Immunopotentiating and immunotherapeutic effects of thymic hormones and factors with special emphasis on thymic humoral factor THF-gamma2.

The essential role played by the thymus in the development of the immune response was well documented in many publications. These findings prompted a long series of studies devised to define the factors produced and secreted by thymus cells, which are involved in the development and nature of immunological responsiveness. First experiments done with crude thymus extracts were followed by isolation of purified products and finally by chemical characterization and synthesis of immunologically active thymus-derived peptides. In this article we review the various thymic hormones and factors described, that is, thymosin fractions 5, the thymosins, prothymosin alpha, thymulin (FTS-Zn), thymopoietin, thymostimulin (TP-1), Thymic humoral factor (THF), and THF-gamma2. Studies demonstrating the activity of the various thymic factors in increasing the immunocompetence potential in both in vitro and in vivo conditions are discussed. The immunostimulatory potential of thymic factors was also investigated in experimental models where beneficial therapeutic effects were sought in a situation of immunological malfunction. The last part of the review is dedicated to clinical trials with thymic factors that revealed improvement in the immunocompetence potential in cases of immunodeficiencies, viral infections, and cancer and its correlation with therapeutic effectiveness. It seems that more research is required in order to better define conditions for the use of thymic factors in immunotherapy.

Potentiation of myeloid colony-formation in bone marrow of intact and neonatally thymectomized mice by the thymic hormone THF-gamma 2.

The effect of the thymic hormone THF-gamma 2 on committed stem cells of bone marrow (BM) origin was determined using the myeloid progenitor cell clonal assay. Preincubation of normal BM cells with THF-gamma 2 for 1 hour or 18 hours caused a 2- to 6-fold increase in the number of myeloid colonies in the presence of suboptimal concentrations of colony-stimulating factor (CSF). The optimal dose of THF-gamma 2 causing this enhancement was in the range of 25 to 100 ng/mL. THF-gamma 2 was not able to replace CSF as an inducer in these experiments. THF-gamma 2 neither induced IL-6 activity upon 24-hour incubation with bone marrow cells nor enhanced LPS-induced IL-6 secretion by bone marrow cells in vitro. Neonatal thymectomy (NTx) of Balb/c mice caused a decrease in myeloid progenitors, which was repaired by serial injections of THF-gamma 2. The repair of the stem cell compartment in the bone marrow correlated with an increased percentage of Thy1+ cells in the spleen of THF-gamma 2-treated NTx mice. These findings indicate that THF-gamma 2 is able to regulate committed stem cell functions in the bone marrow of immune-deprived NTx and of normal mice.

Thymic humoral factor-gamma 2, an immunoregulatory peptide, enhances human hematopoietic progenitor cell growth.

Thymus humoral factor-gamma 2 (THF gamma 2), an octapeptide important for T-lymphocyte regulation, was assessed for its effect on the in vitro growth of human hematopoietic progenitor cells. This was achieved using a recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF)-stimulated myeloid cell colony formation (granulocyte-macrophage colony-forming cells, GM-CFC) assay as well as a recombinant erythropoietin (rEpo)-stimulated erythroid burst formation (erythroid burst-forming units, BFU-E) assay. Cells were obtained from bone marrow (BM) and peripheral blood (PB) of normal healthy donors and from patients with suppressed bone marrows. The latter group included aplastic anemia, leukemia, and lymphoma patients and patients with solid tumors who responded to intensive chemotherapy with significant pancytopenia. THF gamma 2 significantly enhanced normal BM and PB GM-CFC and PB BFU-E by 2- to 2.5-fold. This effect was totally dependent on the presence of the respective growth factors, that is, rGM-CSF or rEpo, and was specifically reversed by an anti-THF gamma 2 antiserum. Furthermore, although THF gamma 2-induced enhancement of GM-CFC colony formation was not affected by lymphocyte or monocyte depletion, the augmenting effect of the peptide on BFU-E was completely abrogated in the absence of lymphocytes. THF gamma 2-induced augmented growth of progenitor cells derived from severely suppressed marrows was minimal. However, cells from moderately neutropenic patients with leukemia in remission or with lymphoma under chemotherapy responded to the peptide similarly to cells from normal donors. These results suggest a stimulatory role for THF gamma 2 on human myeloid and erythroid hematopoietic progenitor cells. They also suggest the lymphocyte dependence of BFU-E enhancement and lymphocyte independence of GM-CFC stimulation by THF gamma 2. In the former case the thymus-derived peptide may act through the induction of certain erythroid-enhancing lymphokines.

Thymic humoral factor, THF-gamma 2, enhances immunotherapy of murine cytomegalovirus (MCMV) infection by both CD4+ and CD8+ immune T cells.

Infection of mice with murine cytomegalovirus (CMV) presents a model for the study of the role of the immune system in the pathogenesis of human CMV. The contribution of the different spleen cell subsets in conferring curative immunocytotherapy to fatally MCMV-infected immunosuppressed mice was assessed using adoptive immunotherapy. It was found that the efficacy of passively transferred immune spleen cells is dose dependent and that the therapeutic effect can be enhanced considerably by treating donor mice with thymic humoral factor (THF-gamma 2). Polymerase chain reaction (PCR) of the donor spleen population was negative, indicating that no MCMV-DNA was transferred with the immune cells. Analysis of the donor mice after THF-gamma 2 treatment showed increased levels of CMV-neutralizing antibodies, while enhancement of natural killer (NK) activity was transient and lasted only during the early phase of the infection. FACS analysis demonstrated that treatment with THF-gamma 2 restored the size of both cell subsets CD4+ and CD8+ that were decreased following MCMV infection. It is shown that both CD4+ and CD8+ T-cell subsets participate in controlling the development of the fatal disease in MCMV-infected immunosuppressed recipients. It is suggested that the enhancement of the immunocompetence of both populations of spleen cells from treated donors is mediated in part by the restoration of Interleukin-2 (IL-2) production by THF-gamma 2.

A mouse ear reaction for assessment of human lymphocyte immunocompetence.

A mouse ear reaction for testing cell mediated immunity of human lymphocytes using the local graft-versus-host reaction assay is described. Five million human mononuclears were locally injected into the ears of immune suppressed NZW mice. The reaction mounted was quantitated by determining the 125I-Iodo-Deoxyuridine (125I-UdR) incorporation in both ears. The ratio of 125I-UdR incorporation, of the injected to that of the non-injected ear (GVHR index), 7 days after lymphocyte injection, served as an accurate measure for the extent of the reaction. Only normal human mononuclears and purified, separated normal human T lymphocytes mounted a local graft-versus-host reaction. Whereas normal human B lymphocytes, chronic lymphatic leukemia B lymphocytes, mononuclears from patients with transitional cell carcinoma of the bladder, irradiated normal human mononuclears, mouse syngeneic mononuclears, or human erythrocytes gave no positive reaction. These experiments demonstrate that this assay can be used to quantitate an in-vivo specific graft-versus-host reaction.

Treatment of murine cytomegalovirus salivary-gland infection by combined therapy with ganciclovir and thymic humoral factor gamma 2.

An optimal therapeutic regimen against primary CMV salivary-gland infection has not yet been developed. We used a murine CMV (MCMV) model system to assess the ability of combined thymic humoral factor THF-gamma 2 immunotherapy and ganciclovir (GCV) antiviral chemotherapy to eliminate detectable viral DNA from salivary glands of infected animals. Mice in different experimental groups were inoculated intraperitoneally with MCMV, treated, and then sacrificed either 2 weeks or 3 months later. To amplify and detect MCMV DNA in infected salivary-gland tissue, we developed a sensitive polymerase chain reaction (PCR) using a glycoprotein B gene primer pair that amplifies a 356 bp segment. During the acute phase of the infection, the detection of high titers of infectious virus in the salivary glands correlated with a strong PCR amplification signal. Although active virions could not be recovered from untreated animals 3 months after viral inoculation, the PCR assay detected a latent MCMV genome. Treatment with either GCV alone or THF-gamma 2 alone had little or no effect on the presence of MCMV DNA. By contrast, combined treatment with THF-gamma 2 and GCV significantly reduced the amount of salivary-gland MCMV DNA to below the limit of PCR detection. The results presented here, and experimental data from previous MCMV research in our laboratories, imply that elimination of the virus from the salivary glands could be due in part to THF-gamma 2 restoration of the various MCMV-suppressed, cell mediated immune-responses. Combining THF-gamma 2 immunotherapy and GCV antiviral chemotherapy may be an important step toward an effective therapeutic regimen that has the potential to prevent the establishment of viral latency ensuing from primary MCMV salivary-gland infection.

Immune derangements in asymptomatic male homosexuals in Israel: a pre-AIDS condition?

We have described several immune derangements found in a clinically asymptomatic group of 117 male homosexuals (MHS) in Israel. These consisted of a marked decrease in TH and TS cells, decreased NK and allogeneic response, and increased levels of acid labile alpha-interferon and circulating immune complexes (CIC). Most of these alterations were found in approximately 40% of the subjects, with no simple correlation between them. Since Israel is a low incidence area for AIDS and related syndromes, it is suggested that this situation reflects a common situation among asymptomatic MHS in general, although the reasons for these impairments are not clear. This situation may therefore explain susceptibility of the male homosexual for developing AIDS, given the appropriate circumstances, environment, and genetic background.

The activities of enzymes of purine metabolism in murine thymus dependent lymphocytes.

Measurement of the activities of purine metabolizing enzymes in murine T cell subpopulations showed that these activities differed markedly among T cells of different levels of functional maturity. The activities of adenosine deaminase and deoxyadenosine phosphorylation were highest in immature, PNA + thymocytes, while the activities of purine nucleoside phosphorylase, ecto-5'-nucleotidase and deoxyguanosine phosphorylation were highest in mature, splenic T cells. These enzymes' activities can be used as biochemical markers for T cell of different degree of maturation.

Immunocompetence status as related to growth progression of mouse MOPC-315 plasmacytoma.

The immunocompetence status of mice bearing MOPC-315 plasmacytoma was determined at various days after tumor inoculation. Changes in T and B-cell functions appeared gradually. The allogeneic response of spleen cells from BALB/c tumor-bearing mice against C57BL spleen cells was impaired from the 4th day after the tumor inoculation (nonpalpable tumor stage). The primary antibody response in vitro against SRBC was depressed at 18 days, and the mitogenic response of splenic cells to PHA and to LPS was depressed at 25 days after the tumor inoculation. T cells taken from day 18 tumor-bearing mice partially suppressed the MLR response of normal splenocytes. Mice bearing large MOPC-315 tumors responded less to SRBC immunization than normal, noninoculated mice. The relative percentage of Lyt 1, Lyt 2 and L3T4 T-cell subsets decreased starting from the 11th day after tumor inoculation.

Correlation between emotional reaction to loss of an unborn child and lymphocyte response to mitogenic stimulation in women.

PIP: The effect of emotional stress due to the loss of a loved object such as an unborn child on some parameters of cell-mediated immunity was studied in 77 women who had undergone abortions. Based on their psychiatric reaction to this loss, these women were subdivided into 2 groups: 1) Group NA--those not accepting this loss and 2) Group A--those appearing to accept this loss. The response of lymphocytes from peripheral blood taken during the psychiatric interview to the mitogens phytochemagglutinin (PHA) and concanavalin-A (Con-A) in Group NA was compared with the lymphocyte response in blood from Group A. The percent of T-cells was also measured in the 2 groups. It was found that nonacceptance of the loss of a fetus correlated with a significant reduction in the proliferative response to both PHA and Con-A. The percent of T-cells remained constant across both groups. When each of the psychiatric parameters of anxiety, guilt, and despression were correlated separately with the immune response, the most marked effect was found in those women who had the highest score on the depression scale.^ieng

Efficacy of Ledermix paste in eliminating Staphylococcus aureus from infected dentinal tubules in vitro.

The efficacy of Ledermix paste in disinfection of dentinal tubules was studied in a model developed by Haapasalo and Orstavik with some modifications. Ledermix and 3% Tetracycline in a hydrous base were effective in reducing the amount of Staphylococcus aureus in dentinal tubules after 7 days of incubation and also after recontamination. They were not effective after 24 h.

Adaptation of an Aedes aegypti mosquito cell line to growth at 15 degrees C and its response to infection by Sindbis virus.

Aedes aegypti mosquito cells, usually cultured at 28 to 30 degrees C, were adapted to grow at 15 degrees C. They were designated A. aegypti (c) cells, and had an estimated doubling time of 10 days. Sindbis virus (SV) replicated in these cells to peak titres of over 1.0 x 10(9) p.f.u./ml 8 to 10 days after inoculation. These, or about 10-fold lower titres, continued to be produced over a 130 day test period without causing visible cell damage. Continuous virus proliferation and the yield of uniformly large plaque forming progeny viruses are the two most important features which differentiate infection with this virus in A. aegypti (c) cells from that of A. aegypti cells grown at 28 degrees C (Peleg & Stollar, 1974). Absence of homologous interference vis-à-vis cell-virus coexistence suggests that homologous interference is not a prerequisite for maintaining cell-virus coexistence. Preinoculation of A. aegypti (c) cultures with a small plaque forming Sindbis virus (SV-S) leads, under certain conditions, to the establishment of homologous interference.

THF, a thymic hormone, promotes interleukin-2 production in intact and thymus-deprived mice.

T cells stimulated by mitogen or antigen produce a T cell mediator, interleukin-2 (IL-2), which induces clonal expansion of activated target cells. The activity of IL-2 obtained from cell-free supernatants of spleen cells after 24-h incubation with concanavalin A was tested in an IL-2-dependent blast cell proliferation assay. IL-2 production was found to be diminished in neonatally thymectomized (NTx) mice. Treatment of NTx mice with a series of 14 daily injections of thymic hormone, THF (5-10 ng), resulted in reconstitution of IL-2 activity to the level found in intact, untreated mice. The rise in IL-2 activity was observed to be part of a general reconstitution of the immune capacity in the THF-treated NTx mice, manifested by elevated responses to mitogenic stimulation by T lectins and elevated mixed lymphocyte reactivity and cell-mediated cytotoxicity. Elevation of IL-2 activity could also be detected in vitro when spleen cells from intact mice were preincubated with THF for 24 h. THF did not enhance the production of interleukin-1 (IL-1) by bone marrow-derived macrophages, and in experiments in which T cells and macrophages were separately exposed to THF, augmentation of IL-2 activity was found only when the T cell-enriched spleen fraction was pretreated with THF. This was taken as proof that THF acted exclusively on T cells capable of producing IL-2, and that neither macrophages nor IL-1 were involved in the T helper activity induced by THF. Although THF could induce and/or augment IL-2 production by helper T cells, THF lacked IL-1 and IL-2 characteristics, since it could not maintain IL-2-dependent clonal expansion of target blasts nor induce IL-2 production by activated T cells.

Thymic humoral factor gamma 2: purification and amino acid sequence of an immunoregulatory peptide from calf thymus.

Thymic humoral factor gamma 2 (THF-gamma 2), an octapeptide essential for immune regulation, was purified from calf thymus. The purification of THF-gamma 2, monitored in vitro and in vivo in mouse splenocyte proliferation assays, was achieved by gel filtration of low molecular weight thymus extracts followed by ion-exchange chromatography and sequential reversed-phase high-performance liquid chromatography. The process yielded 5 micrograms of THF-gamma 2/1000 kg of thymus tissue. The concentration of THF-gamma 2 required for augmentation of lymphocyte proliferation and interleukin 2 production was 5 ng/mL in vitro and 10 ng/kg per mouse in vivo. THF-gamma 2 has the amino acid sequence Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu. The proposed structure has been confirmed because a peptide was synthesized on the basis of this sequence that showed activity identical with that of the biological molecule. It shows no homology to the amino acid sequence of other thymic hormones nor is it part of any peptide or protein of known sequence. THF-gamma 2 retains essentially all of the biological activity of the thymus extract from which it is derived.

DNA sequence of a class I pseudogene from the Tla region of the murine MHC: recombination at a B2 alu repetitive sequence.

We have determined the DNA sequence of a BALB/c Tla region class I gene from the major histocompatibility complex (MHC) that had been identified previously as encoding a murine antigen by DNA-mediated gene transfer. Analysis of the DNA sequence shows, however, that this gene, the T1c gene from the Tlac genotype, could not encode a TL antigen or any other functional class I molecule due to the presence of numerous stop codons and frameshift mutations in the coding regions. This result suggests that the earlier transformation data may have been incorrect or perhaps that the clone containing the T1c gene contains sequences that induced expression of a serologically reactive Tla gene in the genome of the recipient L cell. The T1c gene is structurally related to the previously sequenced T13c gene that encodes a serologically defined TL antigen. The 3' half of the T1c gene including exons 4, 5, 6, and the 3' untranslated region has about 85% nucleotide similarity (including introns) with the corresponding parts of the T13c gene; however, the 5' half of the T1c gene has little homology with the T13c gene. There is a sharp line of demarcation between the homologous and nonhomologous regions, and this border occurs precisely at a B2 Alu repeat sequence present in the T13c gene. This suggests that a recombination event took place here and that an Alu repeat sequence that is known to have characteristics of transposable elements played some role in a recombination or gene conversion event.