Effects of synbiotic-based Bifidobacterium animalis in female rats experimentally infected with Toxoplasma gondii

The aim of this study was to assess the effects of a synbiotic composed of Bifidobacterium animalis and fructooligosaccharides on female rats infected with Toxoplasma gondii. Female Wistar rats, treated or not with dexamethasone, were daily supplemented with synbiotics for 21 days. After 15 days of supplementation, the rats were orally infected with 10(4) T. gondii bradyzoites. Blood samples were collected to measure the levels of IFN-gamma, IL-10 and T. gondii antibodies. All synbiotic-supplemented rats survived until the end of the experiment; however, non-supplemented dexamethasone-treated rats died between the fifth and the eighth days after T. gondii infection. Dexamethasone-treated rats supplemented with synbiotics (P < 0.05) were capable of synthesizing IFN-gamma, and this immunological response was essential to ensure their survival. In addition, brain cysts were found in one rat not supplemented with synbiotics. Results suggest that the synbiotic composed of B. animalis and fructooligosaccharides may be beneficial to toxoplasmosis control. (C) 2010 Elsevier Ltd. All rights reserved.

Cell-mediated immune response to Leishmania chagasi experimental infection of BALB/c immunosuppressed mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of periodontal treatment on metabolic control, systemic inflammation and cytokines in patients with type 2 diabetes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Correlation between Clinical/Radiographic Features and Inflammatory Cytokine Networks Produced by Macrophages Stimulated with Endodontic Content

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production of pro- and anti-inflammatory cytokines by monocytes from patients with paracoccidioidomycosis

Monocytes and macrophages can produce a large repertoire of cytokines and participate in the pathogenesis of granulomatous diseases. We investigated the production of pro- and anti-inflammatory cytokines by monocytes from patients with active paracoccidioidomycosis. Peripheral blood monocytes from 37 patients and 29 healthy controls were cultivated with or without 10 mug/ml of lipopolysaccharide (LPS) for 18 h at 37 degreesC, and the cytokine levels were determined in the culture supernatants by enzyme immunoassay. The results showed that the endogenous levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10 and transforming growth factor beta detected in the supernatant of patient monocytes cultivated without stimulus were significantly higher than those produced by healthy controls. These data demonstrated that monocytes from patients with active paracoccidioidomycosis produce high levels of cytokines with both inflammatory and anti-inflammatory activities. However, patient monocytes produced significantly lower TNF-alpha and IL-6 levels in response to LPS when compared to normal subjects, suggesting an impairment in their capacity to produce these cytokines after LPS stimulation. Concentrations of IL-1beta, IL-8 and IL-10 in cultures stimulated with LPS were higher in patients than in controls. These results suggest that an imbalance in the production of pro- and anti-inflammatory cytokines might be associated with the pathogenesis of paracoccidioidomycosis. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Cytokine production in lungs and adrenal glands of high and low antibody producing mice infected with Paracoccidioides brasiliensis

Mice genetically selected for high (H) and low (L) antibody production (HIV-A and L-IV-A) were used in an experimental model of paracoccidioidomycosis. In a previous work, it was observed that male HIV-A animals were more susceptible to the infection due to adrenal gland damage. Male HIV-A and LIV-A animals were intravenously inoculated with Paracoccidioides brasiliensis (strain 18) and sacrificed 2, 4, 6, 8 and 10 weeks after inoculation. At each time interval, lungs and adrenals were removed to estimate recoverability of the fungus, as well as to determine Th1 (IFN-gamma, TNF-alpha) and Th2 (IL-4 and IL-10) cytokine profiles. While viable fungi recoverability from the lungs of HIV-A mice was higher after 4 and 8 weeks, there was less fungal recovery from the adrenals of LIV-A animals after the 2nd week, with total fungal elimination after the 8th week. With regard to Th2 cytokines, there was an inhibition in IL-4 production in the organs from infected animals, the extent of which varied according to the organ and the time period after initiation of infection. IL-10 production was found to be lower in both organs. Determination of Th1 cytokines revealed that IFN-gamma production increased in both organs, mainly in the adrenal of LIV-A after 8 and 10 weeks, when these animals showed a total fungal elimination. A significant difference was observed between HIV-A and LIV-A concerning TNF-alpha production in both organs and at all recovery times, in that LIV-A produced a higher level of this cytokine, mainly in the adrenal. These results may explain the high susceptibility of HIV-A to P. brasiliensis infection, is due, at least in part, to adrenal involvement. The higher production of Th1 cytokines by LIV-A in comparison to HIV-A mice may account for LIV-A resistance to P. brasiliensis infection. Our data reveal the importance of this experimental model in the study of the adrenal involvement in paracoccidioidomycosis, since this gland may be highly compromised in the patients, leading to the development of Addison's Disease.

Th1 polarized response induced by intramuscular DNA-HSP65 immunization is preserved in experimental atherosclerosis

We previously reported that a DNA vaccine constructed with the heat shock protein (HSP65) gene from Mycobacterium leprae (DNA-HSP65) was protective and also therapeutic in experimental tuberculosis. By the intramuscular route, this vaccine elicited a predominant Th1 response that was consistent with its protective efficacy against tuberculosis. It has been suggested that the immune response to Hsp60/65 may be the link between exposure to microorganisms and increased cardiovascular risk. Additionally, the high cholesterol levels found in atherosclerosis could modulate host immunity. In this context, we evaluated if an atherogenic diet could modulate the immune response induced by the DNA-HSP65 vaccine. C57BL/6 mice (4-6 animals per group) were initially submitted to a protocol of atherosclerosis induction and then immunized by the intramuscular or intradermal route with 4 doses of 100 µg DNA-HSP65. on day 150 (15 days after the last immunization), the animals were sacrificed and antibodies and cytokines were determined. Vaccination by the intramuscular route induced high levels of anti-Hsp65 IgG2a antibodies, but not anti-Hsp65 IgG1 antibodies and a significant production of IL-6, IFN-g and IL-10, but not IL-5, indicating a Th1 profile. Immunization by the intradermal route triggered a mixed pattern (Th1/Th2) characterized by synthesis of anti-Hsp65 IgG2a and IgG1 antibodies and production of high levels of IL-5, IL-6, IL-10, and IFN-g. These results indicate that experimentally induced atherosclerosis did not affect the ability of DNA-HSP65 to induce a predominant Th1 response that is potentially protective against tuberculosis.

Interleukin-15 augments oxidative metabolism and fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tobacco Smoke Induces Ventricular Remodeling Associated with an Increase in NADPH Oxidase Activity

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Modulatory effects of tuberculosis vaccines on experimental autoimmune encephalomyelitis

Although BCG is the only accepted vaccine against tuberculosis (TB), its protective ability is very limited. Therefore, many new vaccines are being evaluated. Our group has been working on DNAhsp65 - a genetic construction containing the hsp65 gene from Mycobacterium leprae. In previous experimental works, we demonstrated that both DNAhsp65 alone or associated with BCG, in a prime-boost regimen, were effective to control TB. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 alone or associated with BCG in a rat model of multiple sclerosis. Female Lewis rats were immunized with three doses of DNAhsp65 or primed with BCG followed by two DNAhsp65 boosters. The animals were, then, immunized with myelin associated with complete Freund's adjuvant to develop experimental autoimmune encephalomyelitis (EAE). The following parameters were evaluated: weight loss, clinical score, central nervous system (CNS) inflammation and anti-myelin immune response. No deleterious effect was associated with these immunizations schedules. Immunized animals equally lost weight, the clinical scores were similar and CNS inflammation did not increase. Interestingly, both procedures determined decreased inflammation in the brain and lumbar spinal cord. This was concurrent with a modulatory effect over cytokine production by peripheral lymphoid organs. Cell cultures from spleen and lymph nodes in vitro stimulated with myelin produced less IFN-gamma and IL-10, respectively. This phenomenon was more clear in rats immunized with the genetic vaccine alone than with the prime-boost strategy. Together the results suggest that these strategies for TB prophylaxis would not accelerate or aggravate multiple sclerosis, being therefore, safe in this aspect. In addition, they indicate that these vaccination regimens have a potential anti-inflammatory activity that could be better explored in the future.

Th1/Th2 cytokines' expression and production by propolis-treated mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The inhibitory effect of MK886 on the inflammatory process caused by Paracoccidioidomycosis brasiliensis infection in genetically selected mice

Leukotrienes are classic inflammatory response mediators considered chemotactic agents and microbicidal activity regulators in cells of the innate immune system, playing a protective role against different infectious agents. In this study, we investigated the involvement of leukotrienes in the course of murine paracoccidioidomycosis based on the following immunologic parameters: cell influx, mieloperoxydase activity, NO production, cytokine production, and fungal recovery in lungs of mice selected according to the intensity of their low (AIRmin) and high (AIRmax) acute inflammatory response. Infection by P. brasiliensis induced considerable production of IL-6, IL-10, IFN-gamma and TNF-alpha cytokines, and led to cell recruitment, as well as NO production in lungs at different study periods. In animals treated with MK886, a leukotriene biosynthesis inhibitor, IFN-gamma, IL-6 and TNF-alpha production was lower, while neutrophil influx and NO production decreased. These results may explain the higher fungal load in lungs of animals in which leukotriene synthesis was inhibited, suggesting that leukotrienes have a possible protective role in experimental paracoccidioidomycosis. AIRmax animals had lower fungal load in comparison with AIRmin ones, which can be related to the AIR phenotype regarding neutrophil migration, besides lower production of NO and pro-inflammatory cytokines. Thus, mice presenting AIRmax background are more resistant to infection by P. brasiliensis.

Clove and eugenol in noncytotoxic concentrations exert immunomodulatory/anti-inflammatory action on cytokine production by murine macrophages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytokine production in experimental paracoccidioidomycosis of murine selected lineages for acute inflammatory response (air)

Paracoccidioidomycosis is a systemic human mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), an imperfect dimorphic fungus whose conidia are its infective form. Mice genetically selected for maximum (AIRmax) and minimum (AIRmin) acute inflammatory response were used as experimental paracoccidioidomycosis models. The animals were intraperitoneally inoculated with P. brasiliensis (strain 18) and killed 6, 12 and 24 hours or 3, 7 and 14 days after infection. In these periods, fragments from their spleen, liver and lung were removed for evaluation of the infection level by fungal cells, assessment of macrophagic activity by peritoneal and splenic macrophages - through the determination of nitric oxide (NO) concentrations and production of pro- and anti-inflammatory cytokines of lung and spleen homogenate supernatants. In the present study, it was observed that AIRmax lineages presented greater control of the infectious process than the AIRmin ones. Regarding NO production, AIRmax animals produced more metabolites in late periods, what may help control the infectious process. Concerning cytokine production, it was observed that the production of INF-gamma, TNF-alpha, IL-1, IL-6, IL-8 and IL-12 were increased in AIRmax lineages in most analyzed organs and periods, thus contributing to the greater resistance exhibited by such lineages against infection, except for IL-4 and IL-10 that showed decreased production in AIRmax lineage, reproducing its suppressive biological effect. From these results, it was observed that the AIRmax lineage was more effective in controlling the infectious process, with an important involvement of the analyzed cytokines. These findings are probably related to the genetically selected factors involved in the acute inflammatory response.

ROLE of TLR2 and TLR4 on human neutrophil functions against Paracoccidioides brasiliensis

In paracoccidioidomycosis, a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), studies have focused on the role of neutrophils that are involved in the primary response to the fungus. Neutrophil functions are regulated by pro- and anti-inflammatory cytokines. Molecular mechanisms involved in this process are not fully understood, but there are strong evidences about the involvement of toll-like receptors (TLRs). We aimed at evaluating TLR2 and TLR4 expression on human neutrophils activated by GM-CSF, IL-15, TNF-alpha or IFNgamma and challenged with a virulent strain of P. brasiliensis (Pb18). Moreover, we asked if these receptors have a role on fungicidal activity, H(2)O(2) and IL-6, IL-8, TNFalpha and IL-10 production, by activating and challenging cells. All cytokines increased TLR2 and TLR4 expression. Pb18 also increased TLR2 expression, inducing an additional cytokine effect. on the contrary, it inhibited TLR4 expression. All cytokines increased neutrophil fungicidal activity and H(2)O(2) production; however, this process was not associated with TLR2 or TLR4. Neutrophil activation by GMCSF and TNF-alpha resulted in a significant increase of IL-8 production, while IL-15 and IFN-alpha have no effect. Pb18 also augmented IL-8 expression, inducing an additional effect to that of cytokines. None of the cytokines activated neutrophils by releasing IL-10. This cytokine was only detected after Pb18 challenge. Interestingly, IL-8 and IL-10 production involved TLR2 and mainly TLR4 modulation. The present results suggest that Pb18 interaction with neutrophils through TLR2 and TLR4 with consequent IL-8 and IL-10 production may be considered a pathogenic mechanism in paracoccidioidomycosis.