Overexpression of the anti-apoptotic oncogene, bcl-2, in the thymus does not prevent thymic atrophy induced by estradiol or 2,3,7, 8-tetrachlorodibenzo-p-dioxin.

Dexamethasone (Dex), estradiol (E2), and 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) all affect the immune system, causing immunosuppression and thymic atrophy. It is still uncertain how and where these compounds act to induce thymic atrophy. However, it has been suggested that these compounds may have similar actions and targets, i.e., apoptosis of immature thymocytes for Dex and TCDD and preferential targeting of double-positive cells by Dex and E2. The lckpr-bcl-2 transgenic mouse has been shown to be protected against Dex-induced thymic atrophy. We used this murine model to determine if bcl-2 expression would also protect against E2- and TCDD-induced thymic atrophy. Our results indicate that, although the bcl-2 transgenic (TG+) mice were fully protected from atrophy induced by a single dose of Dex, atrophy was still induced in these mice following treatment with E2 or TCDD. Phenotypic analysis of thymocytes from TG- and TG+ mice also showed distinct consequences of atrophy induced by Dex, E2, and TCDD. Finally, since there are alternative pathways for apoptosis that are bcl-2 independent, both TG- and TG+ thymocytes were examined directly for indications of apoptosis using the TUNEL assay. After TCDD and E2 treatment there were no detectable signs of apoptosis in either TG- or TG+ mice even at early time points and at elevated dose levels. These results indicate that there are distinct mechanisms for the actions of Dex, E2, and TCDD in the thymus and that apoptosis is not a key mechanism of E2- and TCDD-induced thymic atrophy.

Pharmacokinetics/dynamics of 5c8, a monoclonal antibody to CD154 (CD40 ligand) suppression of an immune response in monkeys.

The pharmacokinetics and pharmacodynamics (PK/PD) of chimeric (Ch5c8) and humanized (Hu5c8) 5c8, a monoclonal antibody that binds CD154 (CD40 ligand), thus blocking the interaction between CD40 and CD154, were investigated in cynomolgus monkeys. Single-dose groups (n = 3 animals per dose) received saline, 0.2, 1, 5 or 20 mg/kg i.v. doses of Hu5c8. The repeat-dose groups (n = 4 animals) received 0 or 5 mg/kg i.v. doses of Ch5c8 or Hu5c8 on days 1, 2, 3, 5, 7 and 9. The single-dose PK parameters showed dose proportionality, with a terminal half-life of 300 h, a volume of distribution at steady state of 73 ml/kg and clearance of 0.2 ml.h-1.kg-1. The repeat-dose regimen produced a longer terminal half-life (500 h) and lower clearance (0.13 ml.h-1.kg-1) than in the single-dose groups. The antibody titer to tetanus toxoid (ATT) challenge served as the immunodynamic marker. The primary ATT response consisted of a latent phase of approximately 10 days, during which the immune system was processing antigen but not yet producing antibody, a rise to an antibody maximum titer at approximately 18 days and a decline toward baseline by approximately 40 days in controls. The 5c8 produced a log(dose)-proportional reduction in the area under the curve of ATT. An indirect PK/PD model based on the kinetics of tetanus toxoid exposure and inhibition of ATT production in relation to 5c8 concentrations was developed. A median inhibitory concentration of 0.84 microg/ml and a efficacy of 0.84 reflected marked inhibition of ATT response by 5c8. The model provides quantitation of reduced ATT responses after 5c8 and was applicable to primary and secondary immune responses and to both single-dose and multiple-dose treatments. The monoclonal antibody 5c8 blocks the CD40 and CD154 interaction, producing consistent and substantive reduction in antibody formation after administration of tetanus toxoid, which can be characterized with PK/PD modeling. It is anticipated that 5c8 may have utility in the treatment of antibody-mediated autoimmune disease.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced thymic atrophy and lymphocyte stem cell alterations by mechanisms independent of the estrogen receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has both agonist and antagonist effects on estrogen-mediated activities and estrogen receptor (ER) levels in epithelial tissues following exposure. We previously demonstrated that TCDD alters bone marrow lymphocyte stem cells, including prothymocytes, as measured by functional assays and alterations in the lymphocyte stem cell-specific markers terminal deoxynucleotidyl transferase (TdT) and recombinase activating gene-1 (RAG-1). We have also shown that 17 beta-estradiol valerate (E2V) affects lymphocyte stem cells by reducing TdT and RAG-1 mRNA. It has been suggested that the effect of TCDD on these lymphocyte stem cells may be mediated directly or indirectly through estrogenic action and/or the ER. Studies were designed to evaluate whether endogenous estrogens or the ER mediate TCDD-elicited bone marrow alterations and thymic atrophy. Ovariectomy did not alter the sensitivity of mice to TCDD-induced thymic atrophy or to a reduction in TdT biosynthesis in bone marrow cells compared with either intact or sham-operated mice. The pure estrogen antagonist ICI 164,384 blocked E2V-induced uterine hypertrophy, thymic atrophy and reductions in lymphocyte stem cell markers. However, the antiestrogen failed to protect against TCDD-elicited thymic atrophy or bone marrow alterations in intact animals. The results are consistent with the hypothesis that the effects of TCDD on the thymus and/or bone marrow are mediated by mechanisms independent of estrogens or the ER.

Dexamethasone, beta-estradiol, and 2,3,7,8-tetrachlorodibenzo-p-dioxin elicit thymic atrophy through different cellular targets.

The effects of single doses of dexamethasone (DEX), beta-estradiol-17-valerate (E2), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the kinetics of thymic atrophy and related bone marrow and thymocyte phenotype alterations were examined. The results imply differences in the mechanisms by which these compounds act. Of the three compounds, DEX induced maximal atrophy by 3 days with complete recovery by Day 12. At the point of maximal atrophy, the RAG-1+TdT+CD4+8+3int thymocyte population was proportionately the most depleted. In contrast, TCDD and E2 caused maximal thymic atrophy by Day 12. E2 treatment, like DEX, resulted in a preferential decrease in the RAG-1+TdT+CD4+8+3int population, but unlike DEX, this decrease persisted. TCDD-induced thymic atrophy resulted from a proportional loss of all classes of thymocytes. There was no significant relative reduction of TdT+RAG-1+ cells by TCDD in the thymus. A slow and persistent reduction of TdT and RAG-1 in bone marrow by both TCDD and E2 contrasted with the rapid reduction and quick recovery of these markers in marrow from DEX-treated animals. Additional studies showed that only DEX-induced atrophy was accompanied by the induction of thymocyte apoptosis, as detected by multiple nucleosomal length DNA fragments within the first 24 hr. The different kinetics and proportions of subsets in the atrophied thymuses, as well as the distinct patterns of alterations of RAG and TdT expression, and the presence or the absence of apoptosis provide evidence for different mechanisms of thymic atrophy by these agents. The slow induction and longer persistence of thymic atrophy induced by E2 and TCDD, as well as their effects on bone marrow stem cell markers, suggest that bone marrow thymocyte precursors are major targets for these agents.

The thymus does not mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin-elicited alterations in bone marrow lymphocyte stem cells.

Our previous studies have shown that bone marrow lymphocyte stem cells are affected following perinatal or adult exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These alterations may, in part, be responsible for thymic atrophy that is also observed following TCDD exposure. However, other investigators have suggested that the thymus or thymic-derived lymphocytes can affect bone marrow stem cell development. The purpose of these studies was to determine whether the TCDD-elicited effects that we have observed on lymphocyte stem cells in bone marrow were secondary to the actions of this chemical on the thymus. A single intraperitoneal dose of TCDD (30 micrograms/kg) to sham-operated or neonatally thymectomized female BALB/c mice reduced the levels of mRNA in the bone marrow for the lymphocyte stem cell-specific enzymes terminal deoxynucleotidyl transferase (TdT) and recombinase activating gene (RAG-1). TdT biosynthesis was also reduced by TCDD treatment. Thus, neonatal thymectomy had no effect on the TCDD-elicited reduction of TdT or RAG-1 mRNAs or TdT biosynthesis. Genetically athymic (nu/nu) mice were used to further determine if the actions of TCDD on the thymus or long-lived T-cells altered lymphocyte stem cell development. As observed in BALB/c mice, TCDD treatment decreased the expression of TdT and RAG-1 mRNAs in bone marrow from athymic nu/nu and intact nu/+ littermates. We conclude that TCDD-elicited alterations in bone marrow lymphocyte stem cells are not secondary to any actions, direct or indirect, that TCDD has on the thymus or thymic-derived T-cells.

Alternate immune system targets for TCDD: lymphocyte stem cells and extrathymic T-cell development.

We here summarize evidence that thymic atrophy induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can be mediated, at least in part, by damage to extrathymic T-cell precursors in bone marrow and fetal liver. This atrophy induction does not involve apoptotic mechanisms in thymocytes affected by the bcl-2 proto-oncogene. TCDD mediates atrophy induction through its specific receptor (the AhR) and not through effects on the estrogen receptor. Both TCDD and estradiol induce extrathymic T-cell differentiation in the liver. These extrathymic T-cell populations include cells expressing elevated levels of V beta T-cell receptors that are normally deleted in thymic development.

Evaluation of murine lymphocyte membrane potential, intracellular free calcium, and interleukin-2 receptor expression upon exposure to 1,1-dimethylhydrazine.

The effects of 1,1-dimethylhydrazine (UDMH) on several early events associated with lymphocyte activation were examined. The concentration of intracellular calcium ([Ca2+]i) and membrane potential of murine lymphocytes were found to be altered upon exposure to UDMH; [Ca2+]i was increased in murine thymocytes, while splenocytes exhibited membrane hyperpolarization. In addition, interleukin-2 receptor expression induced by in-vitro concanavalin A stimulation of murine splenocytes at 24 and 48 h in the presence of UDMH was not affected. UDMH may interfere with the ability of these two distinct lymphocyte populations to regulate normal ionic fluctuations, thus contributing to altered immune responsiveness.

The effects of perfluorodecanoic acid (PFDA) on humoral, cellular, and innate immunity in Fischer 344 rats.

The in vivo effects of perfluorodecanoic acid (PFDA) exposure on antibody production, delayed type hypersensitivity (DTH), and natural killer (NK) cell activity were determined. Fischer 344 rats were injected with PFDA (20 mg/kg or 50 mg/kg, 8 days or 30 days prior to sacrifice) and were immunized with keyhole limpet hemocyanin (KLH). Pair-fed and ad libitum-fed control rats were included to evaluate effects of PFDA-induced anorexia. KLH-specific IgG2a production was significantly decreased (p < 0.05) in PFDA-treated rats when compared to ad libitum-fed and pair-fed controls at 8 days but not at 30 days following PFDA treatment. The DTH response of PFDA-treated rats was decreased 8 days and 30 days after PFDA treatment when compared to ad libitum-fed and pair-fed controls, however, the decrease was not statistically significant. NK activity 30 days after PFDA treatment was significantly elevated (p < 0.05) when compared to ad libitum-fed controls, but pair-fed controls had similarly elevated NK activity. NK activity at 8 days after PFDA treatment was not significantly altered. In conclusion, PFDA has been demonstrated to have immunomodulatory effects, some of which may be associated with drug-induced anorexia.

Effect of 1,1-dimethylhydrazine (UDMH) on Corynebacterium parvum-associated immunosuppression in mice.

These studies further investigate the immunoenhancement properties of UDMH by utilizing Corynebacterium parvum-induced immunosuppressed mice as well as evaluating activated macrophage production of reactive oxygen intermediates or their effects. Forty-eight hour Con A-induced lymphoblastogenic responses from splenocytes isolated from C. parvum and UDMH-treated Balb/C mice were significantly increased compared with C. parvum alone, although less than normal control mice (no treatment). In vitro bioassay of IL-2 production in cell culture supernatant isolated from these same treatment groups exhibited a pattern of stimulation similar to that of lymphocyte blastogenesis. In addition, UDMH did not interfere with H2O2-mediated suppression of either Con A- or LPS-induced lymphocyte blastogenesis and actually enhanced suppression of Con A-induced lymphocyte cultures at 25 micrograms/ml. We also report that production of superoxide anion from TPA-activated peritoneal macrophages exposed to various concentrations of UDMH in vitro was not affected. Although in vivo exposure to UDMH partially reversed C. parvum-induced immunosuppression in mice, the exact mechanism by which UDMH acts to reverse this immune suppression is not clear. UDMH does not appear to interfere with either activated peritoneal macrophage production of superoxide anion or H2O2-induced suppression of lymphocyte blastogenesis to elicit immune enhancement.

The in vitro and in vivo effects of 1,1-dimethylhydrazine (UDMH) on murine lymphocyte subsets and Ia antigen expression.

1,1-Dimethylhydrazine or unsymmetrical dimethylhydrazine (UDMH) is a highly volatile and reactive compound used primarily as a liquid rocket propellant. Previous studies found UDMH to possess immunomodulatory activity similar to other hydrazine derivatives. Modulation of T lymphocyte subpopulations and Major Histocompatibility Complex Class II or Ia antigen were evaluated as possible mechanisms for this UDMH-induced immunomodulation. Murine lymphoid cell populations were examined by flow cytometry for changes in their cell surface marker percentages or relative number upon exposure to UDMH either in vitro or in vivo. The results show UDMH caused significant suppression of the T helper cell population derived from the thymus at the 75 mg/kg dose in vivo, but did not affect other lymphocyte subpopulations isolated from mesenteric lymph node, spleen or thymus at this or any other dose. In vivo exposure of mice at all doses of UDMH did not significantly alter expression of Ia antigens on adherent cell populations and expression of the Ia antigen following in vitro UDMH exposure was not affected as well. Results indicate that the immunomodulatory effects of UDMH are not mediated by phenotypic alteration of T lymphocyte subpopulations or Ia antigen.

Subunit vaccine protects Macaca nemestrina (pig-tailed macaque) against simian T-cell lymphotropic virus type I challenge.

Five macaques received two vaccinations consisting of soluble human T-cell lymphotropic virus type I proteins from a cell/serum-free human T-cell lymphotropic virus type I-producing cell line. Five other macaques were vaccine controls. All were challenged with a simian T-cell lymphotropic virus type I-producing cell line. The vaccinated macaques generated a strong serological response to challenge as opposed to the control macaques. Western blot analysis of the sera showed that both groups recognized gag and env proteins, but the vaccinate's sera reacted better to the env proteins. Additionally, the antibody produced by both groups had antibody-dependent, complement-mediated cytotoxic activity toward both human and simian T-cell lymphotropic virus type I-infected target cells. The responses of lymphocytes and neutrophils, as measured by lymphocyte blast transformation and chemiluminescence response, respectively, showed no apparent difference between the vaccinates and controls. Testing for reverse transcriptase in lymphocyte supernatants revealed that the controls contained reverse transcriptase activity, while the vaccinates remained negative. The data presented here demonstrate that the vaccine was successful in protecting Macaca nemestrina from simian T-cell lymphotropic virus type I infection.

Efficacy of an HTLV-1 subunit vaccine in prevention of a STLV-1 infection in pig-tailed macaques.

Three pig-tailed macaques were vaccinated twice, four weeks apart, with a human T-cell lymphotropic virus type 1 (HTLV-1) subunit vaccine. Four weeks post-vaccination, the vaccinated macaques and two control monkeys were challenged with a simian T-cell lymphotropic virus type 1 (STLV-1)-infected cell line. Following the first vaccination, an antibody response developed to HTLV-1 and STLV-1 viral proteins as visualized by Western blot. The antibody recognized both gag and env protein precursors and the titer remained elevated after the second vaccination. After challenge, the antibody titers of the vaccinates increased. The controls also developed HTLV-1 and STLV-1 specific antibody which recognized the gag precursor and to a lesser extent the putative tax protein. Immunized monkeys also produced syncytium inhibiting antibody primarily toward HTLV-1-infected cells and to a lesser extent toward STLV-1-infected cells. Control monkeys produced negligible amounts of syncytium inhibiting antibody. Additionally, both polymorphonuclear (PMN) and mononuclear (MNC) cells were examined. The PMN population was tested for its ability to generate an oxidative burst. Neither vaccination nor challenge had any significant effect on the PMN CL response. The MNCs were tested for their capability to proliferate after stimulation. Again, no significant change occurred. However, when examined for cell-mediated cytotoxicity, the MNCs from immunized monkeys produced greater cytotoxic activity against the HTLV-1-infected cell line than the MNCs from control monkeys. In order to determine if the immunized monkeys were protected by the subunit vaccine against the STLV-1 challenge, reverse transcriptase (RT) activity was measured in the five monkey's MNCs. RT activity was exclusively present in the non-immunized, control monkeys implying that the HTLV-1 subunit vaccine was successful in protecting the pig-tailed macaques from the STLV-1 infection.

Evaluation of a HTLV-1 subunit vaccine in prevention of experimental STLV-I infection in Macaca nemestrina.

Pig-tailed macaques were vaccinated with a human T-cell lymphotropic virus type I (HTLV-I) subunit vaccine. Vaccinates and controls were challenged with simian T-cell lymphotropic virus type I (STLV-I)-infected cells. Vaccination yielded antibody responses to HTLV-I and STLV-I gag and env precursors. Controls developed HTLV-I and STLV-I antibody to gag and tax protein. Immunization produced syncytium inhibiting antibody and cellular cytotoxicity to virus-infected cells. Reverse transcriptase activity was present in control macaques only, implying that the subunit vaccine was protective against STLV-I infection.

Health and disease profile of the island of Bimini based upon physical and multi-phasic screening of 530 residents - abstract

In collaboration with the Ministry of Health of the Bahamas, the Department of Family Medicine, University of Miami School of Medicine, through its residency programme, has been providing primary care to the island of Bimini for one year. In order to extend this care to include comprehensive medical care, a health and ecology fair was planned by the Medical School, the Ministry of Health and a Health Committee representative of the island community. The islanders themselves raised most of the funds to cover transportation, accommodations and cost of laboratory tests. Additional support was provided by G. D. Searle International Company and Biochemistry Associates International. Approximately 15 persons from the Department of Family Medicine provided the health manpower on a voluntary basis. The objectives of the study were: (1) to obtain prevalence information on biochemical and pesticide residues of the inhabitants, (2) to upgrade medical records of the community, and (3) establish a baseline profile of the population and biochemical characteristics of the individual against which future medicine could be practiced. Approximately one-third of the island (530) of the 1,500 inhabitants, mostly children and adolescents, participated in this fair. Physical and visual screening were conducted and serum was obtained from 254 islanders. SMA-12, VDRL, PAP smears, imminizations, TB tests, and serum organochlorine studies were completed. The prevalence of these biochemical test are presented and distribution frequences of selected biochemistries are discussed. Abnormal findings and the most suitable thyroid tests in a populatioin favouring fish in their diet is described. Plans for completion of a total island survey are under way for 1972 (AU)