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In vivo effects of thymosin on cellular immunity
In Vivo Effects of Thymosin on Cellular Immunity Samuel B. Salvin, Ph.D. Department of Microbiology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania Impairment of thymic function may result in a reduction in the capacity to develop immune responses. It has been demonstrated that soluble thymic factors can restore immunologic reactivity in experiments involving (a) activity of thymus grafts in diffusion chambers, (b) restoration of immunocompetency of neonatally thymectomized adult female mice by pregnancy, and (c) action of thymic extracts on immunologic reactivity (13, 15-17, 2 4 - 2 6 , 29, 39). Although a large number of thymic extracts have been reported to have immunologic activity, at the present time probably thymosin and its derivatives have been examined most extensively (35). Thus, with regard to cellular-immune responses in vivo, thymosin was shown to restore the capacity of spleen cells from neonatally thymectomized mice to produce graft-versus-host (GVH) reactions (14) and to restore the capacity of neonatally thymectomized mice to reject skin allografts (9). The purpose of this article is to review the effects of thymosin and its component peptides on immune responses of the cellular type in vivo. Three major effects of thymosin in vivo will be discussed, namely, resistance to infection, rejection of grafts or tumors, and correction of immunodeficiency states. These effects will be examined first in experimental animals and then, to a more limited ex.tent, in humans.
Use of Thymosin in Experimental Animals Resistance to Infection Defenses against such organisms as Candida albicans, Mycobacterium tuberculosis, and Listeria mono~'togenes primarily involve cell-mediated re-
sponses on the part of the host. Since macrophages and thymus-derived lymphocytes are primarily involved in this type of specific resistance, a substance such as thymosin may have a profound effect on the host response. Studies have been reported wherein inbred murine strains were challenged intravenously with a sublethal dose of C. albicans; at intervals thereafter their kidneys were cultured quantitatively for growth of the yeast (31, 32). The strains varied greatly in their capacity to resist infection: Some strains, such as C57B1/6J, C57B1/10SNJ, and C57BI/KsJ were highly resistant while other strains, such as C3H/HeJ, CBA/ CaJ, AKR/J, and DBA/IJ were highly susceptible. Those strains that were resistant to infection were able to release high titers of migration inhibitory factor (MIF) and interferon-gamma (IFN-7) in vivo into the circulation on stimulation and to develop footpad reactions of the delayed type. In contrast, those strains that were susceptible tO infection with C. albicans were able to release only low titers, if any at all, of MIF and IFN-7 into the circulation and to develop relatively poor footpad reactions of the delayed type (22, 23). Both the resistant and susceptible strains were administered daily doses of 5 ~g thymosin fraction 5 (TF5) from the time of challenge with C. albicans to the time of sacrifice. The resistant strains became more susceptible to infection with C. albicans, while the susceptible strains became more resistant (31). TF5 also had different effects on the in vivo release of MIF and IFN-~, into the circulation, in that the titers of the two lymphokines were decreased in the circulation of the resistant strains but were markedly increased in three of the five susceptible strains (22). Thus, a parallelism existed in the response of a given strain to thymosin with regard to resistance to intravenous infection with C. albicans and the in vivo release of MIF and IFN- 7 into the circulation. With regard to the effect of TF5 on
delayed footpad hypersensitivity, the responses of resistant-sensitized mice to specific antigen were enhanced, whereas, the responses of the susceptible strains were not affected. The administration of TF5 to alloxan-diabetic mice affects their resistance to infection with C. albicans (28, 32). When mice of resistant, high-responder strains such as C57B1/ 10SNJ and C57BI/KsJ became hyperglycemic after treatment with alloxan, a marked reduction occurred in the resistance to infection with C. albicans, the in vivo release of MIF into the circulation, and the expression of delayed hypersensitivity in a sensitized mouse on challenge of the footpads with specific antigen. The effect of one of the components of TF5, namely thymosin cq, was examined in mice infected with a lethal dose of C. albicans (2). Some prolongation of survival time was noted in (Balb/cCr x DBA/2 Cr)F 1 and cyclophosphamide-treated (BalblcCr × DBA/2 Cr)F 1 mice after administration of the peptide a I at optimal dose and schedule. Studies also have been carried out on the in vivo effects of thymosin treatment of mice infected with Mycobacterium bovis strain BCG (5, 6, 21). When adult thymectomized, lethally irradiated, bone marrow-reconstituted mice were injected daily for 16 days with thymosin (8 days before challenge) and infected intravenously with 4 × 106 living BCG, 80% of the controls were dead by day 100 postinfection. In contrast, those mice treated with thymosin did not have any deaths during that interval. These thymosintreated mice also had enhanced delayed hypersensitivity reactions to "old tuberculin." The administration of thymosin did not inhibit the growth of BCG in vitro or in vivo, but did result in the reduction of the number of foamy macrophages in the lung. When thymectomized, irradiated, bone marrow-reconstituted (THXB) B6D2 mice were challenged intravenously with 1 × 106 viable BCG and treated with doses of thymosin from 20 to 100 mg/Kg body weight, a 10to 100-fold drop in bacterial counts
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was noted in both the lungs and spleen, in comparison with untreated control animals. The control THXB mice, which were depleted in T lymphocytes, had a slow gradual increase in the number of viable BCG over this period (5). Treatment of mice with thymosin also affected their cell-mediated immune (CMI) responses to infection with L. monocytogenes (27). In this study, female Balb/c mice, either 6 wk-of-age (young) or I I mo-of-age (old), were fed either a control diet of 20% casein or a protein-deficient (MPD) diet of 4% casein, but equal in calories. The mice were treated with 100 I-tg TF5 every other day for i or 2 wk. One day after a total treatment of 400 or 700 p.g of thymosin, the mice were infected intraperitoneally ,/¢ith I × 104 cells of L. monocytogenes. The effect of the thymosin depended on the age as well as the diet of the mice. Young mice fed an MPD diet did not have their resistance to L. monocytogenes altered by treatment with thymosin, whereas, young mice fed a control diet had their resistance significantly suppressed by the thymosin treatment. Old mice also showed different responses to treatment with thymosin, depending on the diet. Here, however, resistance against L. monocytogenes by the old mice fed the MPD diet was enhanced by treatment with thymosin, whereas, the resistance of old mice fed the control diet was impaired at all times after thymosin treatment. Thus, both young and old control mice of the Balb/c strain became more susceptible to an intraperitoneal infection with L. monocytogenes after treatment with TF5, whereas, only the old MPD mice had an-increase in resistance after treatment with TF5. Thymosin also improved the resistance of experimental animals to some virus infections. In C57B1 and CC57W mice, for example, chronic infections by influenza virus were prevented (33). When 7-day-old mice were treated with thymosin after they had been infected with reovirus type 3 at age 2 4 - 3 6 hr, a substantial enhancement of survival followed (41). Such infected mice, however, failed to
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exhibit increased T-lymphocyte responses to concanavalin A (Con A) and elevated delayed-type hypersensitivity to oxazolone sensitization. Normal uninfected 7-day-old mice treated in an identical manner with thymosin exhibited an increase in spleen-lymph response to Con A and an increase of the delayed-type response to oxazolone. Thus, the capacity of mice to survive challenge with reovirus type 3 was enhanced by treatment with thymosin; however; this increase in resistance apparently was not paralleled by their capacity to respond to Con A or to develop increased delayed-type hypersensitivity to oxazolone. Treatment of C57B1/6 mice with TF5 modulated the production of IFN2¢ (12). Subcutaneous injection of a single dose of 100 or 200 ttg of TF5 about 40 hr before the induction of IFN with Newcastle's disease virus (NDV) significantly increased serum IFN titers, in comparison with the IFN titers induced in untreated mice. Maximum enhancement was observed when a single injection of TF5 was administered 6 - 1 2 hr before the NDV. Increased IFN production of IFN-',/also was observed in cultures of spleen cells stimulated with Con A, where the cells were from mice treated in vivo with 150 lxg TF5. Two purified thymosin polypeptides were found to have opposite effects: a l, which induced maturation of helper T-cells, and ct7, which induced maturation of suppressor T-ceils. Three injections each of 5 ~g thymosin cq administered before NDV enhanced production of IFN, while three daily injections each of 1 I-tg thymosin et7 suppressed the responsiveness of mice to NDV. Resistance to T u m o r s Thymosin has the capacity to induce an altered host response to tumorcausing viruses. Thus, thymosin altered the responses of AKR or adult thymectomized Balb/c mice exposed to Rauscher leukemia virus (10). Depending on the timing of the administration of thymosin, the hormone was found to either reduce or enhance virus titer or tumor cell proliferation.
© 1984 Elsevier Science Publishing Co., Inc.
Thymosin also affected the response of mice to Moloney sarcoma virus (43, 11). In one set of experiments (43), mice received three injections of TF3 before challenge with the virus. Examination of the mice after 60 days indicated that thymosin had a protective effect, since 14:36 thymosin-treated mice survived and of the controls 0:30 survived. The incidence of tumors was the same in both groups. The Immunodeficiency State and Thymosin Evidence that CMI is enhanced by thymosin has been presented not only in immunodificient mice (1, 9, 10, 36), but also in inbred Weimaraner dogs with small thymuses and wasting syndrome (30). The administration of thymosin to neonatally thymectomized CBA mice reduced the incidence of wasting disease and death (10). These mice also exhibited enhanced lymphocytopoiesis, and restoration of such cell-mediated functions as rejection of skin allografts and elicitation of GVH reactions. The Weimaraner pups had a marked absence of thymic cortex, as well as abnormalities in T-dependent immune functions such as lymphocyte blastogenesis to PHA (30). Two pups from a sire and dam that were known to have produced affected offspring were chosen for detailed study. Both pups developed a severe wasting syndrome at age 5 - 1 0 wk, at which time they were administered TF5 1 mg/kg/day subcutaneously for 6 - 7 days. There was good clinical response, with weight gain and increased vigor. Although both dogs responded to therapy, neither displayed an increased in vitro lymphocyte response to PHA. T-cell functions in old mice were enhanced when they were injected with synthetic thymosin cq (8). When old mice of the (C57B1/10 × DBA/ 2)F t strain were inoculated with 1-10 txg of the thymosin for 5 days, the helper activity of their T ceils was efficiently repaired. Studies have been presented that show that thymosin may alter the course of autoimmunity in NZB mice (38). Here, TF5 was administered intraperitoneally into NZB mice in 3 - 9
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injections totaling 0.3-3.0 mg. DNA synthesis was studied in the splenic and lymph node cells after transfer of the NZB thymocytes into lethally irradiated C57BI/6J mice. This treatment of 8-wk-old donor NZB mice with thymosin corrected the abnormal DNA proliferation of NZB thymocytes. Some of the thymosin fractions, therefore, have the definitive capability of enhancing immunologic responses, especially those of CMI in vivo in laboratory animals. This increased resistance has been demonstrated with respect to infectious agents, tumor growth, and immunoregulation, but has been most striking in infectious diseases, such as experimental candidiasis, where cellular immune responses can be most effective.
Use of Thymosin in Humans TF5 has been administered to humans who have had any one of several possible immunologic defects, such as cancer, autoimmune disease, malnutrition, or immunodeficiency. The results have not been consistent, possibly because of the differences in the conditions of experimentation or because of the differences in the physical condition of the patients. General beneficial effects of treatment with thymosin, however, have occurred. These effects are illustrated as follows. Thymosin may improve the clinical and immunologic condition of patients with immunodeficiency syndromes. For example, a patient with thymic hypoplasia was selected to receive thymosin in vivo, since her T-cell rosettes had increased from 15% to 48% after incubation of her lymphocytes with thymosin in vitro (40). She improved clinically during the immunotherapy, the percentage of her T-cell rosettes gradually increased to normal, and positive skin tests of delayed-type hypersensitivity developed. Thus, thymosin may be useful clinically for partial reconstitution of cellular immunity. An increase percentage of T-cell rosettes after incubation with thymosin in vitro may predict which patients will respond to thymosin therapy in vivo. In another case, an infant with hy-
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poparathyroidism, severe T-cell deficiency, and hypoimmunoglobulinemia with an increased number of B-lymphocytes was treated with thymosin (3). The initial proportion of lymphocytes forming spontaneous rosettes with E-rosettes was 12%, while a response of lymphocytes to PHA was absent. Two months after treatment with thymosin, 68% of lymphocytes formed rosettes with sheep erythrocytes, and the response to PHA was normal. Thymosin was found to improve the immunologic condition in patients with malignancies. In one study involving ten patients with disseminated malignancies (7), administration of thymosin was associated with an increase in Erosette-forming capacity of the patients' lymphocytes, and in the development of new delayed skin test reactions to recall antigens. Thymosin treatment of patients with head and neck squamous carcinoma resuited in improvement of parameters of CMI, such as leukocyte migration inhibition (42). Here, the leukocytes of cancer patients had significantly lower migration inhibition to streptokinase-streptodomase (SK-SD) than did the leukocytes of normal patients. Thymosin increased the degree of migration inhibition to SK-SD in the cancer patients to levels similar to those of normal subjects. These and other studies (19, 20, 34) indicate that thymosin may aid in the restoration of parameters of CMI in patients with cellular immunodeficiencies. In studies on patients with small cell bronchogenic carcinoma, individuals received intensive chemotherapy with TF5 60 mg/m2 (Tro), 20 mg/m2, or 0 mg/m2 (P) twice weekly during the initial 6 wk of therapy. Chemotherapy was then continued for 2 yr (4, 18). The overall growth of the tumors did not differ significantly among the three treatment groups. Survival of the 'F6o group, however, was significantly greater than that of the P group. The results from experiments involving treatment of patients with thymosin support the hypothesis that patients with relatively low levels of immunity benefit most from administration of thymosin. The mechanism
© 1984 Elsevier Science Publishing Co., Inc.
of action, therefore, is reconstitution of immune defects rather than augmentation of relatively normal levels of cell-mediated resistance.
Conclusions That the immunologic activity of TF5 is not definitive may be related to the fact that it is not a single entity but a mixture of peptides. In addition, these peptides may not only reinforce each other's immunologic activities, but may actually interfere with or counteract each other's activities. An indication of this possible situation was shown when C57BI/10SNJ and C3H/HeJ mice were treated with TF5 and then challenged with C. albicans (31). Normally, mice of the C57BI/ 10SNJ inbred strain are resistant to challenge with C. albicans, whereas, mice of the C3H/HeJ strain are highly susceptible. When these strains are treated with TF5 at the time of challenge with C. albicans, however, the C57Bl/10SNJ mice become more susceptible, while the C3H/HeJ mice become more resistant. Whether the strains are responding to different peptides in the TF5 or whether they are responding differently to the same peptide is not known. It seems possible, therefore, that one single naturally occurring peptide may be present, which is able to enhance CMI and, thus, also enhance resistance to infection. Such a peptide may be available, since a new naturally occurring peptide recently has been isolated; it has high potency in inhibiting the growth of C. albicans in susceptible mouse inbred strains and does not enhance the growth in resistant strains (Salvin and Horecker, unpublished data). Another thymic peptide may be present that is able to do the reverse (i.e., suppress CMI responses and, thereby, enhance susceptibility to infection). This thought was presented many years ago by Szent-Gyorgi, when he stated that his thymus "extracts contained two active substances, the one promoting, the other inhibiting malignant growth, and the result depended on their balance. It is a common experience that two unknown variables make results messy" (37).
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The author expresses thanl~s and appreciation to Dr. Ruth Neta for her critical review of the manuscript. References 1. Asanuma, Y., A. L. Goldsteln, and A. White (1970). Reduction in the incidence of wasting disease in neonatally thymectomized CBAJW mice by the injection of thymosin. Endocrinology 86:600-610. 2. Biston|, F. el al. (1982). Increase of mouse resistance to Candida albicans infection by thymosin alpha 1. Infect. Immun. 36:609-614. 3. Bonagura, V. R. and J. Pitt (1981). Hypoparathyroidism with T-cell deficiency and hypoimmunoglobulinemia: Response to thymosin therapy. Clin. lmmunol. Immunopathol. 18:375386. 4. Cohen, M. H. et al. (1979). Thymosin fraction 5 and intensive combination therapy. J.A.M.A. 241:18131815. 5. Collins, F. M. and L. K. Auclair (1979). Effect of thymosiu treatment on antituberculous immunity in immunosuppressed mice. J. Reticuloendothel. Soc. 26:143-153. 6. Collins, F. M. and N. E. Morrison (1979). Restoration of T-cell responsiveness by thymosin: Expression of anti-tuberculous inmaunity in mouse lungs. Infect. Immun. 23:330-335. 7. Costanzi, J. J. et al. (1977). The effect of thymosin on patients with disseminated malignancies. A phase I study. Cancer 40:14-19. 8. Frasca, D., M. Garavini, and G. Doria. (1982). Recovery of T-cell functions in aged mice injected with synthetic thymosin-alpha I. Cell. Immunol. 72:384-391. 9. Goldstein, A. L. et al. (1970). Influence of thymosin on cell-mediated and humoral immune responses in normal and in immunologically deficient mice. J. Immunol. 104:359-366. 10. Goldstein, A. L. et al. (1976). Use of thymosin in the treatment of primary immunodeficiency diseases and cancer. Med. Clin. 60:591-606. 11. Hardy, M. A. et al. (1971). Reversal by thymosin of increased susceptibility of immunosuppressed mice to Moloney sarcoma virus. Trans. Proc. 3:926928. 12. Huang, K.-Y. et al. (1981). Thymosin treatment modulates production of interferon. J. Interferon Res. 1:411-420. 13. Law, L. W. et al. (1964). In The Thymus, Wistar Institute Symposium
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Monograph No. 2. Wistar Institute Press, Philadelphia, pp. 105-117. Law, L. W., A. L. Goldstein, and A. White (1968). Influence of thymosin on immunological competence of lymphoid cells from thymectomized mice. Nature 219:1391-1392. Law, L. W. et al. (1964). Humoral thymic factor in mice: Further evidence. Science 143:1049-1051. Levey, R. H., N. Tralnin, and L. W. Law (1963). Evidence for function of thymic tissue in diffusion chambers implanted in neonatally thymectomized mice. Preliminary report. J. Natl. Cancer Inst. 31"199-206. Levey, R. H. et al. (1963). Lymphocytic chorimeningitis infection in neonatally thymectomized mice bearing diffusion chambers containing thymus. Science 142:483-485. Lipson, D. S. et al. (1979). Thymosin immunotherapy in patients with small cell carcinoma of the lung. Correlation of in vitro studies with clinical course. Cancer 43:863-870. Marshall, G. D., J r . et al. (1980). Thymosin: Basic properties and clinical application in the treatment of immunodefieiency diseases and cancer. Recent results. Cancer Res. 7 5 : I 0 0 105. Mawhlnney, H., V. F. Gleadhill, and S. MeCrea (1979). In vitro and in vivo responses to thymosin in severe combined immunodeficiency. Clin. Immunol. Immunopathol. 14:196203. Morrison, N. E. and F. M. Collins (1976). Restoration of T-cell responsiveness by thymosin: Development of antituberculous resistance in BCG-infected animals. Infect. Immun. 13:554-563. Neta, R. and S. B. Saivin (1983). Resistance and susceptibility to infection in inbred murine strains. II. Variations in the effect of treatment with thymosin fraction 5 on the release of lymphokines, in vivo. Cell. Immunol. 75:173-180. Neta, R., S. B. Salvin, and M. Sabaawi (1981). Mechanisms in the/n vivo release of lymphokines. I. Comparative kinetics in the release of six lymphokines in inbred strains of mice. Cell. Immunol. 64:203-219. Osoba, D. (1965). Immune reactivity in mice thymectomized soon after birth: Normal response after pregnancy. Science 147:298-299. Osoba, D. and J. F. A. P. Miller (1963). Evidence for a humoral thymus factor responsible for the maturation of immunological faculty. Nature (Aug. 17):653-654.
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26. Osoba, D. and J. F. A. P. Miller (1964). The lymphoid tissues and immune responses of neonatally thymectomized mice bearing thymus tissue in Millipore diffusion chambers. J. Exp. Med. 119:177-194. 27. Petro, T. M., G. Chien and R. R. Watson (1982). Alteration of cell-mediated immunity to Listeria monocTtogenes in protein-malnourished mice treated with thymosin fraction V. Infect. Immun. 37:601-608. 28. Raffel, L. et al. (1981). Experimental Candida albicans, Staphylococcus aureus, and Streptococcus faecalis pyelonephritis in diabetic rats. Infect. Immun. 34:773-779. 29. Roberts, S. and A. White (1949). Biochemical characterization of lymphoid tissue proteins. J. Biol. Chem. 178:151-162. 30. Roth, J. A. et al. (1980). Thymic abnormalities and growth hormone deficiency in dogs. Am. J. Vet. Res. 41:1256-1262. 31. Salvin, S. B. and R. Neta. (1983). Resistance and susceptibility to infection in inbred murine strains. I. Variations in the response to thymic hormones in mice infected with Candida albicans. Cell. Immunol. 75:160-172. 32. Salvin, S. B. and E. P. Tanner. (1983). Resistance and susceptibility to infection in inbred murine strains. III. Effect of thymosin on cellular immune responses in alloxan diabetic mice. Clin. Exp. Immunol. 54:133-139. 33. Savtsova, A. D. et al. (1982). Role of immune cytolysis and its thymosin stimulation in experimental influenza. Zh. Milrobiol. Epidemiol. Immunobiol. 3:55-58. 34. Schafer, L. A. et ai. (1976). In vitro and in vivo studies with thymosin in cancer patients. Ann. N.Y. Acad. Sci. 277:609-620. 35. Schulof, T. S. et al. (1981). b7 K'. W. Sells and W. V. Miller (eds.), The Lymphocyte. Alan R. Liss, New York, pp. 191-215. 36. Splitter, G. A., T. C. McGuire, and W. C. Davis (1976). Ability of thymus, thymus epithelium and thymus extracts to cause allograft rejection in nude and AT × BM mice. Fed. Proc. 35:593 (abst.). 37. Szent-Gyorgi, A. (1960). On the chemistry of the thymus gland, pp. 123-125, In Introduction to a Submolecular Biology. Academic Press, New York. 38. Talal, N. et al. (1975). Effect of thymosin on thymocyte proliferation and autoimmunity in NZB mice. Ann. N.Y. Acad. Sci. 249:438-450.
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39. Trainin, N. et al. (1966). A thymic factor preventing w/~sting and influencing lymphopoiesis in mice. Israel J. Med. Sci. 2:549-559. 40. Wara, D. W. et ai. (1975). Thymosin activity in patients with cellular immunodeficiency. N. Engl. J. Med 292:70-74.
41. Willey, D. E. and R. N. Ushijima. (1981). The effect of thymosin on mufine lymphocyte responses and corticosteroid levels during acute reovirus type 3 infection of neonatal mice. Clin. Immunol. Immunopathol. 19:35-43. 42. Wolf, G. T., S. E. Kerney, and P. A. Chretien (1980). Improvement
Thymosin oq-Induced Modulation of Immunoregulatory T-Lymphocyte Activities Gino Doria, M.D., Daniela Frasca, M.D., and Luciano Adorini M.D. Laboratory of Pathology, ENEA, C.R.E. Casaccia Rome, Italy Thymic factors are known to play a crucial role in the differentiation and maturation of T lymphocytes. Several peptides with thymic hormone-like activity have been isolated from thymus extracts and, among the different preparations, thymosins present in fraction 5 have been thoroughly studied and characterized. Fraction 5, a partially purified extract from bovine thymus (8), contains 4 0 - 5 0 peptide components identified by isoelectrofocusing, and exhibits a wide range of biologic activities in extensive animal and clinical studies. Among these peptides, thymosin otI has been the first biologically active polypeptide to be isolated, sequenced (4), and synthesized (13). Thymosin ct1 consists of 28 amino acid residues wtih a molecular weight of 3108, and has been shown to be 101000 times more active than thymosin fraction 5 (TF5) in promoting phenotypic expression of T-cell markers and helper T-cell functions.
Summary of Biologic Studies and Clinical Trials with Thymosin cq Thymosin ct I has been shown to be an effective modulator of helper T-cell activity in several systems using both normal or immunodeficient experimental models (11,15). Mice injected with ett show enhanced T-cell responses to mitogens, and increased. lymphokine and prostaglandin produc-
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tion. Thymosin ett also prolongs the survival of tumor-bearing mice or of immunosuppressed mice infected with Candida spp., BCG, or Cryptococcus spp. In mice, studies in vitro indicate that thymosin eq induces the development of Thy l-positive as well as Lyt 1,2,3-positive cells from T-cell precursors, it modulates terminal deoxynucleotidyl transferase (TdT) activity in bone marrow cells, thymocytes, and splenocytes, and it increases the percentage of cortisone-resistant thymocytes. Moreover, it enhances the production of both interferon-alpha and -gamma (IFN-et, -~) and of macrophage inhibitory factor, T-cell dependent IgG, IgA, and IgM secondary antibody responses, helper T-cell activities, and E-rosette formation. In humans, as well, incubation in vitro of peripheral blood lymphocytes (PBL) with thymosin al increases the percentage of E-rosette-forming cells and autologous rosette-forming cells in patients with primary immunodeficiency, cancer, viral infection, or autoimmune pathology. Clinical trials with etI alone or in conjunction with conventional chemotherapy have been performed on cancer patients. Preliminary results indicate that administration in vivo of et 1 enhances T-cell number and functions in advanced cancer patients with low pretreatment T-cell values. Beneficial effects of this thymic hormone at the clinical level, however, are much more evident when eq is administrated in conjunction with chemotherapy, rather than alone.
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of impaired leukocyte migration inhibition in patients with head and neck squamous carcinoma. Am. J. Surg. 140:531-537. 43. Zisblatt, M. et al. (1970). Acceleration by thymosin in the development of resistance to murine sarcoma virusinduced tumor in mice. Proc. Natl. Acad. Sci. 66:1170-1174.
Recently, it was demonstrated that markedly elevated serum levels of ff-I represent one of the early signals for identifying patients with acquired immunodeficiency syndrome (AIDS) and asymptomatic individuals at high risk (6). These patients also show markedly depressed OKT4:OKT8 ratios. In spite of the elevated levels of et 1, T4 cells isolated from PBL of AIDS patients exhibit enhanced helper T-cell functions after incubation in vitro with etI.
Role of Thymosin cq in Aging of the Immune S y s t e m Thymosin ~l serum levels, as detected by radioimmunoassay, decrease with advancing age in humans and in mice (5) as a result of thymus involution (7). Since thymic factors are known to promote the differentiation of immature T lymphoeytes into functional cells, the decline in serum concentration of these factors is likely to play an important role in age-associated immunodeficiencies. Thus, during senescence, thymus-dependent immunity is more strongly compromised than thymus-independent immunity. The decline of T-cell mediated antibody response associated with advancing age (2) can be partially or fully restored by injection of thymic hormone preparations (1, 3, 14). In recent studies carded out in our laboratory, immunodeficient aged mice were injected with synthetic thymosin ctI in order to assess whether or not this peptide is able to induce restoration of helper T-cell activity in vivo. (C57B1/10 × DBA/2)F 1 mice, age 3 24 mo, were left uninjected or received one intraperitoneal injection of 1, 10, or 100 I.tg synthetic ct I (gift of Dr. A. L. Goldstein). After 3 days,
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Use of Thymosin in Experimental Animals Resistance to Infection Defenses against such organisms as Candida albicans, Mycobacterium tuberculosis, and Listeria mono~'togenes primarily involve cell-mediated re-
sponses on the part of the host. Since macrophages and thymus-derived lymphocytes are primarily involved in this type of specific resistance, a substance such as thymosin may have a profound effect on the host response. Studies have been reported wherein inbred murine strains were challenged intravenously with a sublethal dose of C. albicans; at intervals thereafter their kidneys were cultured quantitatively for growth of the yeast (31, 32). The strains varied greatly in their capacity to resist infection: Some strains, such as C57B1/6J, C57B1/10SNJ, and C57BI/KsJ were highly resistant while other strains, such as C3H/HeJ, CBA/ CaJ, AKR/J, and DBA/IJ were highly susceptible. Those strains that were resistant to infection were able to release high titers of migration inhibitory factor (MIF) and interferon-gamma (IFN-7) in vivo into the circulation on stimulation and to develop footpad reactions of the delayed type. In contrast, those strains that were susceptible tO infection with C. albicans were able to release only low titers, if any at all, of MIF and IFN-7 into the circulation and to develop relatively poor footpad reactions of the delayed type (22, 23). Both the resistant and susceptible strains were administered daily doses of 5 ~g thymosin fraction 5 (TF5) from the time of challenge with C. albicans to the time of sacrifice. The resistant strains became more susceptible to infection with C. albicans, while the susceptible strains became more resistant (31). TF5 also had different effects on the in vivo release of MIF and IFN-~, into the circulation, in that the titers of the two lymphokines were decreased in the circulation of the resistant strains but were markedly increased in three of the five susceptible strains (22). Thus, a parallelism existed in the response of a given strain to thymosin with regard to resistance to intravenous infection with C. albicans and the in vivo release of MIF and IFN- 7 into the circulation. With regard to the effect of TF5 on
delayed footpad hypersensitivity, the responses of resistant-sensitized mice to specific antigen were enhanced, whereas, the responses of the susceptible strains were not affected. The administration of TF5 to alloxan-diabetic mice affects their resistance to infection with C. albicans (28, 32). When mice of resistant, high-responder strains such as C57B1/ 10SNJ and C57BI/KsJ became hyperglycemic after treatment with alloxan, a marked reduction occurred in the resistance to infection with C. albicans, the in vivo release of MIF into the circulation, and the expression of delayed hypersensitivity in a sensitized mouse on challenge of the footpads with specific antigen. The effect of one of the components of TF5, namely thymosin cq, was examined in mice infected with a lethal dose of C. albicans (2). Some prolongation of survival time was noted in (Balb/cCr x DBA/2 Cr)F 1 and cyclophosphamide-treated (BalblcCr × DBA/2 Cr)F 1 mice after administration of the peptide a I at optimal dose and schedule. Studies also have been carried out on the in vivo effects of thymosin treatment of mice infected with Mycobacterium bovis strain BCG (5, 6, 21). When adult thymectomized, lethally irradiated, bone marrow-reconstituted mice were injected daily for 16 days with thymosin (8 days before challenge) and infected intravenously with 4 × 106 living BCG, 80% of the controls were dead by day 100 postinfection. In contrast, those mice treated with thymosin did not have any deaths during that interval. These thymosintreated mice also had enhanced delayed hypersensitivity reactions to "old tuberculin." The administration of thymosin did not inhibit the growth of BCG in vitro or in vivo, but did result in the reduction of the number of foamy macrophages in the lung. When thymectomized, irradiated, bone marrow-reconstituted (THXB) B6D2 mice were challenged intravenously with 1 × 106 viable BCG and treated with doses of thymosin from 20 to 100 mg/Kg body weight, a 10to 100-fold drop in bacterial counts
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was noted in both the lungs and spleen, in comparison with untreated control animals. The control THXB mice, which were depleted in T lymphocytes, had a slow gradual increase in the number of viable BCG over this period (5). Treatment of mice with thymosin also affected their cell-mediated immune (CMI) responses to infection with L. monocytogenes (27). In this study, female Balb/c mice, either 6 wk-of-age (young) or I I mo-of-age (old), were fed either a control diet of 20% casein or a protein-deficient (MPD) diet of 4% casein, but equal in calories. The mice were treated with 100 I-tg TF5 every other day for i or 2 wk. One day after a total treatment of 400 or 700 p.g of thymosin, the mice were infected intraperitoneally ,/¢ith I × 104 cells of L. monocytogenes. The effect of the thymosin depended on the age as well as the diet of the mice. Young mice fed an MPD diet did not have their resistance to L. monocytogenes altered by treatment with thymosin, whereas, young mice fed a control diet had their resistance significantly suppressed by the thymosin treatment. Old mice also showed different responses to treatment with thymosin, depending on the diet. Here, however, resistance against L. monocytogenes by the old mice fed the MPD diet was enhanced by treatment with thymosin, whereas, the resistance of old mice fed the control diet was impaired at all times after thymosin treatment. Thus, both young and old control mice of the Balb/c strain became more susceptible to an intraperitoneal infection with L. monocytogenes after treatment with TF5, whereas, only the old MPD mice had an-increase in resistance after treatment with TF5. Thymosin also improved the resistance of experimental animals to some virus infections. In C57B1 and CC57W mice, for example, chronic infections by influenza virus were prevented (33). When 7-day-old mice were treated with thymosin after they had been infected with reovirus type 3 at age 2 4 - 3 6 hr, a substantial enhancement of survival followed (41). Such infected mice, however, failed to
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exhibit increased T-lymphocyte responses to concanavalin A (Con A) and elevated delayed-type hypersensitivity to oxazolone sensitization. Normal uninfected 7-day-old mice treated in an identical manner with thymosin exhibited an increase in spleen-lymph response to Con A and an increase of the delayed-type response to oxazolone. Thus, the capacity of mice to survive challenge with reovirus type 3 was enhanced by treatment with thymosin; however; this increase in resistance apparently was not paralleled by their capacity to respond to Con A or to develop increased delayed-type hypersensitivity to oxazolone. Treatment of C57B1/6 mice with TF5 modulated the production of IFN2¢ (12). Subcutaneous injection of a single dose of 100 or 200 ttg of TF5 about 40 hr before the induction of IFN with Newcastle's disease virus (NDV) significantly increased serum IFN titers, in comparison with the IFN titers induced in untreated mice. Maximum enhancement was observed when a single injection of TF5 was administered 6 - 1 2 hr before the NDV. Increased IFN production of IFN-',/also was observed in cultures of spleen cells stimulated with Con A, where the cells were from mice treated in vivo with 150 lxg TF5. Two purified thymosin polypeptides were found to have opposite effects: a l, which induced maturation of helper T-cells, and ct7, which induced maturation of suppressor T-ceils. Three injections each of 5 ~g thymosin cq administered before NDV enhanced production of IFN, while three daily injections each of 1 I-tg thymosin et7 suppressed the responsiveness of mice to NDV. Resistance to T u m o r s Thymosin has the capacity to induce an altered host response to tumorcausing viruses. Thus, thymosin altered the responses of AKR or adult thymectomized Balb/c mice exposed to Rauscher leukemia virus (10). Depending on the timing of the administration of thymosin, the hormone was found to either reduce or enhance virus titer or tumor cell proliferation.
© 1984 Elsevier Science Publishing Co., Inc.
Thymosin also affected the response of mice to Moloney sarcoma virus (43, 11). In one set of experiments (43), mice received three injections of TF3 before challenge with the virus. Examination of the mice after 60 days indicated that thymosin had a protective effect, since 14:36 thymosin-treated mice survived and of the controls 0:30 survived. The incidence of tumors was the same in both groups. The Immunodeficiency State and Thymosin Evidence that CMI is enhanced by thymosin has been presented not only in immunodificient mice (1, 9, 10, 36), but also in inbred Weimaraner dogs with small thymuses and wasting syndrome (30). The administration of thymosin to neonatally thymectomized CBA mice reduced the incidence of wasting disease and death (10). These mice also exhibited enhanced lymphocytopoiesis, and restoration of such cell-mediated functions as rejection of skin allografts and elicitation of GVH reactions. The Weimaraner pups had a marked absence of thymic cortex, as well as abnormalities in T-dependent immune functions such as lymphocyte blastogenesis to PHA (30). Two pups from a sire and dam that were known to have produced affected offspring were chosen for detailed study. Both pups developed a severe wasting syndrome at age 5 - 1 0 wk, at which time they were administered TF5 1 mg/kg/day subcutaneously for 6 - 7 days. There was good clinical response, with weight gain and increased vigor. Although both dogs responded to therapy, neither displayed an increased in vitro lymphocyte response to PHA. T-cell functions in old mice were enhanced when they were injected with synthetic thymosin cq (8). When old mice of the (C57B1/10 × DBA/ 2)F t strain were inoculated with 1-10 txg of the thymosin for 5 days, the helper activity of their T ceils was efficiently repaired. Studies have been presented that show that thymosin may alter the course of autoimmunity in NZB mice (38). Here, TF5 was administered intraperitoneally into NZB mice in 3 - 9
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injections totaling 0.3-3.0 mg. DNA synthesis was studied in the splenic and lymph node cells after transfer of the NZB thymocytes into lethally irradiated C57BI/6J mice. This treatment of 8-wk-old donor NZB mice with thymosin corrected the abnormal DNA proliferation of NZB thymocytes. Some of the thymosin fractions, therefore, have the definitive capability of enhancing immunologic responses, especially those of CMI in vivo in laboratory animals. This increased resistance has been demonstrated with respect to infectious agents, tumor growth, and immunoregulation, but has been most striking in infectious diseases, such as experimental candidiasis, where cellular immune responses can be most effective.
Use of Thymosin in Humans TF5 has been administered to humans who have had any one of several possible immunologic defects, such as cancer, autoimmune disease, malnutrition, or immunodeficiency. The results have not been consistent, possibly because of the differences in the conditions of experimentation or because of the differences in the physical condition of the patients. General beneficial effects of treatment with thymosin, however, have occurred. These effects are illustrated as follows. Thymosin may improve the clinical and immunologic condition of patients with immunodeficiency syndromes. For example, a patient with thymic hypoplasia was selected to receive thymosin in vivo, since her T-cell rosettes had increased from 15% to 48% after incubation of her lymphocytes with thymosin in vitro (40). She improved clinically during the immunotherapy, the percentage of her T-cell rosettes gradually increased to normal, and positive skin tests of delayed-type hypersensitivity developed. Thus, thymosin may be useful clinically for partial reconstitution of cellular immunity. An increase percentage of T-cell rosettes after incubation with thymosin in vitro may predict which patients will respond to thymosin therapy in vivo. In another case, an infant with hy-
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poparathyroidism, severe T-cell deficiency, and hypoimmunoglobulinemia with an increased number of B-lymphocytes was treated with thymosin (3). The initial proportion of lymphocytes forming spontaneous rosettes with E-rosettes was 12%, while a response of lymphocytes to PHA was absent. Two months after treatment with thymosin, 68% of lymphocytes formed rosettes with sheep erythrocytes, and the response to PHA was normal. Thymosin was found to improve the immunologic condition in patients with malignancies. In one study involving ten patients with disseminated malignancies (7), administration of thymosin was associated with an increase in Erosette-forming capacity of the patients' lymphocytes, and in the development of new delayed skin test reactions to recall antigens. Thymosin treatment of patients with head and neck squamous carcinoma resuited in improvement of parameters of CMI, such as leukocyte migration inhibition (42). Here, the leukocytes of cancer patients had significantly lower migration inhibition to streptokinase-streptodomase (SK-SD) than did the leukocytes of normal patients. Thymosin increased the degree of migration inhibition to SK-SD in the cancer patients to levels similar to those of normal subjects. These and other studies (19, 20, 34) indicate that thymosin may aid in the restoration of parameters of CMI in patients with cellular immunodeficiencies. In studies on patients with small cell bronchogenic carcinoma, individuals received intensive chemotherapy with TF5 60 mg/m2 (Tro), 20 mg/m2, or 0 mg/m2 (P) twice weekly during the initial 6 wk of therapy. Chemotherapy was then continued for 2 yr (4, 18). The overall growth of the tumors did not differ significantly among the three treatment groups. Survival of the 'F6o group, however, was significantly greater than that of the P group. The results from experiments involving treatment of patients with thymosin support the hypothesis that patients with relatively low levels of immunity benefit most from administration of thymosin. The mechanism
© 1984 Elsevier Science Publishing Co., Inc.
of action, therefore, is reconstitution of immune defects rather than augmentation of relatively normal levels of cell-mediated resistance.
Conclusions That the immunologic activity of TF5 is not definitive may be related to the fact that it is not a single entity but a mixture of peptides. In addition, these peptides may not only reinforce each other's immunologic activities, but may actually interfere with or counteract each other's activities. An indication of this possible situation was shown when C57BI/10SNJ and C3H/HeJ mice were treated with TF5 and then challenged with C. albicans (31). Normally, mice of the C57BI/ 10SNJ inbred strain are resistant to challenge with C. albicans, whereas, mice of the C3H/HeJ strain are highly susceptible. When these strains are treated with TF5 at the time of challenge with C. albicans, however, the C57Bl/10SNJ mice become more susceptible, while the C3H/HeJ mice become more resistant. Whether the strains are responding to different peptides in the TF5 or whether they are responding differently to the same peptide is not known. It seems possible, therefore, that one single naturally occurring peptide may be present, which is able to enhance CMI and, thus, also enhance resistance to infection. Such a peptide may be available, since a new naturally occurring peptide recently has been isolated; it has high potency in inhibiting the growth of C. albicans in susceptible mouse inbred strains and does not enhance the growth in resistant strains (Salvin and Horecker, unpublished data). Another thymic peptide may be present that is able to do the reverse (i.e., suppress CMI responses and, thereby, enhance susceptibility to infection). This thought was presented many years ago by Szent-Gyorgi, when he stated that his thymus "extracts contained two active substances, the one promoting, the other inhibiting malignant growth, and the result depended on their balance. It is a common experience that two unknown variables make results messy" (37).
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The author expresses thanl~s and appreciation to Dr. Ruth Neta for her critical review of the manuscript. References 1. Asanuma, Y., A. L. Goldsteln, and A. White (1970). Reduction in the incidence of wasting disease in neonatally thymectomized CBAJW mice by the injection of thymosin. Endocrinology 86:600-610. 2. Biston|, F. el al. (1982). Increase of mouse resistance to Candida albicans infection by thymosin alpha 1. Infect. Immun. 36:609-614. 3. Bonagura, V. R. and J. Pitt (1981). Hypoparathyroidism with T-cell deficiency and hypoimmunoglobulinemia: Response to thymosin therapy. Clin. lmmunol. Immunopathol. 18:375386. 4. Cohen, M. H. et al. (1979). Thymosin fraction 5 and intensive combination therapy. J.A.M.A. 241:18131815. 5. Collins, F. M. and L. K. Auclair (1979). Effect of thymosiu treatment on antituberculous immunity in immunosuppressed mice. J. Reticuloendothel. Soc. 26:143-153. 6. Collins, F. M. and N. E. Morrison (1979). Restoration of T-cell responsiveness by thymosin: Expression of anti-tuberculous inmaunity in mouse lungs. Infect. Immun. 23:330-335. 7. Costanzi, J. J. et al. (1977). The effect of thymosin on patients with disseminated malignancies. A phase I study. Cancer 40:14-19. 8. Frasca, D., M. Garavini, and G. Doria. (1982). Recovery of T-cell functions in aged mice injected with synthetic thymosin-alpha I. Cell. Immunol. 72:384-391. 9. Goldstein, A. L. et al. (1970). Influence of thymosin on cell-mediated and humoral immune responses in normal and in immunologically deficient mice. J. Immunol. 104:359-366. 10. Goldstein, A. L. et al. (1976). Use of thymosin in the treatment of primary immunodeficiency diseases and cancer. Med. Clin. 60:591-606. 11. Hardy, M. A. et al. (1971). Reversal by thymosin of increased susceptibility of immunosuppressed mice to Moloney sarcoma virus. Trans. Proc. 3:926928. 12. Huang, K.-Y. et al. (1981). Thymosin treatment modulates production of interferon. J. Interferon Res. 1:411-420. 13. Law, L. W. et al. (1964). In The Thymus, Wistar Institute Symposium
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Monograph No. 2. Wistar Institute Press, Philadelphia, pp. 105-117. Law, L. W., A. L. Goldstein, and A. White (1968). Influence of thymosin on immunological competence of lymphoid cells from thymectomized mice. Nature 219:1391-1392. Law, L. W. et al. (1964). Humoral thymic factor in mice: Further evidence. Science 143:1049-1051. Levey, R. H., N. Tralnin, and L. W. Law (1963). Evidence for function of thymic tissue in diffusion chambers implanted in neonatally thymectomized mice. Preliminary report. J. Natl. Cancer Inst. 31"199-206. Levey, R. H. et al. (1963). Lymphocytic chorimeningitis infection in neonatally thymectomized mice bearing diffusion chambers containing thymus. Science 142:483-485. Lipson, D. S. et al. (1979). Thymosin immunotherapy in patients with small cell carcinoma of the lung. Correlation of in vitro studies with clinical course. Cancer 43:863-870. Marshall, G. D., J r . et al. (1980). Thymosin: Basic properties and clinical application in the treatment of immunodefieiency diseases and cancer. Recent results. Cancer Res. 7 5 : I 0 0 105. Mawhlnney, H., V. F. Gleadhill, and S. MeCrea (1979). In vitro and in vivo responses to thymosin in severe combined immunodeficiency. Clin. Immunol. Immunopathol. 14:196203. Morrison, N. E. and F. M. Collins (1976). Restoration of T-cell responsiveness by thymosin: Development of antituberculous resistance in BCG-infected animals. Infect. Immun. 13:554-563. Neta, R. and S. B. Saivin (1983). Resistance and susceptibility to infection in inbred murine strains. II. Variations in the effect of treatment with thymosin fraction 5 on the release of lymphokines, in vivo. Cell. Immunol. 75:173-180. Neta, R., S. B. Salvin, and M. Sabaawi (1981). Mechanisms in the/n vivo release of lymphokines. I. Comparative kinetics in the release of six lymphokines in inbred strains of mice. Cell. Immunol. 64:203-219. Osoba, D. (1965). Immune reactivity in mice thymectomized soon after birth: Normal response after pregnancy. Science 147:298-299. Osoba, D. and J. F. A. P. Miller (1963). Evidence for a humoral thymus factor responsible for the maturation of immunological faculty. Nature (Aug. 17):653-654.
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26. Osoba, D. and J. F. A. P. Miller (1964). The lymphoid tissues and immune responses of neonatally thymectomized mice bearing thymus tissue in Millipore diffusion chambers. J. Exp. Med. 119:177-194. 27. Petro, T. M., G. Chien and R. R. Watson (1982). Alteration of cell-mediated immunity to Listeria monocTtogenes in protein-malnourished mice treated with thymosin fraction V. Infect. Immun. 37:601-608. 28. Raffel, L. et al. (1981). Experimental Candida albicans, Staphylococcus aureus, and Streptococcus faecalis pyelonephritis in diabetic rats. Infect. Immun. 34:773-779. 29. Roberts, S. and A. White (1949). Biochemical characterization of lymphoid tissue proteins. J. Biol. Chem. 178:151-162. 30. Roth, J. A. et al. (1980). Thymic abnormalities and growth hormone deficiency in dogs. Am. J. Vet. Res. 41:1256-1262. 31. Salvin, S. B. and R. Neta. (1983). Resistance and susceptibility to infection in inbred murine strains. I. Variations in the response to thymic hormones in mice infected with Candida albicans. Cell. Immunol. 75:160-172. 32. Salvin, S. B. and E. P. Tanner. (1983). Resistance and susceptibility to infection in inbred murine strains. III. Effect of thymosin on cellular immune responses in alloxan diabetic mice. Clin. Exp. Immunol. 54:133-139. 33. Savtsova, A. D. et al. (1982). Role of immune cytolysis and its thymosin stimulation in experimental influenza. Zh. Milrobiol. Epidemiol. Immunobiol. 3:55-58. 34. Schafer, L. A. et ai. (1976). In vitro and in vivo studies with thymosin in cancer patients. Ann. N.Y. Acad. Sci. 277:609-620. 35. Schulof, T. S. et al. (1981). b7 K'. W. Sells and W. V. Miller (eds.), The Lymphocyte. Alan R. Liss, New York, pp. 191-215. 36. Splitter, G. A., T. C. McGuire, and W. C. Davis (1976). Ability of thymus, thymus epithelium and thymus extracts to cause allograft rejection in nude and AT × BM mice. Fed. Proc. 35:593 (abst.). 37. Szent-Gyorgi, A. (1960). On the chemistry of the thymus gland, pp. 123-125, In Introduction to a Submolecular Biology. Academic Press, New York. 38. Talal, N. et al. (1975). Effect of thymosin on thymocyte proliferation and autoimmunity in NZB mice. Ann. N.Y. Acad. Sci. 249:438-450.
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39. Trainin, N. et al. (1966). A thymic factor preventing w/~sting and influencing lymphopoiesis in mice. Israel J. Med. Sci. 2:549-559. 40. Wara, D. W. et ai. (1975). Thymosin activity in patients with cellular immunodeficiency. N. Engl. J. Med 292:70-74.
41. Willey, D. E. and R. N. Ushijima. (1981). The effect of thymosin on mufine lymphocyte responses and corticosteroid levels during acute reovirus type 3 infection of neonatal mice. Clin. Immunol. Immunopathol. 19:35-43. 42. Wolf, G. T., S. E. Kerney, and P. A. Chretien (1980). Improvement
Thymosin oq-Induced Modulation of Immunoregulatory T-Lymphocyte Activities Gino Doria, M.D., Daniela Frasca, M.D., and Luciano Adorini M.D. Laboratory of Pathology, ENEA, C.R.E. Casaccia Rome, Italy Thymic factors are known to play a crucial role in the differentiation and maturation of T lymphocytes. Several peptides with thymic hormone-like activity have been isolated from thymus extracts and, among the different preparations, thymosins present in fraction 5 have been thoroughly studied and characterized. Fraction 5, a partially purified extract from bovine thymus (8), contains 4 0 - 5 0 peptide components identified by isoelectrofocusing, and exhibits a wide range of biologic activities in extensive animal and clinical studies. Among these peptides, thymosin otI has been the first biologically active polypeptide to be isolated, sequenced (4), and synthesized (13). Thymosin ct1 consists of 28 amino acid residues wtih a molecular weight of 3108, and has been shown to be 101000 times more active than thymosin fraction 5 (TF5) in promoting phenotypic expression of T-cell markers and helper T-cell functions.
Summary of Biologic Studies and Clinical Trials with Thymosin cq Thymosin ct I has been shown to be an effective modulator of helper T-cell activity in several systems using both normal or immunodeficient experimental models (11,15). Mice injected with ett show enhanced T-cell responses to mitogens, and increased. lymphokine and prostaglandin produc-
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tion. Thymosin ett also prolongs the survival of tumor-bearing mice or of immunosuppressed mice infected with Candida spp., BCG, or Cryptococcus spp. In mice, studies in vitro indicate that thymosin eq induces the development of Thy l-positive as well as Lyt 1,2,3-positive cells from T-cell precursors, it modulates terminal deoxynucleotidyl transferase (TdT) activity in bone marrow cells, thymocytes, and splenocytes, and it increases the percentage of cortisone-resistant thymocytes. Moreover, it enhances the production of both interferon-alpha and -gamma (IFN-et, -~) and of macrophage inhibitory factor, T-cell dependent IgG, IgA, and IgM secondary antibody responses, helper T-cell activities, and E-rosette formation. In humans, as well, incubation in vitro of peripheral blood lymphocytes (PBL) with thymosin al increases the percentage of E-rosette-forming cells and autologous rosette-forming cells in patients with primary immunodeficiency, cancer, viral infection, or autoimmune pathology. Clinical trials with etI alone or in conjunction with conventional chemotherapy have been performed on cancer patients. Preliminary results indicate that administration in vivo of et 1 enhances T-cell number and functions in advanced cancer patients with low pretreatment T-cell values. Beneficial effects of this thymic hormone at the clinical level, however, are much more evident when eq is administrated in conjunction with chemotherapy, rather than alone.
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of impaired leukocyte migration inhibition in patients with head and neck squamous carcinoma. Am. J. Surg. 140:531-537. 43. Zisblatt, M. et al. (1970). Acceleration by thymosin in the development of resistance to murine sarcoma virusinduced tumor in mice. Proc. Natl. Acad. Sci. 66:1170-1174.
Recently, it was demonstrated that markedly elevated serum levels of ff-I represent one of the early signals for identifying patients with acquired immunodeficiency syndrome (AIDS) and asymptomatic individuals at high risk (6). These patients also show markedly depressed OKT4:OKT8 ratios. In spite of the elevated levels of et 1, T4 cells isolated from PBL of AIDS patients exhibit enhanced helper T-cell functions after incubation in vitro with etI.
Role of Thymosin cq in Aging of the Immune S y s t e m Thymosin ~l serum levels, as detected by radioimmunoassay, decrease with advancing age in humans and in mice (5) as a result of thymus involution (7). Since thymic factors are known to promote the differentiation of immature T lymphoeytes into functional cells, the decline in serum concentration of these factors is likely to play an important role in age-associated immunodeficiencies. Thus, during senescence, thymus-dependent immunity is more strongly compromised than thymus-independent immunity. The decline of T-cell mediated antibody response associated with advancing age (2) can be partially or fully restored by injection of thymic hormone preparations (1, 3, 14). In recent studies carded out in our laboratory, immunodeficient aged mice were injected with synthetic thymosin ctI in order to assess whether or not this peptide is able to induce restoration of helper T-cell activity in vivo. (C57B1/10 × DBA/2)F 1 mice, age 3 24 mo, were left uninjected or received one intraperitoneal injection of 1, 10, or 100 I.tg synthetic ct I (gift of Dr. A. L. Goldstein). After 3 days,
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