- Email: [email protected]
Thymostimulin (TP-1)
20. Hersh, E. M. (1983). Elevated serum thymosin a-1 levels associated with evidence of immune dysregulation in male homosexuals with a history of infectious diseases or Kaposi's syndrome. N. Engl. J. Med. 308:45-46. 21. ltirokawa, K., G. E. MeLure, and A. L. Goldstein. (1982). Age related changes in localization of thymosin in the human thymus. Thymus 4:19-29. 22. Incefy, G. S., et al. (1977). Thymic activity in severe combined immunodeficiency diseases. Proc. Natl. Acad. Sci. USA 74:1250- 1253. 23. lwata, T., et al. (1979). Circulating thymic hormone levels in zinc deficiency. Cell. Immunol. 47:100-105. 24. Iwata, T., et ai. (1981). Circulating thymic hormone activity in patients with primary and secondary immunodeficiency diseases. Amer. J. Med. 71:385-394. 25. Kirpatrick, H., et al. (1978). Plasma thymic hormone activity in patients with chronic mucocutaneous candi-
Thymostimulin (TP-I) Fabrizio Spinozzi, M.D. Pietro Rambotti, M.D. Fausto Grignani, M.D. Stephen Davis, M.D. Instituto di Clinica Medica Universit(~ di Perugia Perugia, Itah
The thymus gland and its numerous soluble factors, widely referred to as "thymic hormones," play a fundamental role in the maturation of T lymphocytes (1,7). Over the past few years, a great number of these hormones, obtained both from humans and animals, have been isolated, characterized, and adopted in an attempt to correct the various T lymphocyte defects found in primary and secondary immunodeficiency syndromes associated with infectious, neoplastic, or immune disease. Thymostimulin (TP-I, Serono, Italy) is extracted from calf thymus with ammonium acetate (4). It is then precipitated with ammonium sulfate and subjected to ultrafiltration and Sephadex G-50 chromatography. On poly-
36
0197-1859/86/$0.00
+ 02.20
diasis. Clin. Exp. lmmunol. 34:311317. 26. Lewis, V., et al. (1977). Circulating thymic hormone activity in congenital immunodeficiency. Lancet ii:475-478, 27. Lewis, V., et al. (1978). Age, thymic involution and circulating thymic hormone activity. J. Clin. Endocrinol. Metabolism 47:145- 150. 28. McClure, J. E., et al. (1982). Immuno-chemical studies on thymosin, radioimmunoassay of thymosin alpha 1. J. lmmunol. 128:368-375. 29. Pleau, J. M., et al. (1978). Dosage radio-immunologique du facteur thymique s6rique (FTS). In Radioimmunoassay and related procedures in Medicine, Vol. 2. Vienna. 30. Savino, W., et al. (1982). Characterization and localization of serum thymic factor in young mouse thymus mediated by monoclonal antibodies. J. Exp. Med. 186:628-633. 31. Savino, W., M. Dardenne, and J. F.
acrylamide gel electrophoresis, this extract shows two characteristic (pH 8.6) bands with an Rf of 0.22 and 0.42. The molecular weight of the proteins is less than 10,000 daltons. The first studies carried out in order to assess its in vitro activity showed an increase in the nonimmune rosette formation with sheep red blood cells (E-RFC) by cord blood lymphocytes, an increase in the blastogenic response to mitogens (PHA and LPS) (Table 1) by mouse spleen cells, and a stimulation of immature mouse lymphoid cells in an in vivo induced graft versus host reaction (GvHR). Conversely, a thymic secretory activity susceptible to inhibition via negative feedback after prolonged TP-I administration was found to exist in mice. This would support its role as a hormonal substance excreted by thymic epithelial cells. Immunologic studies on laboratory animals have also revealed a marked rise in the antibody-forming activity; an increase in the mitogenic response after immunization with E-RFC and treatment with TP-1, and an improvement in the survival rate, both with and without chemotherapy, after neoplastic cell inoculation.
(c;~1986 Elsevier Science Publishing Co.. lnc~
Bach. (1983a). Thymic hormone containing cells. II. Evolution of cells containing the serum thymic factor (FTS or thymulin) in normal and autoimmunc mice, as revealed by anti-FTS monoclonal antibodies. Relationship with la bearing cells. Clin. Exp. lmmunol. 52:1--6. 32. Savino, W., and M. Dardenne. (1984). Thymic hormone-containing cells. VI. Immunohistologic evidence for the simultaneous presence of thymulin, thymoipoietin and thymosin al in normal and pathological human thymuses. Eur. J. Immunol. 11:987-991. 33. Stutman, O., E. J. Yunis, and R. A. Good. (1968). Carcinogen-induced tumours of neonatally thymectomized mice with a functional thymoma. J. Natl. Cancer Inst. 41:1431 - 1435. 34. Twomey, J. J., et al. (1977). Bioassay determinations of thymopoietin and thymic hormone levels in human plasma. Proc. Natl. Acad. Sci. USA 6:2541-2545.
I m m u n o l o g i c S t u d i e s in H u m a n s The enhancement of E-rosette receptors in vitro by TP-I has been studied in circulating lymphocytes obtained from both healthy adults and umbilical cord blood. Incubation in a human AB serum-supplemented medium gave rise to increases in the T lymphocyte E-rosetting capacity and a significant rise of active rosettes in both groups. Our group confirmed and extended these data on cord lymphocytes after incubation in vitro with TP-I. The results seem to indicate an immunostimulating effect of TP- 1, which acts to induce T lymphocyte maturation; TP-1 increases the percentage of Erosettes and cells bearing a helper/inducer phenotype (T4) in human cord blood. Other authors have observed an increase blast transformation induced both with mitogens and the mixed lymphocyte reaction (MLR) in normal circulating lymphocytes using Raji lymphoblastoid cell lines. These data suggest that the existing mature cell subpopulations, not immature cells, are subject to an action that makes them responsive to the hormone, because the effect is produced only with optimal doses of TP- 1, rather than with the ex-
Clinical Immunology Newsletter 7:3, 1986
pected linear dose-response. Further experiments have shown how TP-1 increases the production of immune interferon in normal lymphocytes after stimulation with ConA and Raji cells, but not with phytohemagglutinin (PHA). The stimulating effect becomes evident when interferon production, in basal conditions, is low and does not appear to depend on the blastogenic stimulation no matter how it is experimentally induced. Indeed, it is seen to take place prior to cell proliferation. Thus it is likely that TP-1 may exert selective activity on specific interferonproducing cell subsets.
Clinical Studies Primary Immunodeficiencies The first therapeutic applications of TP-I in vivo in primary immunodeficient patients involved the administration of the drug at a dose of 1 mg/kg/ day for i week, then twice weekly for 3 months. Eight of the 13 immunodeficient patients showed marked clinical improvement, which, in five patients, corresponded to better immunologic parameters in vitro (E-RFC percentage, positive HTLA, and mitogenic response) and in vivo (appearance of skin response to intradermic antigens). Treatment was successful in patients with thymic hypoplasia, whereas no immunologic or clinical improvement was observed in patients with thymic aplasia, combined immunodeficiency syndrome (SCID), or Wiskott-Aldrich syndrome. Clinical responses, along with a total absence of side effects, have been described in studies in children affected with SCID and thymic hypoplasia associated with herpes simplex viral infections. It is interesting to note that very low serum thymic hormone levels were found in those patients who responded to TP-1 immunostimulation therapy. Moreover, a parallel improvement of their clinical picture, such as weight gain, normalization of their general condition, and remission of infections was observed. A hypothesis that the drug may affect prethymic cell maturation or that residual thymic stromal cells may be stimulated, thus acquiring immunocompetence, again was forwarded.
Clinical ImmunologyNewsletter7:3. 1986
This means that overall cellular immunity can be normalized, because there is limited evidence of a more specific effect of TP- 1 on any T lymphocyte subpopulations e.g., T 4, T 8.
Secondary Immunodeficiencies Infectious Diseases Studies have been carried out on the use of TP-1 in infections caused by herpes viruses (variceila zoster, HSV, and CMV), which are commonly observed in patients who are immunodepressed due either to primary abnormalities of the immune system or to immunosuppressive therapy. Of particular interest are the investigations of Businco (3) performed on a group of 13 children with primary and secondary immunodeficiencies. The primary diseases covered a wide spectrum of disorders: ataxia-telangiectasia (2 cases), pure T cell defect (2 cases), common variable hypo-~/-globulinemia (2 cases), thalassemia major (1 case), and Hodgkin's disease (1 case). The most interesting clinical manifestations were chickenpox in the Hodgkin's disease patient approximately 2 years after undergoing thymectomy and splenectomy; chronic diarrhea and retarded statural growth in one patient with hypo-~/globulinemia and evidence of clinical atopy, namely asthma caused by Dermatophagoides peteronissimus ; and disseminated eczema secondary to milk intolerance in the remaining five. The viral diseases with which the 13 children presented were hemorrhagic chickenpox in four, herpes encephalitis in one, CMV interstitial pneumonia in four, and recurrent herpes stomatitis and keratitis in the remaining four. Thymostimulin 1 was given in a 75 mg/m2/day dose for 7 days with a follow-up dose twice weekly for a period of 3 months. These investigators observed an improvement in the hemorrhagic skin lesions in the patients with chickenpox after their first injection. Positive results were also achieved in the encephalitic patient and in those with recurrent herpes stomatitis and keratitis. However, there was no appreciable improvement in the cases with CMV pneumonia. One patient developed an anaphylactic reaction to
© 1986ElsevierSciencePublishingCo., Inc.
TP-1, and therapy was therefore interrupted. Immunologic profiles showed a significant increase in the percentage of E-RFC and of the absolute number of lymphocytes, as evaluated with heteroantisera. Delayed hypersensitivity skin tests became positive in five of the 13 patients, but the in vitro response to mitogens remained unchanged. The therapeutic effect again may be best explained by the differentiation and maturation of the prethymic precursors and/ or the release of lymphokines or immune interferon caused by activated macrophages, lymphocytes, or both. Analogous results on immunostimulation have been reported in patients with chronic uremia and in patients with various types of tumors associated with herpes virus superinfections. An increase of E-RFC and the development of skin test positivity to DNCB were obtained in 3/10uremic patients, whereas in ~J2 of patients with cancer (all on TP- 1, 0.5-1.5 mg/kg/day for 3 - 8 days), a regression of fever and herpes skin lesions were observed along with a rise in the T lymphocyte count in five cases and an increase in total granulocytes in six. Similar data were obtained in a randomized study on patients affected with recurrent herpes labialis, including 21 patients with immunologic defects (IgA selective defects, common variable hypo-'y-globulinemia, hyper-IgE syndrome, and drug-induced immunosuppression). The first randomized group received 1 mg/kg/day of TP-1 for 1 week, then twice weekly for 7 weeks, after which they were given 0.5 mg/kg twice weekly over a period of 6 months. The second group was given a placebo. Immunologic assays were carried out prior to therapy, then after 15 days, 3 months, and 6 months. The results showed a dramatic reduction in the occurrence and severity of the sto-
Table 1 List of Abbreviations PHA LPS ConA HSV CMV DNCB
Phytohemagglutinin Lipopolysaccharide Concanavalin A Herpes simplex virus Cytomegalovirus Dinitrochlorobenzine
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37
matitis episodes, as well as an increase in leukocytes, T cells, and the in vitro mitogenic response to viral antigens. There have been descriptions of favorable clinical and immunologic changes during the course of acute viral hepatitis, type B and non-A/non-B. Specifically, a more rapid and constant fall in GPT and a marked rise in total T lymphocytes, which involved both the helper/inducer and suppressor/cytotoxic subpopulations, as evaluated by OKT monoclonal antibodies. These patients were.receiving TP-1, 1 mg/kg/day for l0 days, the twice a week for a period of 4 weeks.
Neoplastic Disorders Gastrointestinal Tumors Shoham undertook a study on patients with disseminated gastrointestinal tumors, randomly applying chemotherapy with 5-fluorouracil, vincristine, and CCNU, with and without TP-1 administered in two different doses (6). In the three groups of patients (chemotherapy alone, chemotherapy plus 0.1 mg/kg/day TP-I, and chemotherapy plus 0.5 mg/kg/day TP-I) evaluation of immunocompetence was based on the capacity at form E-rosettes or active Erosettes and the skin response to DNCB. The results show a rise in the E-rosetting capacity of T lymphocytes in the two groups receiving TP-1 compared to the chemotherapy alone group; in addition, most patients receiving TP-1 reacted positively to the delayed hypersensitivity test for DNCB. No survival advantage was seen in the TP- 1 groups. The same workers also examined the in vitro effects of TP- 1 in a mixed allogeneic lymphocyte culture using continuous cell lines (Raji or lg R3 cells) as stimulators, and on the lymphocyte response to mitogens, in patients with solid tumors and normal subjects. They demonstrated that TP-1 is capable of enhancing the blastogenic response in MLR in both cancer-bearing and normal control patients, whereas the response to PHA and ConA remained unchanged.
Malignant Melanoma It has been reported that normal T lymphocyte function may positively in-
38
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fluence the clinical course of malignant melanomas in patients whose tumors have been surgically excised. The onset of immunodepression has occasionally been observed to precede clinically demonstrable lesions. Research on the in vitro effects of TP-1 on lymphocytes in 100 patients with melanoma showed a significant rise in the E-rosette forming capacity with E-RFC, and in patients with metastatic and non-metastatic disease. Sixteen immunodepressed patients, ten with and six without metastases, were given TP-1 alone (25 rag/week for a period of 5 months, 10 patients). The remaining six patients were given TP-1 with DTIC (200 mg/m2/day a month for 6 months). The following results were obtained: • Normal values in the absolute number and percentage of E-RFC were reached in 8A0 with metastases. • The absolute number and percentage of active E-rosettes in five of the patients with metastatic disease with low values, were raised to normal. • A complete normalization of the number of E-rosettes at 4°C and active E-rosettes was achieved in all six patients without clinically overt metastases. Three of the six stage IV patients died within 164 days of therapy. Two patients showed a regression of the number of skin metastases. All patients in stages I and lib receiving TP-1 alone were free of metastases after 6 months of therapy. Therefore, it was postulated that there was a positive effect exerted by TP-1 on cell immunity and that this fact significantly contributed to the favorable clinical evolution observed in certain patients on combined chemoimmunotherapy. Further studies by the same group on 52 patients with malignant melanomas have provided supporting evidence for their previous results. Thirty-two patients without clinically overt metastases were given either TP-I (8 patients); DTIC (8 patients), or were surgically treated (16 patients). All had low E-RFC levels that rose to normal in patients receiving TP-I. Thirty-four months later, six of these showed no clinical evidence of metastatic lesions,
Ct) 1986 Elsevier Science Publishing Co., Inc.
whereas seven of the eight patients on DTIC and 13 of the 16 who underwent surgery alone developed metastases.
Hodgkin's Disease From its early stages, Hodgkin's disease (HD) is characterized by an impairment of cell-mediated immunity, expressed by a hyporesponsivenss to skin tests with recall antigens, and a reduced capacity of peripheral blood lymphocytes to form spontaneous rosettes with sheep red blood and to incur a blastic transformation after in vitro stimulus with mitogens (2). Clinically, such abnormalities are responsible for the impaired rejection capacity of skin allotransplants, and perhaps the susceptibility to viral, mycotic, and mycobacterial infections. In addition, the imbalance in the immune system is characterized by the presence of an elevated number of circulating immune complexes, anti-lymphocyte antibodies, and the common finding of a raised in vitro immunoglobulin synthesis. In theory, the use of thymic hormones in this disorder would be justified by the successful results already obtained in other cellular immunodeficiency diseases, such as solid tumors, and the documentation, albeit scarce, of thymic functional defects in HD. In 1973, Rzepecky noted that a thymus transplant produced a beneficial effect in one patient with HD, and later Aiuti et al. observed a rise in the percentage of E-rosettes in two of three HD patients whose lymphocytes had been incubated with pig thymus extract. Our group evaluated the in vitro effect of TP-I on lymphocyte functional capacity to form E-rosettes and to respond to mitogens. A group of 19 patients were studied at the time of diagnosis. After 1-hour incubation with TP-1 concentration, 50 ~g/ml, the percentage of E-RFC increased markedly in patients with very low T lymphocyte levels. A significant rise in the blastogenic response was observed in 13 patients with very low PHA stimulation index. However, only three reached the normal range. On the other hand, no significant change was induced in the cases with normal E-RFC levels and stimulation indices, nor in the control group.
Clinical Immunology Newsletter 7:3, 1986
An in vivo study was carried out in 19 patients on diagnosis, using a TP-I dose scheme with 1 mg/kg/day for 1 week followed by two doses a week for 2 more weeks. The results obtained confirmed the immunomodulating effect of TP-1 on the E-rosetting capacity of peripheral blood lymphocytes and their capacity to respond in vitro to mitogens. Additionally, after TP- 1 therapy, serum LIF activity appeared, and the skin tests became positive to recall antigens in initially anergic patients. Serologically, a rise in the lysozyme levels, associated with a fall in complement-fixing and noncomplementfixing circulating immune complexes 'was noted. The overall results obtained in the in vitro and in vivo studies in HD patients seem to indicate a TP-1 immunomodulating effect that may act on T lymphocyte maturation, augmenting
its functional activity, and, simultaneously, on the monocyte-macrophage system, improving its clearance for immune complexes.
Stress and the Immune Response
48 or 77 hours of noise and sleep deprivation. Decrements in immune function were also associated with bereavement after the death of a spouse (12, 14). In each of these studies, researchers found that the response of lymphocytes to mitogen stimulation was lower after the stressful event. Similarly, lymphocytes from patients with acute necrotizing ulcerative gingivitis (trenchmouth), an infection associated with normally nonpathogenic indigenous oral bacteria, showed a poorer blastogenic response than healthy controis (4). The patients were also more depressed and anxious. The primary focus of work from our laboratory has been the investigation of the effects of relatively commonplace stressful events on immune function. We reasoned that if stress-related immunosuppression were indeed a risk factor of any importance in the incidence of infectious disease (and perhaps malignant disease as well), then there should be measurable immunologic changes associated with more commonplace stressful events, as well as very novel and intense events. In our first study (8), we obtained blood samples two times from 75 first-
Ronald Glaser, Ph.D. Janice K. Kiecolt-Glaser, Ph.D. Department of Medical Microbiology and Immunology, and Comprehensive Cancer Center and Department of Psychiatry The Ohio State University Columbus, Ohio
Life Events, Stress, a n d Immune Function There is strong evidence that stressful events may have adverse effects on health. The immune system is thought to be the critical mediating link between stress and an increased incidence of infectious (and perhaps malignant) disease (1). Although relatively few critical human studies on stress and immunity have been performed, there are now data suggesting that such a connection exists. Earlier studies assessed possible stress-related changes in immune function using small subject samples and rare events, such as changes in immune function in astronauts after spaceflights, or immunologic changes after
Clinical immunologyNewsletter7:3, 1986
References 1. Aiuti, F., R. D. Amelio, and G. Giunchi. (1980). Thymic hormones and their Clinical use in immunodeficiencies, pp. 375-389. In F. Aiuti and H. Wigzell (eds.), Thymus, thymic hormones and T lymphocytes. Academic Press, London. 2. Bernengo, M. G., C. Barbera, A. De Matteis, M. Meregalli, and G. Zina. (1980). In vitro effects of a calf thymus extract in one hundred melanoma patients and preliminary results in vivo, pp. 313-322. In F. Aiuti and H. Wigzell (eds.), Thymus, thymic hormones and T lymphocytes. Academic Press, London. 3. Buscinco, L., P. Rossi, I. Qninti, and R. Perlini. (1980). Therapy with TP- I of viral diseases in immunodeficient patients, pp. 295-305. In F. Aiuti and
© 1986ElsevierSciencePublishingCo., Inc.
4.
5.
6.
7.
H. Wigzell (eds.), Thymus, thymic hormones and T lymphocytes. Academic Press, London. Falchetti R., et aL (1977). Pharmacological and biological properties of a calf thymus extract. Drugs Exp. Clin. Res. 3:39. Martelli, M. F., A. Velardi, P. Rambotti, C. Cernetti, A. M. Bracaglia, E. Ballatori, and S. Davis. (1982). The in vitro effect of a calf thymus extract on immunologic parameters of patients with untreated Hodgkin's disease. Cancer 49:245250. Shoham, J., E. Theodor, H. J. Benner, B. Goldman, A. Lusky, and S. Chaitchick. (1980). Enhancement of the immune system of chemotherapytreated cancer patients by simultaneous treatment with TP-1. Cancer Immunol. Immunother. 9:173-180. Shoham, J., E. Ben-David, and U. Sandbank. (1982). Feedback inhibition of thymic secretory activity. Immunology 45:31-38.
year medical students. Baseline blood samples were drawn 1 month before final examinations, and acute stress blood samples were obtained on the first day of final examinations. Natural killer (NK) cell activity decreased significantly during examinations when compared to NK activity obtained at baseline. Lonelier students (those who scored above the median on a loneliness scale) also had lower levels of NK activity.
Stress a n d H e r p e s v i r u s L a t e n c y Stress-related changes in herpesvirus infections have been the subject of considerable anecdotal speculation. There are data from animal and human studies implicating stress as a risk factor in the development of primary herpesvirus infections, the duration of the acute episode or primary lesion, and the frequency with which subsequent lesions (Herpes simplex virus [HSV] types 1 and 2) reappear after the primary lesion. There is strong evidence that the competency of the cellular immune response is important in limiting primary herpesvirus infections and in the control of latent herpesviruses (6). Dysfunc-
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39
Thymostimulin (TP-I) Fabrizio Spinozzi, M.D. Pietro Rambotti, M.D. Fausto Grignani, M.D. Stephen Davis, M.D. Instituto di Clinica Medica Universit(~ di Perugia Perugia, Itah
The thymus gland and its numerous soluble factors, widely referred to as "thymic hormones," play a fundamental role in the maturation of T lymphocytes (1,7). Over the past few years, a great number of these hormones, obtained both from humans and animals, have been isolated, characterized, and adopted in an attempt to correct the various T lymphocyte defects found in primary and secondary immunodeficiency syndromes associated with infectious, neoplastic, or immune disease. Thymostimulin (TP-I, Serono, Italy) is extracted from calf thymus with ammonium acetate (4). It is then precipitated with ammonium sulfate and subjected to ultrafiltration and Sephadex G-50 chromatography. On poly-
36
0197-1859/86/$0.00
+ 02.20
diasis. Clin. Exp. lmmunol. 34:311317. 26. Lewis, V., et al. (1977). Circulating thymic hormone activity in congenital immunodeficiency. Lancet ii:475-478, 27. Lewis, V., et al. (1978). Age, thymic involution and circulating thymic hormone activity. J. Clin. Endocrinol. Metabolism 47:145- 150. 28. McClure, J. E., et al. (1982). Immuno-chemical studies on thymosin, radioimmunoassay of thymosin alpha 1. J. lmmunol. 128:368-375. 29. Pleau, J. M., et al. (1978). Dosage radio-immunologique du facteur thymique s6rique (FTS). In Radioimmunoassay and related procedures in Medicine, Vol. 2. Vienna. 30. Savino, W., et al. (1982). Characterization and localization of serum thymic factor in young mouse thymus mediated by monoclonal antibodies. J. Exp. Med. 186:628-633. 31. Savino, W., M. Dardenne, and J. F.
acrylamide gel electrophoresis, this extract shows two characteristic (pH 8.6) bands with an Rf of 0.22 and 0.42. The molecular weight of the proteins is less than 10,000 daltons. The first studies carried out in order to assess its in vitro activity showed an increase in the nonimmune rosette formation with sheep red blood cells (E-RFC) by cord blood lymphocytes, an increase in the blastogenic response to mitogens (PHA and LPS) (Table 1) by mouse spleen cells, and a stimulation of immature mouse lymphoid cells in an in vivo induced graft versus host reaction (GvHR). Conversely, a thymic secretory activity susceptible to inhibition via negative feedback after prolonged TP-I administration was found to exist in mice. This would support its role as a hormonal substance excreted by thymic epithelial cells. Immunologic studies on laboratory animals have also revealed a marked rise in the antibody-forming activity; an increase in the mitogenic response after immunization with E-RFC and treatment with TP-1, and an improvement in the survival rate, both with and without chemotherapy, after neoplastic cell inoculation.
(c;~1986 Elsevier Science Publishing Co.. lnc~
Bach. (1983a). Thymic hormone containing cells. II. Evolution of cells containing the serum thymic factor (FTS or thymulin) in normal and autoimmunc mice, as revealed by anti-FTS monoclonal antibodies. Relationship with la bearing cells. Clin. Exp. lmmunol. 52:1--6. 32. Savino, W., and M. Dardenne. (1984). Thymic hormone-containing cells. VI. Immunohistologic evidence for the simultaneous presence of thymulin, thymoipoietin and thymosin al in normal and pathological human thymuses. Eur. J. Immunol. 11:987-991. 33. Stutman, O., E. J. Yunis, and R. A. Good. (1968). Carcinogen-induced tumours of neonatally thymectomized mice with a functional thymoma. J. Natl. Cancer Inst. 41:1431 - 1435. 34. Twomey, J. J., et al. (1977). Bioassay determinations of thymopoietin and thymic hormone levels in human plasma. Proc. Natl. Acad. Sci. USA 6:2541-2545.
I m m u n o l o g i c S t u d i e s in H u m a n s The enhancement of E-rosette receptors in vitro by TP-I has been studied in circulating lymphocytes obtained from both healthy adults and umbilical cord blood. Incubation in a human AB serum-supplemented medium gave rise to increases in the T lymphocyte E-rosetting capacity and a significant rise of active rosettes in both groups. Our group confirmed and extended these data on cord lymphocytes after incubation in vitro with TP-I. The results seem to indicate an immunostimulating effect of TP- 1, which acts to induce T lymphocyte maturation; TP-1 increases the percentage of Erosettes and cells bearing a helper/inducer phenotype (T4) in human cord blood. Other authors have observed an increase blast transformation induced both with mitogens and the mixed lymphocyte reaction (MLR) in normal circulating lymphocytes using Raji lymphoblastoid cell lines. These data suggest that the existing mature cell subpopulations, not immature cells, are subject to an action that makes them responsive to the hormone, because the effect is produced only with optimal doses of TP- 1, rather than with the ex-
Clinical Immunology Newsletter 7:3, 1986
pected linear dose-response. Further experiments have shown how TP-1 increases the production of immune interferon in normal lymphocytes after stimulation with ConA and Raji cells, but not with phytohemagglutinin (PHA). The stimulating effect becomes evident when interferon production, in basal conditions, is low and does not appear to depend on the blastogenic stimulation no matter how it is experimentally induced. Indeed, it is seen to take place prior to cell proliferation. Thus it is likely that TP-1 may exert selective activity on specific interferonproducing cell subsets.
Clinical Studies Primary Immunodeficiencies The first therapeutic applications of TP-I in vivo in primary immunodeficient patients involved the administration of the drug at a dose of 1 mg/kg/ day for i week, then twice weekly for 3 months. Eight of the 13 immunodeficient patients showed marked clinical improvement, which, in five patients, corresponded to better immunologic parameters in vitro (E-RFC percentage, positive HTLA, and mitogenic response) and in vivo (appearance of skin response to intradermic antigens). Treatment was successful in patients with thymic hypoplasia, whereas no immunologic or clinical improvement was observed in patients with thymic aplasia, combined immunodeficiency syndrome (SCID), or Wiskott-Aldrich syndrome. Clinical responses, along with a total absence of side effects, have been described in studies in children affected with SCID and thymic hypoplasia associated with herpes simplex viral infections. It is interesting to note that very low serum thymic hormone levels were found in those patients who responded to TP-1 immunostimulation therapy. Moreover, a parallel improvement of their clinical picture, such as weight gain, normalization of their general condition, and remission of infections was observed. A hypothesis that the drug may affect prethymic cell maturation or that residual thymic stromal cells may be stimulated, thus acquiring immunocompetence, again was forwarded.
Clinical ImmunologyNewsletter7:3. 1986
This means that overall cellular immunity can be normalized, because there is limited evidence of a more specific effect of TP- 1 on any T lymphocyte subpopulations e.g., T 4, T 8.
Secondary Immunodeficiencies Infectious Diseases Studies have been carried out on the use of TP-1 in infections caused by herpes viruses (variceila zoster, HSV, and CMV), which are commonly observed in patients who are immunodepressed due either to primary abnormalities of the immune system or to immunosuppressive therapy. Of particular interest are the investigations of Businco (3) performed on a group of 13 children with primary and secondary immunodeficiencies. The primary diseases covered a wide spectrum of disorders: ataxia-telangiectasia (2 cases), pure T cell defect (2 cases), common variable hypo-~/-globulinemia (2 cases), thalassemia major (1 case), and Hodgkin's disease (1 case). The most interesting clinical manifestations were chickenpox in the Hodgkin's disease patient approximately 2 years after undergoing thymectomy and splenectomy; chronic diarrhea and retarded statural growth in one patient with hypo-~/globulinemia and evidence of clinical atopy, namely asthma caused by Dermatophagoides peteronissimus ; and disseminated eczema secondary to milk intolerance in the remaining five. The viral diseases with which the 13 children presented were hemorrhagic chickenpox in four, herpes encephalitis in one, CMV interstitial pneumonia in four, and recurrent herpes stomatitis and keratitis in the remaining four. Thymostimulin 1 was given in a 75 mg/m2/day dose for 7 days with a follow-up dose twice weekly for a period of 3 months. These investigators observed an improvement in the hemorrhagic skin lesions in the patients with chickenpox after their first injection. Positive results were also achieved in the encephalitic patient and in those with recurrent herpes stomatitis and keratitis. However, there was no appreciable improvement in the cases with CMV pneumonia. One patient developed an anaphylactic reaction to
© 1986ElsevierSciencePublishingCo., Inc.
TP-1, and therapy was therefore interrupted. Immunologic profiles showed a significant increase in the percentage of E-RFC and of the absolute number of lymphocytes, as evaluated with heteroantisera. Delayed hypersensitivity skin tests became positive in five of the 13 patients, but the in vitro response to mitogens remained unchanged. The therapeutic effect again may be best explained by the differentiation and maturation of the prethymic precursors and/ or the release of lymphokines or immune interferon caused by activated macrophages, lymphocytes, or both. Analogous results on immunostimulation have been reported in patients with chronic uremia and in patients with various types of tumors associated with herpes virus superinfections. An increase of E-RFC and the development of skin test positivity to DNCB were obtained in 3/10uremic patients, whereas in ~J2 of patients with cancer (all on TP- 1, 0.5-1.5 mg/kg/day for 3 - 8 days), a regression of fever and herpes skin lesions were observed along with a rise in the T lymphocyte count in five cases and an increase in total granulocytes in six. Similar data were obtained in a randomized study on patients affected with recurrent herpes labialis, including 21 patients with immunologic defects (IgA selective defects, common variable hypo-'y-globulinemia, hyper-IgE syndrome, and drug-induced immunosuppression). The first randomized group received 1 mg/kg/day of TP-1 for 1 week, then twice weekly for 7 weeks, after which they were given 0.5 mg/kg twice weekly over a period of 6 months. The second group was given a placebo. Immunologic assays were carried out prior to therapy, then after 15 days, 3 months, and 6 months. The results showed a dramatic reduction in the occurrence and severity of the sto-
Table 1 List of Abbreviations PHA LPS ConA HSV CMV DNCB
Phytohemagglutinin Lipopolysaccharide Concanavalin A Herpes simplex virus Cytomegalovirus Dinitrochlorobenzine
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matitis episodes, as well as an increase in leukocytes, T cells, and the in vitro mitogenic response to viral antigens. There have been descriptions of favorable clinical and immunologic changes during the course of acute viral hepatitis, type B and non-A/non-B. Specifically, a more rapid and constant fall in GPT and a marked rise in total T lymphocytes, which involved both the helper/inducer and suppressor/cytotoxic subpopulations, as evaluated by OKT monoclonal antibodies. These patients were.receiving TP-1, 1 mg/kg/day for l0 days, the twice a week for a period of 4 weeks.
Neoplastic Disorders Gastrointestinal Tumors Shoham undertook a study on patients with disseminated gastrointestinal tumors, randomly applying chemotherapy with 5-fluorouracil, vincristine, and CCNU, with and without TP-1 administered in two different doses (6). In the three groups of patients (chemotherapy alone, chemotherapy plus 0.1 mg/kg/day TP-I, and chemotherapy plus 0.5 mg/kg/day TP-I) evaluation of immunocompetence was based on the capacity at form E-rosettes or active Erosettes and the skin response to DNCB. The results show a rise in the E-rosetting capacity of T lymphocytes in the two groups receiving TP-1 compared to the chemotherapy alone group; in addition, most patients receiving TP-1 reacted positively to the delayed hypersensitivity test for DNCB. No survival advantage was seen in the TP- 1 groups. The same workers also examined the in vitro effects of TP- 1 in a mixed allogeneic lymphocyte culture using continuous cell lines (Raji or lg R3 cells) as stimulators, and on the lymphocyte response to mitogens, in patients with solid tumors and normal subjects. They demonstrated that TP-1 is capable of enhancing the blastogenic response in MLR in both cancer-bearing and normal control patients, whereas the response to PHA and ConA remained unchanged.
Malignant Melanoma It has been reported that normal T lymphocyte function may positively in-
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fluence the clinical course of malignant melanomas in patients whose tumors have been surgically excised. The onset of immunodepression has occasionally been observed to precede clinically demonstrable lesions. Research on the in vitro effects of TP-1 on lymphocytes in 100 patients with melanoma showed a significant rise in the E-rosette forming capacity with E-RFC, and in patients with metastatic and non-metastatic disease. Sixteen immunodepressed patients, ten with and six without metastases, were given TP-1 alone (25 rag/week for a period of 5 months, 10 patients). The remaining six patients were given TP-1 with DTIC (200 mg/m2/day a month for 6 months). The following results were obtained: • Normal values in the absolute number and percentage of E-RFC were reached in 8A0 with metastases. • The absolute number and percentage of active E-rosettes in five of the patients with metastatic disease with low values, were raised to normal. • A complete normalization of the number of E-rosettes at 4°C and active E-rosettes was achieved in all six patients without clinically overt metastases. Three of the six stage IV patients died within 164 days of therapy. Two patients showed a regression of the number of skin metastases. All patients in stages I and lib receiving TP-1 alone were free of metastases after 6 months of therapy. Therefore, it was postulated that there was a positive effect exerted by TP-1 on cell immunity and that this fact significantly contributed to the favorable clinical evolution observed in certain patients on combined chemoimmunotherapy. Further studies by the same group on 52 patients with malignant melanomas have provided supporting evidence for their previous results. Thirty-two patients without clinically overt metastases were given either TP-I (8 patients); DTIC (8 patients), or were surgically treated (16 patients). All had low E-RFC levels that rose to normal in patients receiving TP-I. Thirty-four months later, six of these showed no clinical evidence of metastatic lesions,
Ct) 1986 Elsevier Science Publishing Co., Inc.
whereas seven of the eight patients on DTIC and 13 of the 16 who underwent surgery alone developed metastases.
Hodgkin's Disease From its early stages, Hodgkin's disease (HD) is characterized by an impairment of cell-mediated immunity, expressed by a hyporesponsivenss to skin tests with recall antigens, and a reduced capacity of peripheral blood lymphocytes to form spontaneous rosettes with sheep red blood and to incur a blastic transformation after in vitro stimulus with mitogens (2). Clinically, such abnormalities are responsible for the impaired rejection capacity of skin allotransplants, and perhaps the susceptibility to viral, mycotic, and mycobacterial infections. In addition, the imbalance in the immune system is characterized by the presence of an elevated number of circulating immune complexes, anti-lymphocyte antibodies, and the common finding of a raised in vitro immunoglobulin synthesis. In theory, the use of thymic hormones in this disorder would be justified by the successful results already obtained in other cellular immunodeficiency diseases, such as solid tumors, and the documentation, albeit scarce, of thymic functional defects in HD. In 1973, Rzepecky noted that a thymus transplant produced a beneficial effect in one patient with HD, and later Aiuti et al. observed a rise in the percentage of E-rosettes in two of three HD patients whose lymphocytes had been incubated with pig thymus extract. Our group evaluated the in vitro effect of TP-I on lymphocyte functional capacity to form E-rosettes and to respond to mitogens. A group of 19 patients were studied at the time of diagnosis. After 1-hour incubation with TP-1 concentration, 50 ~g/ml, the percentage of E-RFC increased markedly in patients with very low T lymphocyte levels. A significant rise in the blastogenic response was observed in 13 patients with very low PHA stimulation index. However, only three reached the normal range. On the other hand, no significant change was induced in the cases with normal E-RFC levels and stimulation indices, nor in the control group.
Clinical Immunology Newsletter 7:3, 1986
An in vivo study was carried out in 19 patients on diagnosis, using a TP-I dose scheme with 1 mg/kg/day for 1 week followed by two doses a week for 2 more weeks. The results obtained confirmed the immunomodulating effect of TP-1 on the E-rosetting capacity of peripheral blood lymphocytes and their capacity to respond in vitro to mitogens. Additionally, after TP- 1 therapy, serum LIF activity appeared, and the skin tests became positive to recall antigens in initially anergic patients. Serologically, a rise in the lysozyme levels, associated with a fall in complement-fixing and noncomplementfixing circulating immune complexes 'was noted. The overall results obtained in the in vitro and in vivo studies in HD patients seem to indicate a TP-1 immunomodulating effect that may act on T lymphocyte maturation, augmenting
its functional activity, and, simultaneously, on the monocyte-macrophage system, improving its clearance for immune complexes.
Stress and the Immune Response
48 or 77 hours of noise and sleep deprivation. Decrements in immune function were also associated with bereavement after the death of a spouse (12, 14). In each of these studies, researchers found that the response of lymphocytes to mitogen stimulation was lower after the stressful event. Similarly, lymphocytes from patients with acute necrotizing ulcerative gingivitis (trenchmouth), an infection associated with normally nonpathogenic indigenous oral bacteria, showed a poorer blastogenic response than healthy controis (4). The patients were also more depressed and anxious. The primary focus of work from our laboratory has been the investigation of the effects of relatively commonplace stressful events on immune function. We reasoned that if stress-related immunosuppression were indeed a risk factor of any importance in the incidence of infectious disease (and perhaps malignant disease as well), then there should be measurable immunologic changes associated with more commonplace stressful events, as well as very novel and intense events. In our first study (8), we obtained blood samples two times from 75 first-
Ronald Glaser, Ph.D. Janice K. Kiecolt-Glaser, Ph.D. Department of Medical Microbiology and Immunology, and Comprehensive Cancer Center and Department of Psychiatry The Ohio State University Columbus, Ohio
Life Events, Stress, a n d Immune Function There is strong evidence that stressful events may have adverse effects on health. The immune system is thought to be the critical mediating link between stress and an increased incidence of infectious (and perhaps malignant) disease (1). Although relatively few critical human studies on stress and immunity have been performed, there are now data suggesting that such a connection exists. Earlier studies assessed possible stress-related changes in immune function using small subject samples and rare events, such as changes in immune function in astronauts after spaceflights, or immunologic changes after
Clinical immunologyNewsletter7:3, 1986
References 1. Aiuti, F., R. D. Amelio, and G. Giunchi. (1980). Thymic hormones and their Clinical use in immunodeficiencies, pp. 375-389. In F. Aiuti and H. Wigzell (eds.), Thymus, thymic hormones and T lymphocytes. Academic Press, London. 2. Bernengo, M. G., C. Barbera, A. De Matteis, M. Meregalli, and G. Zina. (1980). In vitro effects of a calf thymus extract in one hundred melanoma patients and preliminary results in vivo, pp. 313-322. In F. Aiuti and H. Wigzell (eds.), Thymus, thymic hormones and T lymphocytes. Academic Press, London. 3. Buscinco, L., P. Rossi, I. Qninti, and R. Perlini. (1980). Therapy with TP- I of viral diseases in immunodeficient patients, pp. 295-305. In F. Aiuti and
© 1986ElsevierSciencePublishingCo., Inc.
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5.
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H. Wigzell (eds.), Thymus, thymic hormones and T lymphocytes. Academic Press, London. Falchetti R., et aL (1977). Pharmacological and biological properties of a calf thymus extract. Drugs Exp. Clin. Res. 3:39. Martelli, M. F., A. Velardi, P. Rambotti, C. Cernetti, A. M. Bracaglia, E. Ballatori, and S. Davis. (1982). The in vitro effect of a calf thymus extract on immunologic parameters of patients with untreated Hodgkin's disease. Cancer 49:245250. Shoham, J., E. Theodor, H. J. Benner, B. Goldman, A. Lusky, and S. Chaitchick. (1980). Enhancement of the immune system of chemotherapytreated cancer patients by simultaneous treatment with TP-1. Cancer Immunol. Immunother. 9:173-180. Shoham, J., E. Ben-David, and U. Sandbank. (1982). Feedback inhibition of thymic secretory activity. Immunology 45:31-38.
year medical students. Baseline blood samples were drawn 1 month before final examinations, and acute stress blood samples were obtained on the first day of final examinations. Natural killer (NK) cell activity decreased significantly during examinations when compared to NK activity obtained at baseline. Lonelier students (those who scored above the median on a loneliness scale) also had lower levels of NK activity.
Stress a n d H e r p e s v i r u s L a t e n c y Stress-related changes in herpesvirus infections have been the subject of considerable anecdotal speculation. There are data from animal and human studies implicating stress as a risk factor in the development of primary herpesvirus infections, the duration of the acute episode or primary lesion, and the frequency with which subsequent lesions (Herpes simplex virus [HSV] types 1 and 2) reappear after the primary lesion. There is strong evidence that the competency of the cellular immune response is important in limiting primary herpesvirus infections and in the control of latent herpesviruses (6). Dysfunc-
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