Traditional Drugs: Mechanisms of Immunosuppressor and Corticosteroid Therapies for Inflammatory Bowel Diseases

The inflammatory bowel diseases (IBD) such as Crohn’s disease and ulcerative colitis are immunological dysfunctions of the gastrointestinal tract that develop because of multifactorial processes, including genetic predisposition, gut dysbiosis, and excessive inflammation in susceptible subjects. These pathologies affect millions of people worldwide, with substantial impact on healthcare systems and patients’ quality of life. Considering the chronic inflammation that underlies the IBD presentation, the main treatment options are related to the control of patients’ inflammatory response, through immunosuppressor and modulatory therapies. Therefore, in this chapter we reviewed the main mechanisms associated with the treatments that are aimed at suppressing mucosal immunity and the effects of corticosteroid therapies in Crohn’s disease and ulcerative colitis

Toxoplasma gondii chitinase induces macrophage activation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 degrees C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathoge1012112FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNQP - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2013/10741-8]2013/10741-8SEM INFORMAÇÃOThis study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant number 2013/10741-8). Additional financial help was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Fundação de Apoio ao Ensino, Pe

Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection

Immunological Basis for the Gender Differences in Murine Paracoccidioides brasiliensis Infection

This study aimed to investigate the immunological mechanisms involved in the gender distinct incidence of paracoccidioidomycosis (pcm), an endemic systemic mycosis in Latin America, which is at least 10 times more frequent in men than in women. Then, we compared the immune response of male and female mice to Paracoccidioides brasiliensis infection, as well as the influence in the gender differences exerted by paracoccin, a P. brasiliensis component with carbohydrate recognition property. High production of Th1 cytokines and T-bet expression have been detected in the paracoccin stimulated cultures of spleen cells from infected female mice. In contrast, in similar experimental conditions, cells from infected males produced higher levels of the Th2 cytokines and expressed GATA-3. Macrophages from male and female mice when stimulated with paracoccin displayed similar phagocytic capability, while fungicidal activity was two times more efficiently performed by macrophages from female mice, a fact that was associated with 50% higher levels of nitric oxide production. In order to evaluate the role of sexual hormones in the observed gender distinction, we have utilized mice that have been submitted to gonadectomy followed by inverse hormonal reconstitution. Spleen cells derived from castrated males reconstituted with estradiol have produced higher levels of IFN-γ (1291±15 pg/mL) and lower levels of IL-10 (494±38 pg/mL), than normal male in response to paracoccin stimulus. In contrast, spleen cells from castrated female mice that had been treated with testosterone produced more IL-10 (1284±36 pg/mL) and less IFN-γ (587±14 pg/mL) than cells from normal female. In conclusion, our results reveal that the sexual hormones had a profound effect on the biology of immune cells, and estradiol favours protective responses to P. brasiliensis infection. In addition, fungal components, such as paracoccin, may provide additional support to the gender dimorphic immunity that marks P. brasiliensis infection

Quantification of <i>P. brasiliensis</i> through real-time PCR and Colony-forming units (CFU).

<p>Evaluation of the fungal burdens in lungs and liver of male (grey bars) and female (open bars) mice 30 days after intraperitoneal infection with yeast cells. The bars represent the mean±SD obtained from duplicate samples in groups of six animals. * P<0.05, compared with female infected mice.</p

IFN-γ (A) and IL-10 (B) production by spleen cells from male and female infected mice of different treated groups.

<p>Spleen cells from male and female 30 days infected mice from different groups were collected and cultured for 48 hours under paracoccin stimulation (7 µg/ml). The concentration of IFN-γ and IL-10 in supernatants was measured by ELISA. The results represent the mean±SD of six mice per group, from a representative experiment of two assays. * p<0.05 significant when compared with opposite sex of the same group.</p

Phagocytosis of <i>P. brasiliensis</i> yeasts by macrophages from male and female mice.

<p>Macrophages treated or not with paracoccin (50 µg/well) were challenged with Pb <i>FITC</i>- conjugated yeasts for 4 hs. The cells were stained with Evans Blue and counted in a fluorescence microscope (100x) to determine the yeast Phagocitic index (3A). Panel B illustrates the result and allow to observe that no extracellular attachment of yeasts after 4 hs of incubation. The results on panel A represent the mean±SD from a representative experiment of three assays.</p

NO production and viability of Pb yeasts after phagocytosis by macrophages from male and female mice.

<p>Macrophages from male and female mice were incubated with paracoccin (PCN), <i>P. brasiliensis</i> yeasts (Pb), PCN plus Pb, PCN (pre-treated with GlcNac) plus Pb or only medium and nitrite levels were detected by the Griess method (4A). For the evaluation of viability of Pb cells macrophages were stimulated with PCN, PCN (pre-treated with GlcNac) or only medium and challenged with live Pb. The determination of the number of viable fungi was made by CFU counts (4B). The results represent the mean±SD from a representative experiment of three assays. * p<0.05 significant when compared with male of the same group.</p

Spleen cells from male and female infected mice secrete different levels of cytokines after paracoccin stimulus.

<p>Spleen cells of mice of both sexes (n = 6) infected with <i>P.brasiliensis</i> yeast were collected after 7 and 30 days post infection and cultured for 48 hours under stimulation with paracoccin (0.7 µg/ml), LPS (1 µg/ml) or only medium. The concentrations of IL-12p40, TNF-α, IFN-γ and IL-10 in supernatants were measured by ELISA. Each bar represents the mean ± SD of duplicates and is representative of four experiments made in duplicate. * p<0.05 significant female versus male of the same group. All cytokine data were normalized against those of the non-infected male and female mice.</p