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.

Platelet-activating factor modulates a secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum.

In the present work we characterized the secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This housefly parasite hydrolyzed p-nitrophenylphosphate at a rate of 10.26 nmol Pi/mg protein/min. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate promoted a decrease in this phosphatase activity. When the parasites were assayed in the presence of sodium tartrate, an inhibitor of Leishmania spp-secreted acid phosphatases, this activity was drastically diminished. Cytochemical analysis showed the localization of this enzyme on the external surface and in the flagellar pocket of these parasites. Sodium tartrate inhibited this reaction, confirming the biochemical data. Platelet-activating factor (PAF) inhibited the phosphatase activity determined in the supernatant of living H. m. muscarum.

Platelet-activating factor (PAF) modulates peritoneal mouse macrophage infection by Leishmania amazonensis.

The effects of platelet-activating factor (PAF) on the infection of peritoneal mouse macrophages by Leishmania amazonensis were investigated. Prior to the infection, the parasites and/or the macrophages were treated with PAF and/or one of the following modulators: WEB 2086 (PAF antagonist), and the modulators of protein kinase C, phorbol-12-myristate-13-acetate (PMA), and sphingosine. The infection was inhibited when the macrophages or both the parasites and the macrophages were treated with PAF, but stimulated by PAF-treated parasites. WEB 2086 abrogated PAF effects in both systems. The infection was stimulated when the macrophages were treated with sphingosine plus PAF, but inhibited when the macrophages were treated with sphingosine and the parasites with sphingosine plus PAF. The infection was inhibited by sphingosine-treated parasites, either in the presence or in the absence of PAF. Leishmania amazonensis-macrophage infection was inhibited by PMA in all systems tested.

Secreted phosphatase activities in trypanosomatid parasites of plants modulated by platelet-activating factor.

ABSTRACT The secreted phosphatase activities of two trypanosomatid parasites were characterized and compared with supernatants of living cells. The plant parasite Phytomonas françai and the phytophagous hemipteran parasite Herpetomonas sp. hydrolyzed p-nitrophenylphosphate at a rate of 15.54 and 6.51 nmol Pi/mg of protein per min, respectively. Sodium orthovanadate (N(a)VO(3)) and sodium fluoride (NaF) decreased the phosphatase activities. The phosphatase activity of P. françai was drastically diminished (73% inhibition) in the presence of sodium tartrate, whereas the phosphatase activity of Herpetomonas sp. was inhibited by 23%. Cytochemical analysis showed the localization of these enzymes on the external surface and in the flagellar pocket of the two trypanosomatids. Sodium tartrate inhibited this reaction, confirming the biochemical data. Platelet-activating factor modulated the phosphatase activities, inhibiting P. françai activity and stimulating Herpetomonas sp. phosphatase activity.

Characterization of the intracellular Ca(2+) pools involved in the calcium homeostasis in Herpetomonas sp. promastigotes.

Trypanosomatids of the genus Herpetomonas comprises monoxenic parasites of insects that present pro- and opisthomastigotes forms in their life cycles. In this study, we investigated the Ca(2+) transport and the mitochondrial bioenergetic of digitonin-permeabilized Herpetomonas sp. promastigotes. The response of promastigotes mitochondrial membrane potential to ADP, oligomycin, Ca(2+), and antimycin A indicates that these mitochondria behave similarly to vertebrate and Trypanosoma cruzi mitochondria regarding the properties of their electrochemical proton gradient. Ca(2+) transport by permeabilized cells appears to be performed mainly by the mitochondria. Unlike T. cruzi, it was not possible to observe Ca(2+) release from Herpetomonas sp. mitochondria, probably due to the simultaneous Ca(2+) uptake by the endoplasmic reticulum. In addition, a vanadate-sensitive Ca(2+) transport system, attributed to the endoplasmic reticulum, was also detected. Nigericin (1 microM), FCCP (1 microM), or bafilomycin A(1) (5 microM) had no effect on the vanadate-sensitive Ca(2+) transport. These data suggest the absence of a Ca(2+) transport mediated by a Ca(2+)/H(+) antiport. No evidence of a third Ca(2+) compartment with the characteristics of the acidocalcisomes described by A. E. Vercesi et al. (1994, Biochem. J. 304, 227-233) was observed. Thapsigargin and IP(3) were not able to affect the vanadate-sensitive Ca(2+) transport. Ruthenium red was able to inhibit the Ca(2+) uniport of mitochondria, inducing a slow mitochondrial Ca(2+) efflux, compatible with the presence of a Ca(2+)/H(+) antiport. Moreover, this efflux was not stimulated by the addition of NaCl, which suggests the absence of a Ca(2+)/Na(+) antiport in mitochondria.

Characterization of ectophosphatase activities in trypanosomatid parasites of plants.

ABSTRACT In the present work ectophosphatase activities of three trypanosomatid parasites of plants were characterized using intact cells. Phytomonas françai, Phytomonas mcgheei, and Herpetomonas sp. hydrolyzed p-nitro-phenylphosphate at a rate of 5.40, 7.28, and 25.58 nmol Pi/mg of protein per min, respectively. Experiments using classical inhibitors of acid phosphatases such as sodium orthovanadate (NaVO(3)) and sodium fluoride (NaF) showed a decrease in phosphatase activities. Lithium fluoride (LiF) and aluminum chloride (AlCl(3)) were also used. Although AlCl3 had no effect, LiF was able to promote a decrease in the phosphatase activities. Interestingly, the inhibition caused by LiF was enhanced by the addition of AlCl3 during the reaction, probably due to the formation of fluoroaluminate complexes. This effect was confirmed by cytochemical analysis. In this assay, electron-dense cerium phosphate deposits were visualized on the external surface of the three parasites.

Platelet-activating factor induction of secreted phosphatase activity in Trypanosoma cruzi.

The effects of platelet-activating factor (PAF) on the ecto-phosphatase activity of Trypanosoma cruzi were investigated. Living parasites hydrolyzed p-nitrophenyl phosphate (p-NPP) at a rate of 5.71 +/- 0.37 nmol P(i) mg(-1) min(-1). This ecto-phosphatase activity increased to 8.70 +/- 1.12 nmol P(i) mg(-1) min(-1) when the cells were grown in the presence of 10(-9) M PAF. This effect was probably due to stimulation of the release of the ecto-phosphatase and/or the secretion of an intracellular phosphatase to the extracellular medium, as suggested by cytochemical analysis. Modulation of the ecto-phosphatase activity was also observed when PAF was added during the time course of the reaction. WEB 2086, a competitive PAF antagonist, was able to revert PAF effects when both were used at the same concentration. When PAF was added to a membrane enriched fraction preparation of T. cruzi, no alteration on the phosphatase activity was observed. This result suggests an involvement of intracellular signaling, as PAF was only effective on intact cells. Sphingosine and phorbol-12-myristate-13-acetate (PMA) were then used to investigate a possible involvement of protein kinase C (PKC) with PAF-induced phosphatase secretion. Sphingosine by itself stimulated the secretion of a phosphatase but did not significantly interfere with PAF effects on this enzyme. On the other hand, PMA was able to abrogate PAF-induced release of this phosphatase. These data are highly suggestive of a putative involvement of signal transduction mediated by a ligand of mammalian origin (PAF), through PKC and a specific receptor located on the cell surface of the human parasite Trypanosoma cruzi.

A novel ecto-phosphatase activity of Herpetomonas muscarum muscarum inhibited by platelet-activating factor.

In the present work ecto-phosphatase activity in Herpetomonas muscarum muscarum has been characterized using live parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 4.27 nmol Pi/mg of protein.min. A pH curve was generated, in which these intact flagellates showed the highest phosphatase activity at pH 6.5. Classical inhibitors for acid phosphatase, such as sodium orthovanadate, sodium tartrate, and ammonium molybdate, were used in the experiments and showed different patterns of inhibition. Lithium fluoride, aluminum chloride, and fluoroaluminate complexes were also tested. Although lithium fluoride and fluoroaluminate complexes were capable of inhibiting the phosphatase activity, aluminum chloride stimulated this enzyme. Cytochemical analysis showed the localization of this enzyme on the parasite surface. This ecto-phosphatase activity was also significantly diminished when the parasites were treated with 10(-6) M platelet-activating factor (PAF), a potent phospholipid mediator that promoted cellular differentiation in this parasite.

Effect of platelet-activating factor on the process of cellular differentiation of Herpetomonas muscarum muscarum.

The effects of platelet-activating factor (PAF), at doses ranging from 10(-6) M to 10(-10) M, on cell growth and on cell differentiation of Herpetomonas muscarum muscarum were investigated. Cell differentiation was evaluated by both light and electron microscopy. At the concentrations used, PAF did not interfere with the protozoan growth. However, parasites grown in the presence of PAF (10(-6) M) were significantly more differentiated than those grown in the absence of PAF, since the first day of culture. On the first two days of culture, PAF doses ranging from 10(-10) M to 10(-7) M, did not significantly interfere with the differentiation of these parasites, although after the third day of culture, all PAF doses used significantly increased the protozoan differentiation. Specific PAF receptor antagonists totally abrogated (WEB 2086 and WEB 2170) or significantly decreased (BN 52021) PAF effect on cell differentiation. These findings indicate PAF triggers the process of cell differentiation in Herpetomonas muscarum muscarum and suggest these parasites have receptors for PAF.

Mg-dependent ecto-ATPase activity in Leishmania tropica.

ATPase activity has been located on the external surface of Leishmania tropica. Since Leishmania is known to have an ecto-acid phosphatase, in order to discard the possibility that the ATP hydrolysis observed was due to the acid phosphatase activity, the effect of pH in both activities was examined. In the pH range from 6.8 to 8.4, in which the cells were viable, the phosphatase activity decreased, while the ecto-ATPase activity increased. To confirm that the observed ATP hydrolysis was promoted by neither phosphatase nor 5'-nucleotidase activities, a few inhibitors for these enzymes were tested. Vanadate and NaF strongly inhibited the phosphatase activity; however, no effect was observed on ATPase activity. Neither levamizole nor tetramizole, two specific inhibitors of alkaline phosphatases, inhibited this activity. The lack of response to ammonium molybdate indicated that 5'-nucleotidase did not contribute to the ATP hydrolysis. Also, the lack of inhibition of the ATP hydrolysis by high concentrations of ADP at nonsaturating concentrations of ATP discarded the possibility of any ATP diphosphohydrolase activity. The ATPase here described was stimulated by MgCl2 but not by CaCl2. In the absence of divalent metal, a low level of ATP hydrolysis was observed, and CaCl2 varying from 0.1 to 10 mM did not increase the ATPase activity. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.29 +/- 0.02 mM MgCl2. The apparent K(m) for Mg-ATP2- was 0.13 +/- 0.01 mM and free Mg2+ did not increase the ATPase activity. ATP was the best substrate for this enzyme. Other nucleotides such as ITP, CTP, GTP, UTP, and ADP produced lower reaction rates. To confirm that this Mg-dependent ATPase was an ecto-ATPase, an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid was used. This amino/sulfhydryl-reactive reagent did inhibit the Mg-ecto-ATPase activity in a dose-dependent manner (I0.5 = 27.5 +/- 1.8 microM).

Effect of platelet-activating factor on cell differentiation of Trypanosoma cruzi.

The effects of platelet-activating factor (PAF), at (10)-6M and (10)-9M, on cell growth and cell differentiation of Trypanosoma cruzi were investigated. Cell differentiation was evaluated by both light and electron microscopy. At the concentrations used, PAF slightly interfered with the protozoan growth. However, parasites growth in the presence of PAF were significantly more differentiated than those grown in the absence of PAF, beginning on the fourth day of culture. A specific PAF receptor antagonist (WEB 2086) totally abrogated PAF effect on cell differentiation. These findings indicate that PAF triggers the process of cell differentiation in T. cruzi and suggest that these parasites have receptors for PAF.