Sex-based differences in regulatory T cells.

Females have a higher incidence of autoimmune diseases than males for reasons that are currently unknown. CD4+CD25+ regulatory T cells play an important role in the maintenance of immunological homeostasis and self-tolerance by suppressing autoreactive T cells that could potentially cause autoimmune diseases. Given that autoimmune diseases are more prevalent in women compared to men, we hypothesized that sex steroids could influence the incidence and/or progression of autoimmune disease through an effect on CD4+CD25+ regulatory T cell number and/or function. The overall objective of this project was then to determine whether sex steroids mediate sex-based differences in CD4+CD25+ regulatory T cell number, function and phenotype, and through this mechanism influence the differential incidence of systemic lupus erythematosus in females and males. To attain our objectives, we 1) assessed the influence of androgens (dihydrotestosterone) on CD4+CD25+ regulatory T-cell number, phenotype and function; 2) assessed the influence of estrogens (estradiol) on CD4+CD25+ regulatory T cell number and function; 3) assessed CD4+CD25+ T cells and the effects of androgens in an animal model of systemic lupus erythematosus (SLE). We found that androgens increased the numbers of CD4+CD25+, CD4+CD25+CD103+ and CD4+CD25+CTLA4+ cells. Moreover, male CD4+CD25+CD103+ cells expressed more of the regulatory cell-associated transcription factor, Foxp3, than females, which also correlated with an enhancement in in vitro regulatory function, because male CD4+CD25+ and CD4+CD25+CD103+ cells suppressed the proliferation of responder CD4+CD25- cells better than those from females. Conversely, estrogens had a very little effect on regulatory T cell numbers, phenotype and function. Our conclusion was that androgens, but not estrogens, can in fact, have an influence on the numbers, function and phenotype of CD4+CD25+ regulatory T cells. In radiation bone marrow chimera experiments, we determined that androgens increase CD4+CD25+ regulatory cell numbers through an effect on the thymic epithelium, but influence the development of CD4+CD25+ regulatory function through a direct effect on the bone marrow-derived precursor (not mature) cells. In the second part of our project, we assessed CD4+CD25+ T cells and the effects of androgens in a murine model of systemic lupus erythematosus (NZB x NZW) in which only females get disease, and found that female NZB x NZW mice had significantly lower levels of CD4+CD25+ and CD4+CD25+CD103+ T cells compared with male mice. Furthermore, androgen deprivation (castration) led to a reduction in the percentages of CD4+CD25+CD103+ cells in male mice to levels lower than those found in intact male mice, or comparable to levels in intact females. Moreover, male NZB x NZW mice have more potent and greater numbers of CD4+CD25+CD103+ cells. Androgen deprivation in males led to an increase in disease as indicated by the increase in antibodies to dsDNA and a coincident reduction in CD4+CD25+ cell numbers. On the other hand, the administration of androgens to females inhibited disease progression, as reflected in significantly lower levels of dsdNA antibodies compared to placebo-treated females, and this decrease correlated with enhanced CD4+CD25+ cell number and function. Taken together, these data strongly suggest that androgens could influence the incidence and/or severity of disease by affecting the numbers and/or function of regulatory T cells. It also suggests that androgens appear to be associated with an increase in the percentage of CD4+CD25+ cells that express the important trafficking molecule, CD103, which may facilitate the ability of male CD4+CD25+ regulatory cells to reach sites of inflammation. These androgen-mediated changes appear to correlate with prevention of disease, thus androgens may confer, at least in part, resistance to autoimmune disease in males through an enhansive effect on CD4+CD25+ cell numbers and function

High-throughput screening identifies a bisphenol inhibitor of SV40 large T antigen ATPase activity

The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ∼0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC50, cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose-response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC50 of 41 μM in the primary assay and 6.2 μM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo

The effects of Trypanosome factors on Bovine Leukocyte function.

African trypanosomes infect cattle and humans in tsetse fly-infested areas of sub-Saharan Africa, causing sleeping sickness in humans and nagana in livestock. To date, no vaccines are available to combat these economically important diseases. In order to develop safe and protective vaccines, the immunomodulatory role of parasite factors on immune function must be identified. The suppression of cellular responses is a characteristic feature of African trypanosomosis. In mice, certain aspects of immunosuppression are mediated by the generation of suppressive T-cells and by nitric oxide (NO) produced by gamma interferon (IFN-y)-activated macrophages. In order to ascertain which parasite factors induce the immunosuppression observed in bovine trypanosomosis, the effect of trypanosome factors on NO production by activated bovine peripheral blood mononuclear cells (PBMC) and monocytes as well as proliferative responses of stimulated PBMC, was measured. Cells were tested for their capacity to produce NO in response to activation with IFN-y and trypanosome factors and also for their proliferative capacity after stimulation with mitogens, trypanosome antigens and non-trypanosome antigens. Total mononuclear cell populations and adherent cells derived from uninfected N'Dama cattle and activated in vitro with IFN-y and trypanosome antigens, produced NO. Trypanosome antigens induced very low levels of NO production by both monocytes and PBMC. IFN-y acted synergistically with trypanosome antigens to induce higher levels of NO production. Both trypanosome and non-trypanosome antigens significantly decreased or almost completely blocked the proliferation of bovine PBMC to the recall antigen, foot and mouth disease vaccine (FMDV) antigen, but not to concanavalin A (Con A). These results suggest that immunosuppression observed in trypanosome-infected N'Dama cattle might be induced by specific trypanosome factors. However, these observations require further investigation

Contribution of Arbuscular mycorrhizal fungi (AMF), rhizobia and metarhizium anisopliae to cowpea production in Cameroon

The objective of this study was to investigate on how the interactions between the microbial syrnbionts (AMF+rhizobia) and the rnycopesticide M. anisopliae can affect the cowpea production in varied agro ecological zones of Cameroon. Cowpea of the Bafia local cultivar was grown from 1999 to 2004 in the Sudano-sahelian (zone-I), Guineasavannah (zone-II), monomodal (zone-IV) and bimodal humid-forest rainfall (zone-V) of Cameroon. Two cropping seasons were experimented in each zone, but in different years except in zone-IV. Experiments were conducted in a Randomised Block Design (RED) with two levels of inoculation at sowing (uninoculanted seeds and dually inoculated seeds with Arbuscular Mycorrhizal Fungi (AMF) and rhizobia and two levels of spray applications at onset of flowering with the mycopesticide (Metarhizium anisopliae), or the insecticide Deltamethrin ®. Results indicate that inoculation significantly increased cowpea biomass in the first and second cropping years, respectively by 38 and 40% in zone-I, 54 and 43% in zone-II, 55 and 46% in zone-IV, 41 and 51 % in zone-Vat 45 Days After Planting (DAP). Inoculated plants showed a low but significant (p = 0.01) response to AMF colonization in all the trials compared to uninoculated plants. Nodules were formed by native and introduced rhizobia while the number and dry weight of nodules were significantly higher (p<0.0 1) in roots of inoculated than those of uninoculated plants. Inoculated and sprayed treatments significantly produced more pods per plant (p<0.01) and enhanced the dry weight of pods per plant at harvest (p = 0.03) in all trials compared to the control. These results suggest that AMF, rhizobia and M. anisopliae are variously efficient microsyrnbionts and mycopesticides in different Cameroonian soils and may be used as economical and safe bio-inoculants to improve cowpea production in the country

Arbuscular-mycorrhizal fungi, rhizobia and Metarhizium anisopliae enhance P, N, Mg, K and Ca accumulatons in fields grown cowpea

The concentrations ofP, N, K, Mg, Cain cowpea (Vigna unguiculata (L.) Walp.) roots and shoots were assessed at 45 Days After Planting (DAP) in inoculated and uninoculated plants at sowing with rhizobia and Arbuscular-Mycorrhizal Fungi (AMF). Those of harvested seeds from inoculated and uninoculated plants at sowing or sprayed and unsprayed plants at flowering with Metarhizium anisopliae were also assessed. Field trials were carried out in a complete Randomized Block Design with four treatments, in the Sudano-Sahelian (zone I), Guinea-Savannah (zone II), monomodal and bimodal hlllIlid-forest rainfall (zone IV and V) of Cameroon. The contributions of rhizobia and AMF to cowpea shoots and roots nutrients uptake at 45 DAP significantly accOlmted for up to 17% for total N, 52% for available P, 19% for Ca, 55% for Mg, 46% for K. Compared to the control, AMF+rhizobia, M. anisopliae, AMF+rhizobia and M. anisopliae significantly increased (p ~ 0.04) the N, P, Ca, Mg and K seed concentrations in zone-I (2000 and 2001), zone-II (1999), zone-IV (2004) and zone-V (1999 and 2001) at harvest. The two symbionts and M. anisopliae almost had the same influence on plant nutrient uptake within agro ecological zones. These results demonstrate the dependency of cowpea on microbial inoculants for nutrient acclUllulations in cowpea plants. However, more work still need to be camed out to investigate on the mechanisms by which M. anisopliae contributes to the increment of nutrient uptake in harvested cowpea seeds

Management of cowpea flower thrips, Megalurothrips sjostedti (Thysanoptera, thripidae), in Cameroon

A series of experiments were conducted in Cameroon to investigate options for managing cowpea flower thrips, Megalurothrips sjostedti, via arbuscular mycorrhiza fungi, rhizobia and Metarhizium anisopliae. Six cowpea fields were established in three agroecological zones over a 3-year period. The abundance of both larvae and adults of M. sjostedti was assessed on plants grown from (1) seeds co-inoculated at sowing with arbuscular mycorrhiza fungi (AMF) and rhizobia (mycorrhiza/rhizobia); (2) non-inoculated seeds and plants sprayed three times with M. anisopliae (Metarhizium); (3) seeds co-inoculated at sowing with AMF and rhizobia and plants sprayed three times with M. anisopliae (mycorrhiza/rhizobia/Metarhizium); (4) non-inoculated seeds and plants sprayed three times with the synthetic insecticide deltamethrin; and compared with (5) a control consisting of non-inoculated seeds and unsprayed plants. Results indicate that thrips infestation was associated with the flowering cycle and was higher in the first than in the second cropping season in most agroecological zones. In general, M. sjostedti larval and adult counts were significantly higher (Po0.01) in the control than in other treatments. Compared with the control, treatment insecticide obtained the highest reduction of adults (range 52–95%) and larvae thrips population (64–97%), followed by mycorrhiza/rhizobia/Metarhizium (29–56%) and (29–49%), mycorrhiza/rhizobia (31–49%) and (24–52%), and Metarhizium (25–58%) and (5–52%), respectively. In all of the above treatments, the reduction of thrips led to a subsequent increase of seed yield, although it was not always significant. Apart from two cases (Ngaounde´re´ 2000 and Nkolbisson 1999), biological treatments (Metarhizium, mycorrhiza/rhizobia and mycorrhiza/rhizobia/Metarhizium) consistently reduced grain yield loss in the trials compared with the control at Ngaounde´re´ in 1999 and Maroua 2001. These results are discussed in the context of sustainable management of M. sjostedti populations on cowpea. r 2007 Elsevier Ltd. All rights reserved

Novel approach to identify inhibitors of iron acquisition systems of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that has been declared by the World Health Organization as a “priority 1 critical pathogen” needing immediate new strategies for chemotherapy. During infection, P. aeruginosa uses redundant mechanisms to acquire ferric, heme (Hm), or ferrous iron from the host to survive and colonize. Significant efforts have been undertaken to develop siderophore blockers to inhibit ferric iron acquisition by P. aeruginosa, but there is a lack of inhibitors that can block Hm or ferrous iron acquisition by P. aeruginosa. We developed and employed a targeted high-throughput screen (HTS) and identified a molecule(s) that can specifically inhibit the Hm and ferrous iron acquisition systems of P. aeruginosa. Our targeted approach relies on screening a small-molecule library against P. aeruginosa under three growth conditions, where the only variable was the iron source (ferric, Hm, or ferrous iron). Each condition served as a counterscreen for the other, and we identified molecules that inhibit the growth of P. aeruginosa in the presence of only Hm or ferrous iron. Our data indicate that econazole, bithionate, and raloxifene inhibit the growth of P. aeruginosa in the presence of Hm and that oxyquinoline inhibits the growth of P. aeruginosa in the presence of ferrous iron. These iron-specific inhibitors do not interfere with the activity of meropenem, a commercial antipseudomonal, and can also increase meropenem activity. In conclusion, we present a proof of concept of a successful targeted conditional screening method by which we can identify specific iron acquisition inhibitors. This approach is highly adaptable and can easily be extended to any other pathogen. IMPORTANCE Since acquiring iron is paramount to P. aeruginosa’s survival and colonization in the human host, developing novel strategies to block the access of P. aeruginosa to host iron will allow us to starve it of an essential nutrient. P. aeruginosa uses siderophore, heme, or ferrous iron uptake systems to acquire iron in the human host. We have developed a novel approach through which we can directly identify molecules that can prevent P. aeruginosa from utilizing heme or ferrous iron. This approach overcomes the need for the in silico design of molecules and identifies structurally diverse biologically active inhibitor molecules. This screening approach is adaptable and can be extended to any pathogen. Since Gram-negative pathogens share many similarities in iron acquisition at both the mechanistic and molecular levels, our screening approach presents a significant opportunity to develop novel broad-spectrum iron acquisition inhibitors of Gram-negative pathogens.Microbiology and Molecular Genetic