Role of MCP-1 on inflammatory processes and metabolic dysfunction following high-fat feedings in the FVB/N strain.

BACKGROUND: Monocyte chemoattractant protein 1 (MCP-1) is known to be an important chemokine for macrophage recruitment. Thus, targeting MCP-1 may prevent the perturbations associated with macrophage-induced inflammation in adipose tissue. However, inconsistencies in the available animal literature have questioned the role of this chemokine in this process. The purpose of this study was to examine the role of MCP-1 on obesity-related pathologies. METHODS: Wild-type and MCP-1-deficient mice on an friend virus B NIH (FVB/N) background were assigned to either low-fat diet or high-fat diet (HFD) treatment for a period of 16 weeks. Body weight and body composition were measured weekly and monthly, respectively. Fasting blood glucose and insulin, and glucose tolerance were measured at 16 weeks. Macrophages, T-cell markers, inflammatory mediators and markers of fibrosis were examined in the adipose tissue at the time of killing the mice. RESULTS: As expected, HFD increased adiposity (body weight, fat mass, fat percent and adipocyte size), metabolic dysfunction (impaired glucose metabolism and insulin resistance) macrophage number (CD11b(+)F480(+) cells, and gene expression of EMR1 and CD11c), T-cell markers (gene expression of CD4 and CD8), inflammatory mediators (pNFκB and pJNK, and mRNA expression of MCP-1, CCL5, C-X-C motif chemokine-14, tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6)) and fibrosis (expression of IL-10, IL-13, TGF-ß and matrix metalloproteinase-2 (MMP2); P<0.05). However, contrary to our hypothesis, MCP-1 deficiency exacerbated many of these responses resulting in a further increase in adiposity (body weight, fat mass, fat percent and adipocyte size), metabolic dysregulation, macrophage markers (EMR1), inflammatory cell infiltration and fibrosis (formation of type I and III collagens, mRNA expression of IL-10 and MMP2; P<0.05). CONCLUSIONS: These data suggest that MCP-1 may be a necessary component of the inflammatory response required for adipose tissue protection, remodeling and healthy expansion in the FVB/N strain in response to HFD feedings.

Normal Lyt-1+2- T cells have the unique capacity to respond to syngeneic autoreactive T cells. Demonstration of a T cell network.

In the present communication, we describe the unique observation that Lyt-1+2-, L3T4+ T cells but not Lyt-2+ T cells isolated from the spleens of normal, unimmunized H-2d mice proliferate strongly and directly to an irradiated, syngeneic, Lyt-1+2-, L3T4+, Ia- autoreactive T cell line/clone. In contrast, Lyt-1+2- T cells failed to proliferate when stimulated by long-term, antigen-specific, H-2d-restricted T cell lines. Supernatants from the cultures of autoreactive T cells or recombinant interleukin 2 (IL-2) alone, failed to induce proliferation of the Lyt-1+2- T cells, suggesting that cell-cell interaction is essential for growth. In addition, the proliferative response of Lyt-1+2- T cells was independent of Ia+ antigen-presenting cells and was not blocked by either anti-Iad or anti-H-2d antibodies, but was inhibited by anti-L3T4 antibodies. All these observations suggested that the responding Lyt-1+2- T cells were recognizing the anti-self-Ia receptor expressed on autoreactive T cells and that such T cells might therefore express the internal image of self-Ia determinants. We suggest that the T-T interactions observed may represent interactions between two helper T cell subpopulations at the idiotope level and that autoreactive T cells may function as an important feature of the regulatory network and/or lead to the expansion of the T cell repertoire.

Double-negative T cells from MRL-lpr/lpr mice mediate cytolytic activity when triggered through adhesion molecules and constitutively express perforin gene.

The lpr gene induces in mice, accumulation of large numbers of CD4-CD8- (double negative [DN]) T lymphocytes which bear adhesion molecules not characteristic of normal resting T cells. These cells fail to acquire interleukin 2 (IL-2) receptors, produce IL-2, and proliferate when activated with mitogens or monoclonal antibodies (mAbs) against the T cell receptor (TCR). Because of these poor functions in vitro, the nature and significance of DN T cells in the autoimmune disease process is not clear. In the current study, we describe a surprising finding that mAbs against CD3-TCR-alpha/beta complex can strongly trigger the lytic activity of the DN T cells to induce redirected lysis of Fc receptor-positive targets. Similar redirected lysis was also inducible using mAbs against CD44 and gp90MEL-14, molecules involved in the binding of lymphocytes to endothelial cells. The spontaneous cytotoxic potential of the DN T cells was further corroborated by demonstrating that the lpr DN T cells constitutively transcribed perforin gene but failed to express granzyme A. The current study suggests that DN T cells are capable of mediating lysis of autologous cells bearing the specific ligands for adhesion molecules involved in the signaling of cytotoxicity. These findings provide a novel insight into the functional significance of DN T cells in lpr mice and their potential role in the pathogenesis of autoimmune disease.

Decreased AGO2 and DCR1 in PBMCs from War Veterans with PTSD leads to diminished miRNA resulting in elevated inflammation.

Chronic inflammation is a characteristic of post-traumatic stress disorder (PTSD). The initiation of inflammation and molecules involved are not yet clearly understood. Here, we provide compelling evidence that the inflammation seen in PTSD may result from the dysregulated miRNA processing pathway. Using microarray analysis with a discovery group of peripheral blood mononuclear cell (PBMC) samples from War Veterans with PTSD, we found 183 significantly downregulated miRNAs, several of which target numerous genes categorized to be pro-inflammatory in nature. This observation was further confirmed in a replicate group by including more samples. Furthermore, employing RNA-sequencing, quantitative real time PCR (qRT-PCR) and in vitro experiments, we found that Argonaute 2 (AGO2) and Dicer1 (DCR1) were downregulated in PTSD and provided convincing evidence that their downregulation affects mature miRNA generation. In addition, we noted that STAT3 transcript was reduced in PTSD and this was possibly responsible for reduced AGO2 and DCR1, which in turn affected miRNA synthesis. Furthermore, we observed that activation of CD4+ T cells or monocytes led to reduced mature miRNA availability. Finally, the inflammation seen in PTSD was associated with downregulated miRNA profile. Altogether, the current study demonstrates that the chronic inflammation seen in PTSD may be a result of dysregulated miRNA biogenesis pathway due to diminished expression of the key molecules like AGO2, DCR1 and STAT3.

Immunomodulation by various nitrosoureas and its effect on the survival of the murine host bearing a syngeneic tumor.

Chemotherapeutic efficacies of the nitrosoureas 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), chlorozotocin (CLZ), and streptozotocin (STZ) were investigated against the LSA tumor which is syngeneic to C57BL/6 mice. It was observed that a single injection of 20 mg/kg body weight of BCNU or CLZ, even at an advanced stage of tumor growth, completely cured greater than 90% of the tumor-bearing mice. Furthermore, BCNU-cured or CLZ-cured mice could specifically reject secondary rechallenge with the LSA tumor. In contrast, a single dose treatment with STZ at 20-200 mg/kg body weight failed to cure the tumor-bearing mice (0% survival). The failure of STZ to cure tumor-bearing mice was next addressed considering three possible mechanisms: (a) STZ was less tumoricidal; (b) STZ suppressed the immunity of the host; and (c) STZ failed to eliminate tumor-specific suppressor T-cells. The failure of STZ to cure tumor-bearing mice was not totally related to its tumoricidal properties since STZ at higher doses did possess significant tumoricidal activity in vitro and in vivo, comparable to that of BCNU or CLZ. When spleen cells from normal mice treated with BCNU, CLZ, or STZ were assayed for their responsiveness to the T-cell mitogens concanavalin A or phytohemagglutinin, it was observed that STZ was in fact less immunosuppressive than BCNU or CLZ. The fact that STZ did not suppress the immunity of the host was also suggested by the findings that BCNU-cured mice treated with STZ or CLZ could still reject secondary rechallenge with the specific tumor LSA. Following treatment of tumor-bearing mice with BCNU or CLZ, tumor-specific delayed type hypersensitivity responses were demonstrable in these mice but not in STZ-treated mice. The inability of STZ-treated tumor-bearing mice to elicit a delayed type hypersensitivity response was not due to selective depletion of delayed type hypersensitivity-inducing CD4+ T-cells but was probably due to failure of STZ to eliminate tumor-specific suppressor cells. Together these findings suggested that the failure of STZ to cure LSA tumor-bearing mice was not due to lack of tumoricidal activity or related to suppression of tumor-specific effector T-cell function but may be due to the failure of STZ to eliminate tumor-specific T suppressor cells. The present study suggests that the outcome of chemotherapy with nitrosoureas depends, in addition to the tumoricidal activity of the drug, on the immunomodulating action on the immune mechanisms of the host.

Δ(9) Tetrahydrocannabinol attenuates Staphylococcal enterotoxin B-induced inflammatory lung injury and prevents mortality in mice by modulation of miR-17-92 cluster and induction of T-regulatory cells.

BACKGROUND AND PURPOSE: Staphylococcal enterotoxin B (SEB) is a potent activator of Vß8+T-cells resulting in the clonal expansion of ∼30% of the T-cell pool. Consequently, this leads to the release of inflammatory cytokines, toxic shock, and eventually death. In the current study, we investigated if Δ(9) tetrahydrocannabinol (THC), a cannabinoid known for its anti-inflammatory properties, could prevent SEB-induced mortality and alleviate symptoms of toxic shock. EXPERIMENTAL APPROACH: We investigated the efficacy of THC against the dual administration (intranasal and i.p.) of SEB into C3H/HeJ mice based on the measurement of SEB-mediated clinical parameters, including cytokine production, cellular infiltration, vascular leak, and airway resistance. In addition, the molecular mechanism of action was elucidated in vitro by the activation of splenocytes with SEB. KEY RESULTS: Exposure to SEB resulted in acute mortality, while THC treatment led to 100% survival of mice. SEB induced the miRNA-17-92 cluster, specifically miRNA-18a, which targeted Pten (phosphatase and tensin homologue), an inhibitor of the PI3K/Akt signalling pathway, thereby suppressing T-regulatory cells. In contrast, THC treatment inhibited the individual miRNAs in the cluster, reversing the effects of SEB. CONCLUSIONS AND IMPLICATIONS: We report, for the first time a role for the miRNA 17-92 cluster in SEB-mediated inflammation. Furthermore, our results suggest that THC is a potent anti-inflammatory compound that may serve as a novel therapeutic to suppress SEB-induced pulmonary inflammation by modulating critical miRNA involved in SEB-induced toxicity and death.

3,3'-Diindolylmethane attenuates LPS-mediated acute liver failure by regulating miRNAs to target IRAK4 and suppress Toll-like receptor signalling.

BACKGROUND AND PURPOSE: Acute liver failure (ALF) is a severe and potentially lethal clinical syndrome. 3,3'-Diindolylmethane (DIM) is a natural plant-derived compound with anti-cancer activities. Recently, DIM has also been shown to have anti-inflammatory properties. Here, we tested the hypothesis that DIM would suppress endotoxin-induced ALF. EXPERIMENTAL APPROACH: We investigated the therapeutic potential of DIM in a mouse model of D-galactosamine/Lipopolysaccharide (GalN/LPS)-induced ALF. The efficacy of DIM treatment was assessed by survival, liver histopathology, serum levels of alanine transaminase, pro-inflammatory cytokines and number of activated liver macrophages. Effects of DIM on the expression of two miRNAs, 106a and 20b, and their predicted target gene were measured by qRT-PCR and Western blotting. Effects of DIM on the release of TNF-α from RAW264.7 macrophages transfected with mimics of these miRNAs and activated by LPS was assessed by elisa. KEY RESULTS: DIM treatment protected mice from ALF symptoms and reduced the number of activated liver macrophages. DIM increased expression of miR-106a and miR-20b in liver mononuclear cells and decreased expression of their predicted target gene IL-1 receptor-associated kinase 4 (IRAK4), involved in signalling from Toll-like receptor 4 (TLR4). In vitro transfection of RAW264.7 cells using miRNA mimics of miR-106a and 20b decreased expression of IRAK4 and of TNF-α secretion, following LPS stimulation. CONCLUSIONS AND IMPLICATIONS: DIM attenuated GalN/LPS-induced ALF by regulating the expression of unique miRNAs that target key molecules in the TLR4 inflammatory pathway. DIM may represent a potential novel hepatoprotective agent.

Macrophages but not B cells from aged mice are defective in stimulating autoreactive T cells in vitro.

In the present study the effect of aging on the capacity of Ia+ cells to stimulate autoreactive T cells in the syngeneic mixed lymphocyte reaction (SMLR) was investigated. Using young CD4+ T cells as responders, it was observed that unseparated whole spleen cells from aged mice had normal stimulatory activity comparable to that of young spleen cells. Interestingly, however, when purified splenic adherent cells (SAC) enriched for macrophages or splenic B cells were used as stimulators, aged SAC but not aged B cells were found to be defective in stimulating autoreactive T cells. This defect in aged SAC was not due to decreased expression of Ia antigens since the percentage of Ia+ SAC and density of Ia antigen expression was similar in both young and old mice. Also, the B cells from aged mice expressed normal levels of Ia antigens. Aged SAC, when mixed with young SAC could also actively suppress the normal SMLR. However, this suppression was not due to increased prostaglandin production but was found to be associated with interleukin-1 (IL-1) regulation, inasmuch as addition of exogenous IL-1 could completely reconstitute the defective stimulatory activity of aged SAC and also abolished the suppressor activity of the SAC. Aged mice also demonstrated an intrinsic defect in the CD4+ T cells responding in the SMLR. Together, our studies on the SMLR demonstrate an age-related defect in responder autoreactive T cells and in stimulator splenic macrophages but not in the stimulatory activity of B cells.

Role of CD44 in CTL and NK cell activity.

CD44 is a widely distributed cell surface glycoprotein whose principal ligand has been identified as hyaluronic acid (HA), a major component of the extracellular matrix (ECM). Recent studies have demonstrated that activation through CD44 leads to induction of effector function in T cells and macrophages. At sites of chronic inflammation as seen in certain infections, autoimmune diseases, allograft rejection, graft-versus-host (GVH) disease and treatment of cancer patients with high doses of interleukin-2, significant damage to the endothelial cells has been known to occur, which leads to the toxicity or pathogenesis associated with the disease. The exact mechanism of endothelial cell damage is not clear, although, it has been widely speculated that immune cells may play a critical role. Studies from our laboratory have used interleukin-2 (IL-2) induced vascular leak syndrome (VLS) as a model to investigate the role of cytolytic lymphocytes in the direct cytotoxicity of endothelial cells. Cytotoxic T lymphocytes (CTL), double-negative (DN) T cells and natural killer (NK) cells upon activation express high levels of CD44 and mediate efficient MHC-unrestricted TCR-independent lysis following ligation of CD44. Such CD44-mediated cytotoxicity may play an important role in protection against viral infections and cancer. However, it could also cause non-specific tissue injury. For example, dysregulation in the interaction between activated cytotoxic lymphocytes expressing CD44 and endothelial cells bearing the appropriated ligand such as the hyaluronate (HA), could lead to endothelial cell lysis. Furthermore, such endothelial cell injury could lead to the pathogenesis associated with a variety of clinical diseases.

Blood-transfusion-induced suppression of cytotoxic T lymphocyte responses in mice.

B6AF1 (H-2KbkDbd) mice were transfused weekly with 0.1 ml of whole blood from DBA/2 (H-2d) mice. One week after each transfusion, spleen and serum samples were collected. Blood transfusions did not induce blood donor alloantigen-specific cytotoxic T lymphocytes (CTL) in spleens of B6AF1 mice. When spleen cells from transfused mice were sensitized to alloantigens in mixed lymphocyte culture in vitro, it was observed that 1-3 transfusions induced suppression of blood donor-specific CTL activity. No suppression of CTL activity was found after 4 transfusions. The cell-mixing experiments demonstrated that the suppression of CTL activity following initial 2 blood transfusions was due to the presence of suppressor cells. The presence of antibodies in sera of transfused B6AF1 mice capable of inhibiting CTL was investigated using the CTL-inhibition test. In these experiments, cytotoxic T lymphoblasts generated in MLC in vitro by culturing normal B6AF1 spleen cells with x-irradiated DBA/2 cells were treated with serum before testing them for cytotoxicity. The antibodies capable of inhibiting CTL responses were demonstrable in sera from transfused mice. Three and four BT sera caused significant inhibition of CTL responses. The CTL-inhibitory antibodies were specific for effector cells of the B6AF1 mice and for target cells of the blood donor DBA/2 mice. These results suggest that the inhibition of CTL responses is caused by antibodies directed against the recognition sites on effector T lymphocytes. The data from this study, therefore, demonstrate that BT cause suppression of the recipient's CTL responses against alloantigens present on the blood donor, and that this suppression is mediated by suppressor cells after the initial 1 to 2 transfusions and by antibodies directed against the CTL antigen-specific receptors after subsequent transfusions.

Induction of antibodies by blood transfusions capable of inhibiting responses in MLC.

B6AF1 (H-2KbkDbd) mice were transfused weekly with 0.1 ml of whole blood from DBA/2 (H-2d) mice. One week after each transfusion, the mice were bled and the sera were collected and pooled. The presence of antiidiotypic antibodies in these sera was investigated using the mixed-lymphocyte culture (MLC) inhibition test, in which spleen cells from normal B6AF1 mice were treated with sera from transfused B6AF1 mice, washed, and then tested for their responses to DBA/2 stimulator cells. The sera collected following 3 and 4 transfusions caused a significant inhibition of responses in MLC, but sera obtained after 1 and 2 transfusions caused little or no inhibition. This inhibition was specific for stimulator cells from the blood donor (DBA/2) and was not observed against third-party SJL (H-2s) stimulator cells. In addition, the suppressive effect in MLC was specific for responder cells from the recipient B6AF1 mice, and no suppression was observed with responder cells from C3H (H-2k) and SJL mice. Treatment of DBA/2 stimulator cells with the serum caused no inhibition in MLC. The MLC inhibitory activity of the serum decreased gradually from the first to the third week following 4 transfusions, although a significant inhibition was still demonstrable after 3 weeks. These findings suggest that multiple blood transfusions induce antiidiotypic antibodies that can block the T cell antigen-specific receptors and cause suppression of the recipient's responses against the donor's alloantigens in MLC.

Development of a kit for the assay of haemagglutination inhibition antibodies to flaviviruses using formalinized goose erythrocytes.

Haemagglutination inhibition (HI) test using goose erythrocytes (GRBC) is the method of choice for screening antibodies to various arboviruses. This test makes use of fresh GRBC preserved in dextrose-gelatin-veronal (DGV). We have tried formalinizing GRBC and have found that such cells can retain the surface property of being agglutinated by arboviruses and, therefore, can be used in haemagglutination (HA) and HI tests. Unlike fresh cells preserved in DGV, formalinized cells can be preserved for a long time without haemolysis or loss of sensitivity. They can also be frozen or lyophilized. Making use of these properties, a kit has been developed for the assay of HI antibodies to some members of the flavivirus group. Use of this kit simplifies the HI test and enables it to be carried out routinely by any hospital or laboratory to screen arboviral infections.

Differential induction of apoptosis in activated and resting T cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its repercussion on T cell responsiveness.

TCDD is well known for its immunotoxic effects on T cells, although the exact mechanism of toxicity remains unknown. In the current study, we investigated the effect of TCDD administration on resting and activated T cells within the same animal. To this end, C57BL/6 mice were injected intraperitoneally with either TCDD (50 microg/kg body weight) or the vehicle and were injected with anti-CD3 mAbs into the rear footpads to polyclonally activate T cells in the popliteal lymph nodes (LN). Axillary LN cells harvested from the same groups of mice served as a source of resting T cells. When the LN cells were tested for their proliferative responsiveness to stimulation with anti-CD3 mAbs in vitro, the activated popliteal LN, but not the resting axillary LN cells from TCDD-treated mice exhibited a significant decrease in responsiveness when compared to the vehicle controls. Inasmuch as TCDD has been shown to induce apoptosis in thymocytes, we addressed whether TCDD triggered apoptosis in LN cells, using the terminal deoxynucleotidyl transferase (TdT)-mediated FITC-dUTP nick end labeling (TUNEL) method. The axillary and popliteal LN cells from TCDD-treated mice failed to exhibit significant levels of apoptosis when freshly harvested. However, upon in vitro culture for 24 h with either tissue culture medium alone or with anti-CD3 mAbs, activated popliteal LN cells from TCDD-treated mice showed a significant increase in apoptosis when compared to similar cells from vehicle-treated mice. In contrast, resting axillary LN cells from TCDD-treated mice, similarly cultured in vitro, exhibited decreased levels of apoptosis when compared to the controls. Using a double-staining technique, the activated popliteal LN cells undergoing increased apoptosis in TCDD-treated animals were confirmed to be CD3+ T cells. Together, these data demonstrate that TCDD exerts differential effects on activated and resting T cells, even within the same animal, by inhibiting the proliferative responsiveness of activated, but not resting, T cells. Furthermore, this effect may be mediated by the ability of TCDD to induce increased apoptosis in activated, but not resting, T cells.

Role of Fas apoptosis and MHC genes in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity of T cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is well known for its immunotoxic effects particularly on the thymus as well as on T and B lymphocyte functions. Previous studies have suggested that TCDD may induce apoptosis in thymocytes although its demonstration in vivo has met with limited success. TCDD has also been shown to alter the major histocompatability complex- (MHC) encoded molecules, however, its role in immunotoxicity is not clear. In the current study, we investigated the role of Fas (CD95), an important molecule involved in the induction of apoptosis, on TCDD-mediated immunotoxicity using mice bearing homozygous lpr mutation which leads to failure of expression of Fas. When TCDD was administered orally at 0, 0.1, 1.0, or 5.0 micrograms/kg body weight for 11 days, it was found to be less toxic to the thymocytes from C57BL/6 lpr/lpr mice (Ah-responsive, Fas-) when compared to C57BL/6 +/+ mice (Ah-responsive, Fas+). Similar results were obtained when peripheral T cell responsiveness to antigenic challenge with conalbumin was studied in these mice. When mice that differed only at the MHC were compared for immunotoxic effects of TCDD, it was noted that B10.D2 (Ah-responsive, H-2d) were more sensitive to TCDD-mediated thymic atrophy and peripheral T cell dysfunction when compared to B10 mice (Ah-responsive, H-2b). In all TCDD-sensitive strains tested, the thymic atrophy was accompanied by a uniform depletion of all four subset of T cells (CD4+, CD4+CD8+, CD4-CD8-, and CD8+) and the percentage of these subsets was not altered. Furthermore, in these strains, TCDD suppressed the antigen-specific peripheral T cell responsiveness but not the responsiveness of naive resting T cells to polyclonal mitogens. Lastly, using cell-mixing experiments, it was demonstrated that TCDD directly affected the T cells responding to conalbumin but not the antigen presenting cells (APCs). Together, our studies demonstrate that although Ah locus plays the primary role, determining the toxicity of TCDD on the T cells, there are secondary factors such as expression of Fas or the MHC-phenotype which may play an important role in TCDD-mediated immunotoxicity. The role of Fas further suggests that TCDD may induce toxicity in T cells by triggering apoptosis.

Enhanced activation-induced cell death as a mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity in peripheral T cells.

T cells upon activation undergo apoptosis, a process termed activation-induced cell death (AICD). In the current study, we investigated whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases AICD and whether this constitutes one of the mechanisms by which TCDD induces immunotoxicity. To this end, C57BL/6+/+, C57BL/6 gld/gld (Fas ligand-defective) and C57BL/6 lpr/lpr (Fas-deficient) mice were injected with TCDD (50 microg/kg body weight, ip) or the vehicle (corn oil) and with anti-CD3 mAbs into the footpads. 3 days later, inguinal and popliteal lymph node cells were harvested, pooled and enumerated. Cells were cultured in vitro with anti-CD3 mAbs and cell proliferation was measured. Also, such cells were studied for their ability to undergo apoptosis upon in vitro culture with either tissue culture medium alone or with anti-CD3 mAbs. The data demonstrated that lymph nodes from TCDD-treated wild-type (+/+) mice showed a decrease in cellularity and the T cells exhibited decreased responsiveness to anti-CD3 mAbs when compared to the vehicle-treated control group. Furthermore, such cells from TCDD-treated mice exhibited increased levels of apoptosis upon in vitro culture when compared to the cells from vehicle-treated mice. In contrast, activated lymph nodes from TCDD-treated C57BL/6 gld/gld and C57BL/6 lpr/lpr mice showed normal cellularity and T cell responsiveness to anti-CD3 stimulation when compared to the vehicle controls. In addition, the activated lymph node T cells from the TCDD-treated C57BL/6 gld/gld and C57BL/6 lpr/lpr mice failed to exhibit increased apoptosis when compared to the controls. The current study demonstrates that the immunotoxic effects of TCDD in activated peripheral T cells may result from increased AICD mediated through Fas-Fas ligand interactions.

Evidence for estradiol-induced apoptosis and dysregulated T cell maturation in the thymus.

In an attempt to delineate the immunological alterations that may occur following treatment with estrogen, groups of C57BL/6 mice were treated with 75mg/kg body weight of beta-estradiol-17-valerate (E2) or the vehicle. The thymus from these mice were harvested on days 1, 4 and 7 following treatment. The thymocytes from E2-treated mice when cultured in vitro for 24h, showed increased levels of apoptosis when compared to controls. The apoptosis was demonstrable by both TUNEL assay and AnnexinV/propidium iodide (PI) staining. Also, thymic atrophy and increased apoptosis of thymocytes when cultured in vitro were seen when lower doses of E2 (5mg/kg) were administered. The thymus from E2-treated mice on days 4 and 7 also showed a decrease in the percentage of CD4(+)CD8(+) (DP) T cells and an increase in the percentage of CD4(-)CD8(-) (DN), CD4(+) and CD8(+) T cells. However, the total cellularity of all T cell subsets in the thymus was decreased following E2 treatment. Earlier studies from our laboratory and elsewhere have demonstrated that in thymocytes undergoing apoptosis, there is increased expression of surface markers including CD3, alphabetaTCR and CD44 with a simultaneous decrease in the expression of J11d. Similar changes were observed in thymocytes from mice on days 4 and 7 following E2 treatment. These data therefore confirmed that the thymocytes were indeed undergoing apoptosis following E2 treatment. Together, our studies suggest for the first time that estrogen may induce thymic atrophy by triggering apoptosis.

Short-term toxicity studies of O-chlorobenzylidene malononitrile on humoral immunity in mice.

The effect of O-chlorobenzylidene malononitrile (CS) on the immune system was studied in mice given 8 and 16 mg/kg body weight i.p. of the compound, daily for 10 days. The humoral immune response to SRBC was suppressed at both doses, as determined by the antibody producing cells in the spleen. To find out whether the immunosuppression observed was due to the indirect effect of CS on the nutritional status and endocrine balance, serum proteins and corticosterone levels were measured. While the serum proteins did not alter, corticosterone levels rose significantly only in mice receiving higher dose of CS.