Chronic Aerobic Training at Different Volumes in the Modulation of Macrophage Function and in vivo Infection of BALB/c Mice by Leishmania major.

Physical inactivity is one of the main causes of chronic diseases; however, strenuous exercise can induce immunosuppression. Several studies suggest that moderate amounts of exercise lead to a Th1 response, favoring the resolution of infections caused by intracellular microorganisms, while high volumes of exercise tend to direct the response to Th2, favoring infection by them. Leishmaniasis is a parasitic disease promoted by parasites of the Leishmania genus, with clinical manifestations that vary according to the species of the parasite and the immune response of the host. The experimental Leishmania major-BALB/C mouse model provides a good model for the resistance (Th1 response) or susceptibility (Th2 response) that determines the progression of this infection. The aim of this study was to evaluate the effect of aerobic training at different volumes on modulation of in vitro macrophage infection by L. major, as well as to assess the effect of high volume (HV) aerobic training on the development of L. major in vivo in BALB/c mice. Uninfected animals were submitted to various exercise volumes: none (SED), light (LV), moderate (MV), high (HV), very high (VHV), and tapering (TAP). The macrophages of these animals were infected by L. major and the LV and MV groups showed a decrease in the infection factor, while the VHV showed an increase in the infection factor, when treated with LPS. The cytokine concentration pattern measured in the supernatants of these macrophages suggested a predominant Th1 response profile in the LV and MV groups, while the Th2 profile predominated in the VHV and TAP groups. Groups of BALB/C mice infected with L. major were subjected to high volume (iHV) or non-periodized high volume (iNPHV) exercise or kept sedentary (iSED). The exercised animals suffered a significant increase in injuries caused by the parasites. The animals in the group submitted to high volume exercise (iHV) showed visceralization of the infection. These data strongly suggest that a very high volume of aerobic training increased the susceptibility of BALB/C mice to L. major infection, while moderate distribution of training loads promoted immunological balance, better controlling the infection by this parasite.

Exercise improves the Th1 response by modulating cytokine and NO production in BALB/c mice.

Physical exercise can improve health and may lead to changes in the functionality of the immune system. Moderate intensity exercise can reduce the risk of infection by shifting the overall immune response towards a T helper type 1 pattern. This study investigates the effect of 12 weeks of swimming on the cytokine profile of lymph node cells and macrophages and of the nitric oxide production by these cells. BALB/c mice were divided into 2 groups. The exercise group was subjected to swimming exercise. Lymph node cells culture showed that concentrations of interferon-γ and tumour necrosis factor-α were higher in the exercised group, while levels of interleukine-4 and interleukine-10 were significantly decreased in this group. The interleukine-10/interferon-γ ratio tended towards a T helper type 1 profile. Moreover, macrophages isolated from exercised mice produced more interleukine-12 and tumour necrosis factor-α following lipopolysaccharide stimulus. Challenging these macrophages with Leishmania major resulted in higher interleukine-12 production than was observed with macrophages from the control group. Nitric oxide production was increased in macrophages isolated from exercised group following lipopolysaccharide stimulus but not following infection with Leishmania major. These data suggest that exercise biases the immune system towards a T helper type 1 response profile.

Dissimilar peptidase production by avirulent and virulent promastigotes of Leishmania braziliensis: inference on the parasite proliferation and interaction with macrophages.

In the present paper, we have analysed the cellular and extracellular proteolytic activity profiles in 2 distinct Leishmania braziliensis strains: a recently isolated (virulent) and a laboratory-adapted (avirulent) strain. Quantitative and qualitative differences on the peptidase expression were observed in both strains. For instance, low-molecular mass acidic cysteine peptidase activities were detected exclusively in the virulent strain. Similarly, metallopeptidase activities were mainly produced by L. braziliensis virulent promastigotes. Interestingly, metallo- and cysteine peptidase activities were drastically reduced after several in vitro passages of the virulent strain. Western blotting, flow cytometry and fluorescence microscopy analyses were performed to detect homologous of the major leishmania metallopeptidase (gp63) and cysteine peptidase (cpb) in virulent and avirulent strains of L. braziliensis. Our results revealed that the virulent strain produced higher amounts of gp63 and cpb molecules, detected both in the surface and cytoplasm regions, than the avirulent counterpart. Metallo- (1,10-phenanthroline and EGTA) and cysteine peptidase (E-64) inhibitors arrested the growth of L. braziliensis virulent strain in a dose-dependent manner, as well as the association index with peritoneal murine macrophages. Conversely, these peptidase inhibitors did not affect either the proliferation or the cellular interaction of the avirulent strain. Corroborating these findings, the pre-treatment of the virulent strain with both anti-peptidase antibodies promoted a prominent reduction in the interaction with macrophages, while the association index of the avirulent strain to macrophage was only slightly diminished. Moreover, the spent culture medium from virulent strain significantly enhanced the association index between avirulent strain and macrophages, and this effect was reversed by 1,10-phenanthroline. Collectively, the results presented herein suggest that peptidases participate in several crucial processes of L. braziliensis.

Characterization of Ca2+ uptake in a subcellular membrane fraction of Herpetomonas sp. promastigotes.

ATP-dependent Ca2+ uptake was studied in a subcellular fraction from Herpetomonas sp. prepared by mechanical disruption and using 45Ca2+ as a tracer. The uptake was stimulated by Ca2+ with a K0.5 of 0.1 microm and a Hill number (nH)=2.8+/-0.4. The Ca2+-dependent ATP hydrolysis was optimal at pH 7.0 and had a Ca2+ dependence identical to uptake. The uptake was highly stimulated by oxalate whereas calmodulin had no activating effect. ATP stimulated Ca2+ uptake with a biphasic pattern that resembled the curves described for the purified preparations of rabbit sarcoplasmic reticulum. The ATP stimulation is described as the sum of two Michaelis-Menten curves with Km1=0.25+/-0.19 microm and Km2=29.6+/-6.8 microm. GTP or UTP could also promote Ca2+ uptake, but with less efficiency than ATP. Vanadate inhibited the uptake with low apparent affinity. Thapsigargin and cyclopiazonic acid were almost ineffective. The Ca2+ uptake was insensitive to H+ ionophores and to bafilomycin suggesting no participation of acidocalcisomes. The results are comparable to those obtained using cells permeabilized with digitonin and using arsenaze III as Ca2+ indicator. The Ca2+ uptake activity described here seems to belong to the endoplasmic reticulum of Herpetomonas sp. and is suitable for further studies on the mechanisms of calcium homeostasis in parasites.

Characterization of ecto-phosphatase activities of Trypanosoma cruzi: a comparative study between Colombiana and Y strains.

The etiological agent of Chagas disease, Trypanosoma cruzi, is consisted of two phylogenetic lineages. Using live epimastigotes, in this study we have characterized ecto-phosphatase activities of two strains of T. cruzi, one (Y strain) is a member of group T. cruzi I and the other (Colombiana) is a member of group T. cruzi II. About one-third of the total ecto-phosphatase activity from the Y strain was Mg(2+)-dependent, but no such activity was observed with Colombiana. The level of Mg(2+)-independent activity was dramatically different in the two strains, with Colombiana showing more than 15-fold higher activity. Experiments using classical inhibitors of acid phosphatases, as well as inhibitors of phosphotyrosine phosphatase, showed a decrease in these phosphatase activities, with different patterns of inhibition. The Mg(2+)-independent activities of the Colombiana and Y strains decreased inversely with pH, varying from 6.5 to 8.0. On the other hand, the Mg(2+)-dependent activity of the Y strain increased concomitantly with the increase in pH in the same range.

Magnesium-dependent ecto-ATP diphosphohydrolase activity in Herpetomonas muscarum muscarum.

In the present work we characterized the ecto-ATP diphosphohydrolase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This parasite hydrolyzed ATP at a rate of 15.52 nmol Pi/mg protein/min and this activity reached a maximum at pH 7.5. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate presented no effect on this activity. MgCl2, ZnCl2, and MnCl2 stimulated the ATP hydrolysis by H. m. muscarum. The ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase, bafilomycin A1, a V-ATPase inhibitor, ouabain, a Na(+)+K+-ATPase inhibitor and to levamizole, an inhibitor of alkaline phosphatase. An extracellular impermeant inhibitor 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid (DIDS) and a inhibitor of some ecto-ATPases, suramin, which is also a competitive antagonist of P2-purinergic receptors, promoted a great inhibition on the ATP hydrolysis. This enzyme is able to hydrolysis ATP, ADP, UTP, and UDP, but not GTP, GDP, CTP, or CDP. ADP inhibited the enzymatic activity in a concentration dependent manner, reaching 70% inhibition.