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After stroke or traumatic damages, both necrotic and apoptotic neuronal death cause a loss of functions including memory, sensory perception, and motor skills. From the fact that necrosis has a nature to expand, while apoptosis to cease the cell death cascade in the brain, it is considered that the promising target for the rapid treatment for stroke is the necrosis. In this study, I introduce the discovery of prothymosin alpha (ProTalpha), which inhibits neuronal necrosis, and propose its potentiality of clinical use for stroke. First of all, it should be noted that ProTalpha inhibits the neuronal necrosis induced by serum-free starvation or ischemia-reperfusion stress, which causes a rapid internalization of GLUT1/4, leading a decrease in glucose uptake and cellular ATP levels. Underlying mechanisms are determined to be through an activation of Gi/o, phospholipase C and PKCbetaII. ProTalpha also causes apoptosis later through a similar mechanism. However, we found that ProTalpha-induced apoptosis is completely inhibited by the concomitant treatment with neurotrophins, which are up-regulated by ischemic stress in the brain. Of most importance is the finding that the systemic injection of ProTalpha completely inhibits the brain damages, motor dysfunction and learning memory defect induced by cerebral ischemia-reperfusion stress. As ProTalpha almost entirely prevents the focal ischemia-induced motor dysfunction 4 h after the start of ischemia, this protein seems to have a promising potentiality for clinical use.

Anti-CD3 monoclonal antibody (mAb) activates in vitro peripheral blood mononuclear cells (PBMC) to lyse a variety of tumor cell lines in a non-major histocompatibility-complex(MHC)-restricted manner [subsequently referred to as anti-CD3-activated killer (AAK) cytotoxicity]. Prothymosin alpha (ProTalpha) is a biological response modifier that exerts its effects primarily on mononuclear cells, especially when these cells' effector functions are impaired. In this study, we report that ProTalpha enhances the AAK cytotoxicity in PBMC from healthy donors. This effect was more profound with cancer patients' PBMC, which were deficient in their ability to respond with enhanced AAK cytotoxicity upon in vitro stimulation with anti-CD3. Thus, cancer patients' PBMC, activated with a combination of anti-CD3 and ProTalpha, exhibited increased AAK activity and efficiently lysed both autologous tumor and Daudi targets. The ProTalpha effect on PBMC was demonstrated to involve stimulation of adhesion molecules (CD2, CD18, CD54, CD49f) and CD25 expression, up-regulation of perforin mRNA transcription, increased numbers of perforin-positive (+) cells and elevated production of interleukin-2 (IL-2), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha). Moreover, effector CD8+ and CD56+ cells pretreated with anti-CD3 and ProTalpha contained high cytoplasmic perforin levels and increased expression of IL-1beta- and TNFalpha-specific receptors. The induction of autologous-tumor-reactive CD8+ and CD56+ lymphocytes in anti-CD3-activated PBMC by ProTalpha provides an alternative protocol aimed at the improvement of clinical results in cellular adoptive immunotherapy of cancer.

We have recently reported that administration of ProT alpha to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor alpha [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunothera 38:281]. In this report we demonstrated that ProT alpha, when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumor-specific CD8+ cytotoxic T lymphocytes (CTL). The ProT alpha-induced CD8+ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2Kd allelic product could inhibit the cytotoxic response. Mice receiving only ProT alpha developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving ProT alpha and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The ProT alpha-induced CD8+ CTL activity was regulated by ProT alpha-induced L1210-specific syngeneic CD4+ cells. This was shown in two different ways: first, CD8(+)-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC-restricted proliferative responses of syngeneic CD4+ cells and, second, CD4+ cells from mice that had received both ProT alpha and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8+ cells from mice that had received only L1210 cells. Our data suggest that ProT alpha is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since ProT alpha may prove to be useful in clinical protocols aimed at cancer immunotherapy.

We have recently demonstrated that prothymosin alpha (ProT alpha) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor alpha (TNF alpha) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT alpha in vivo. We demonstrate that i.p. injections of ProT alpha enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT alpha-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8+ cells, NK cells and activated CD3+ cells. The ProT alpha-induced effect persisted for 30 days after the end of the ProT alpha treatment period and returned to normal levels 20 days later. Splenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT alpha in vitro. The ProT alpha-induced NK and LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF alpha and IL-2 were generated in response to ProT alpha in LAK cultures. These findings suggest that ProT alpha may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF alpha in the spleen, peritoneal cavity and probably other lymphoid organs.

The effect of prothymosin alpha (ProT alpha) on the survival of DBA/2 mice inoculated with syngeneic tumour cells was studied. DBA/2 mice inoculated intraperitoneally (i.p.) with 2 x 10(5) syngeneic leukaemic L1210 cells developed ascites within 8-12 days and died 10-14 days later. Treatment with ProT alpha consistently inhibited the development of ascites in 20% of the treated animals and prolonged the survival of 40%-60% of the animals up to 70 days. The most effective treatment schedule of ProT alpha was 300 ng/mouse given i.p. at 2-day intervals for 3 weeks followed by a rest period of 7 days, prior to tumour cell inoculation. Peritoneal exudate (PE) cells collected from mice treated with the optimal dose of ProT alpha produced, in the absence of exogenous stimulus, six- to eightfold higher levels of tumour necrosis factor alpha (TNF alpha) than PE cells from control mice. Furthermore these cells exhibited cytotoxic activity against several tumour cell lines including the syngeneic L1210, the TNF-insensitive P815 mastocytoma, the human MOLT-4 lymphoblastic leukaemia, as well as the murine TNF-sensitive L929 fibroblast cell line. Kinetic studies revealed that both production of TNF alpha and tumoricidal activity peaked 7 days after the last injection of ProT alpha and were maintained at high levels over a period of 1 month. Injections with 150 ng ProT alpha slightly improved the survival of mice whereas higher (500 ng and 1000 ng) doses of ProT alpha and a wide range of thymosin alpha 1 doses remained without any effect. PE cells collected from these mice produced extremely low levels of TNF alpha and exhibited negligible tumoricidal activity. Our data demonstrate that ProT alpha has a protective effect in vivo against the growth of adoptively transferred tumour cells and suggest that this effect is, at least in part, mediated by ProT alpha-activated PE cells. These cells were demonstrated to produce high levels of TNF alpha in vitro and to exhibit activity against both TNF-sensitive and TNF-resistant cell lines.

A combination treatment with thymosin alpha 1 (200 micrograms/kg) for 4 days, followed by a single injection of murine interferon alpha/beta (3 x 10(4) international units/mouse). starting 2 days after cyclophosphamide treatment (200 mg/kg, single injection) demonstrated a dramatic and rapid disappearance of tumor burden in mice bearing Lewis lung carcinoma (3LL) tumor. The effectiveness of this new chemoimmunotherapy protocol was evident even on the long-term survival in a high percentage of animals, and was statistically significant when compared to treatment with the single agents in conjunction with chemotherapy or to chemotherapy itself. The same combination immunotherapy treatment strongly stimulated natural killer activity and cytotoxicity against autologus 3LL tumor cells in 3LL-tumor-bearing mice treated with cyclophosphamide, whereas treatments with each agent singly did not alter or only slightly modified the cytotoxic activity towards Yac-1 or 3LL target cells. Selective depletion with antibodies showed that killer cells stimulated by combination chemoimmunotherapy treatment bear phenotypic characteristics of asialo-GM1-positive cells. A histological study has shown a high number of infiltrating lymphoid cells in the tumors obtained from mice treated with combination chemoimmunotherapy.

Thymosin fraction 5 contains a family of polypeptides with varying biological activities. Current efforts in the thymosin research program are involved in further chemical characterization of thymosin peptides and evaluation of clinical immunotherapeutic protocols. Recent clinical studies with thymosin fraction 5 have shown therapeutic potentials for treatment of patients with primary immunodeficiency diseases and cancer.

We have demonstrated that splenic lymphocytes from normal syngeneic animals can stimulate tumor growth. This effect is T-cell dependent. Preincubation of these lymphocytes with thymosin not only blocks facilitated tumor growth but can induce a significant reduction of local tumor growth and number of pulmonary metastases. Furthermore, thymosin can also block the expression of suppressor activity of lymphocytes either stimulated by Con A or originating from various advanced solid tumor bearing patients. These results therefore suggest that thymosin is able to modulate, directly or indirectly, functional expression of suppressor cells.

Thymosin beta 4 was tested for its ability to induce phenotypic changes in the human T-cell line Molt-4. Cells were cultured with nanogram concentrations of thymosin beta 4 for up to 16 days and were analyzed with a panel of monoclonal antibodies, sheep erythrocyte rosetting, peanut agglutinin binding (PNA) and an antibody to the enzyme, terminal deoxynucleotidyl transferase (TdT). Thymosin beta 4 induced Molt-4 cells to reduce the expression of a T-cell lineage specific antigen, with preferential expression on T blast-cells, detected by WT 1 monoclonal antibody. Thymosin beta 4 also induced an increase in sheep erythrocyte rosettes and PNA binding as well as an increased expression of OKT 11 A and OKT 8 in Molt-4 cells. TdT was found to be unchanged, however. Analysis of thymosin beta 4-treated cells with other monoclonal antibodies (OKT 3, OKT 6, OKT 9) showed no change when compared to controls. These results showed that thymosin beta 4 is capable of inducing phenotypic changes in Molt-4 cells. Such changes may represent a differentiation process of these cells through the early stages of the maturation process of thymus-dependent lymphocytes, albeit not to the stage of mature T cells.

The administration of a thymic factor, thymostimulin (TP-1), to mice resulted in considerable augmentation of natural killer (NK) cell activity as measured in a short-term assay against 51Cr-labeled YAC-1 target cells. Conditions suitable for detection of the thymostimulin-induced boosting of NK included multiple daily exposures to TP-1 (50 micrograms/kg), and peak levels of reactivity were observed at 2-4 days after discontinuation of treatment. A strict age-dependency of the effect was also observed, with optimal augmentation of NK-cell activity when TP-1 was administered to mice at 4-6 weeks of age. The effect was not limited to TP-1 treatment but was also observed on administration of another thymic factor (thymosin alpha 1). The activated cells responsible for the increased natural cell-mediated cytotoxicity appeared to be typical murine NK cells, judging by both functional and antigenic criteria.

We found that both thymosin from calf thymus and its constituent peptide alpha 1 prepared by chemical synthesis restore cell-mediated immunity following its suppression in mice by injection of 5-FU. Conditions suitable for assessing the thymosin activity by means of footpad reaction were established in such immunosuppressed mice. In this new animal model, thymosin alpha 1-peptide showed activity at a low dose of 5-50 micrograms/kg, which was 100-1,000 times less than that required for thymosin F-5 preparations. Further studies utilizing the adoptive transfer technique showed that alpha 1-peptide corrects the 5-FU-induced suppression of mature T cells, transferring the DTH response as well as that of macrophage function responsible for the expression of footpad reaction. Furthermore, regeneration of lymph node and bone marrow cells as well as CFU-c (progenitor cells of macrophages and granulocytes) was enhanced by thymosin alpha 1 in the 5-FU-treated mice. All these results indicate that thymosin alpha 1 accelerates the replenishment and maturation of haematopoietic cells, including not only T cells but also macrophages, when they have been severely damaged by the 5-FU treatment.

The effect of thymosin against tumor progression was examined in mice immunosuppressed by cytostatics or X-ray irradiation. When pretreated with cytostatic agents, such as 5-fluorouracil (5-FU) or BCNU, or by X-ray, and then inoculated with P388 or L1210 leukemias, mice died rapidly within a few days. In these systems, thymosin alpha 1 given concomitantly with the cytostatic agents or after X-irradiation prevented rapid death and extended survival, although the mice eventually died with leukemia like normal mice inoculated with cells of the same tumor. Rapid death in the 5-FU-treated mice was also prevented by adoptive transfer of spleen cells from the donor mice if these had been treated with 5-FU plus thymosin alpha 1, but not if they had received 5-FU alone. However, the restorative activity of the donor spleen cells was abrogated by treatment with anti-asialo GM1, but not by treatment with anti-Thy 1 or anti-mouse Ig serum, suggesting that the effector cells in the spleen are NK cells. In fact, thymosin alpha 1, when given concomitantly with 5-FU or after X-irradiation, maintained the NK activity of spleen, which was damaged by treatment with 5-FU or X-irradiation alone. The present study indicates that thymosin alpha 1 exerts a preventive activity against progression of leukemias at least in part through an effect on NK cells or their progenitor cells.

The influence of two thymosin and two spleen control preparations on the E-rosette formation of peripheral blood lymphocytes and leukemic cells from non-leukemic children with depressed T cell values and leukemic children was investigated. Both thymosin preparations but also one of the control preparations induced a significant increase in the mean percentage of E-rosette forming PBL in the non-leukemic children with depressed T cell values. Thymosin and spleen preparations, however, did not convert the non-erythrocyte binding blasts to erythrocyte binding cells in either common or pre-T-ALL.

A single injection of alpha beta-interferon (alpha beta-IFN) (30000 units/mouse), a major biological modifier of natural killer (NK) cytolytic activity, strongly stimulated NK activity in normal mice, as expected, while the same treatment did not statistically alter the NK response in cyclophosphamide (CY)-suppressed animals. We investigated the possibility of thymosin alpha 1 cooperating with alpha beta-IFN in boosting NK activity in CY-suppressed animals. The results show that treatment with thymosin alpha 1 (200 micrograms/kg) for 4 days, followed by a single injection of alpha beta-IFN 24 h before testing, strongly restored NK activity in CY-suppressed mice. Thymosin alpha 1 was, moreover, able to accelerate the recovery rate of NK activity in bone marrow reconstituted murine chimeras. Taken together the data support the concept that the synergic effect between thymosin alpha 1 and alpha beta-IFN could be the result of effects on differentiation of the NK lineage at different levels.