, New York, 1973. 4. Yoshida, T., and Cohen, S., “Mechanisms of Cell-Mediated Immunity” (R. T. McCluskey and S. Cohen, Eds.), p. 43. Wiley, New York, 1974. 5. Yoshida, T., and Cohen, S., .I. Immunol. 112, 1540, 1974. 6. Cohen, S., Fisher, B., Yoshida, T., and Bettigole, R. E., N. Engl. .I. Med. 290, 882, 1974. 7. Yoshida, T., Edelson, R., Cohen, S., and Green, I., J. Immunol. 114, 915, 1975. 8. Ambrogi, F., Polidori, R., Azzara, A., Petrini, M., and Grassi, B., Eur. J. Cancer 14, 1107, 1978. 9. Fassas, A., Bruley-Rosset, M.. Guibout, C., and Jasmin, C., C/in. Immunnl. fmmunopathol. 14. 368, 1979. 10. Torisu, M., Yoshida, T., and Cohen, S., C/in. Immunol. Immunopathol. 3, 369, 1975. 11. Bruley-Rosset, M., Botto, H. G., and Goutner, A., Eur. J. Cancer 13, 325, 1977.
318
BERTOTTO
ET AL.
12. Bertotto, A., Stagni, G., Sonaglia, F., Caprino, D., and Velardi, A., J. Pediar., in press. 13. Botto, H. G., Gauthier, H., PouilIart, P., Bruley-Rosset, M., and Huguenin, P., Eur. J. Cancer 13, 329, 1977. 14. Fassas, A., and Bruley-Rosset, M., “Adjuvant Therapies and Markers of Post-surgical Minimal Residual Disease” (G. Bonnadona, G. Mathe, and S. Salmon, Eds.), pp. 88-92. Springer-Verlag, Heidelberg, 1978. 15. Palit, J., Bendtzen, K., and Andersen, V., C/in. Exp. Immunol. 31, 66, 1978. 16. Bentzon, J. W., Tubercle 34, 34, 1953. 17. Haider, S., Coutinho, M. DE L., Emond, R. T. D., and Sutton, R. N. P., Lancer 2, 74, 1973. 18. Mangi, R. J., Niederman, J. C., Kelleher, J. E., Dwyer, J. M., Evans, A. S., and Kantor, F. S., N. Engl. J. Med. 291, 1149, 1974. 19. Lai, P. K., Alpers, M. P., and Mac-Kay-Scollay, E. M., Infect. Immun. 17, 28, 1977. 20. Wainwright, W. H., Veltri, R. W., and Sprinkle, P. M., J. Infect. Dis. 140, 22, 1979. 21. Jondal, M., and Klein, G., 1. Exp. Med. 138, 1365, 1973. 22. Epstein, M. A., and Achong, B. G., Lancer 2, 1270, 1977. 23. Papageorgiou, P. S., Sorokin, C. F., and Glade, P. R., J. Immunol. 112, 675, 1974. 24. Cochran, A. J., Klein, G., Kiessling, R., and Gunven, P., J. Nar. Cancer Inst. 51, 1431, 1973. 25. Ortiz-Ortiz, L., Zamacona, G., Garmilla, C., and Arellanomt, T., J. Immunol. 113, 993, 1974. 26. Kotkes, P., andPick, E., C/in. Exp. Immunol. 19, 105, 1975. 27. Cochran, A. J., Mackie, R. M., Ross, C. E., Ogg, L. J., and Jackson, A. M., Inf. J. Cancer 18, 274, 1976. 28. Chess, L., Rocklin, R. E., Macdermott, R. P.. David, J. R.. and Schlossman, S. F., J. Immunol. 115, 315, 1975. 29. Flanagan, T. D., Yoshida, T., and Cohen, S., Infect. Immun. 8, 145, 1973. 30. Flanagan, T. D., Yoshida, T., and Genco, R. J., Fed. Proc. 32, 841, 1973. 31. Tosato, G., Magrath, I., Koski, I., Dooley, N., and Blaese, M., N. Engl. J. Med. 301, 1133. 1979. 32. Johnsen, H. E., Madsen, M., and Kristensen, T., Stand. J. Immunol. 10, 251, 1979. 33. Tardieu, M., and Daguillard, F., Cell. Zmmunol. 19, 148, 1975. 34. Rich, R. R., and Pierce, C. W., J. Immunol. 112, 1360, 1974. 35. David, J. R., and David, R. R., Progr. Allergy 16, 300, 1972. 36. Jirillo, E., and Fumarola, D., Ann. Sclavo (Siena) 21, 573, 1979. 37. Kantor. F. S., N. Engi. J. Med. 292, 629, 1975. 38. Nikoskelainen, J., Ablashi, D. V., Isenberg, R. A., Neel, E. Il., Miller, R. G.. and Stevens. D. A., J. Immunol. 121, 1239, 1978. 39. Claesson, M. H., and Andersen, V., Clin. Exp. Immunol. 38, 483, 1979. 40. Moody, C. E., Casazza, B. A., Christenson, W. N., and Weksler. M. E.. J. Exp. Med. 150, 1448. 1979.