Markers of oxidative stress in adipose tissue during Trypanosoma cruzi infection

The protozoan parasite Trypanosoma cruzi causes Chagas disease. Cardiac and adipose tissues are among the early targets of infection and are sites of persistent infection. In the heart and adipose tissue, T. cruzi infection results in an upregulation of pro-inflammatory mediators. In the heart, infection is associated with an increase in the markers of oxidative stress. To date, markers of oxidative stress have not been evaluated in adipose tissue in this infection. Brown and white adipose tissues were obtained from CD-1 mice infected with the Brazil strain of T. cruzi for 15, 30, and 130 days post infection. Protein carbonylation and lipid peroxidation assays were performed on these samples. There was an upregulation of these markers of oxidative stress at all time-points in both white and brown adipose tissue. Determinants of anti-oxidative stress were downregulated at similar time-points. This increase in oxidative stress during T. cruzi infection most likely has a deleterious effect on host metabolism and on the heart.     

Mouse IgE response against exoantigens of Trypanosoma cruzi

This work describes the occurrence of anaphylactic antibodies against exoantigens of Trypanosoma cruzi in infected or immunized mice. The results obtained show that the exoantigens induce an IgE response when injected into mice with complete Freund's adjuvant or in animals infected with 10(2) trypomastigotes. Among the antigens recognized by anti-exoantigen IgE, two molecules of 27 and 45 kD were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by western blotting. Immunofluorescence analysis located some target antigens on the parasite surface.     

Antioxidant response to oxidative stress induced by exhaustive exercise

The aim of this work was to demonstrate the occurrence of oxidative stress during exhaustive exercise and to determine the antioxidant response. Eight voluntary male subjects participated in this study. The exercise was a cycling mountain stage (171 km) and the cyclists took a mean+/-S.E.M. time of 270+/-12 min to complete it. Blood samples were taken before the cycling stage, immediately after the stage, 3 h after finishing the stage and on the morning of the following day. We determined the activities of erythrocyte antioxidant enzymes, blood levels of oxidised glutathione, plasma levels of antioxidant vitamins and carotenoids, and the serum lipid and cholesterol profile. The mountain cycling stage induced significant increases in catalase and glutathione reductase activities. Significant decreases in glutathione peroxidase activity, both determined with hydrogen peroxide and with cumene hydroperoxide as substrates, were observed. Blood oxidised glutathione and serum uric acid rose after the stage. Plasma vitamin E increased after the stage but dropped to below basal values after 3 h of recovery. Triglycerides and VLDL-cholesterol increased significantly after the stage and remained high 3 h after the cycling stage. The mountain cycling stage induced oxidative stress, as was evidenced by the increases in blood GSSG and in serum urate concentrations and by the pattern of change of erythrocyte antioxidant enzyme activities. A specific utilisation of alpha-tocopherol against oxidative stress during recovery was evidenced.     

Cell culture models for oxidative stress: superoxide and hydrogen peroxide versus normobaric hyperoxia

According to the free radical theory of aging, loss of cellular function during aging is a consequence of accumulating subcellular damage inflicted by activated oxygen species. In cells, the deleterious effects of activated oxygen species may become manifest when the balance between radical formation and destruction (removal) is disturbed creating a situation denoted as ‘oxidative stress’. Cell culture systems are especially useful to study the effects of oxidative stress, in terms of both toxicity and cellular adaptive responses. A better understanding of such processes may be pertinent to fully comprehend the cellular aging process.This article reviews three model systems for oxidative stress: extracellular sources of O₂⁻ and H₂O₂, and normobaric hyperoxia (elevated ambient oxygen). Methodological and practical aspects of these exposure models are discussed, as well as their prominent effects as observed in cultures of Chinese hamster cell lines. Since chronic exposure models are to be preferred, it is argued that normobaric hyperoxia is a particularly relevant oxidative stress model for in vitro cellular aging studies.     

The glucose-6-phosphate dehydrogenase from Trypanosoma cruzi: its role in the defense of the parasite against oxidative stress

The Trypanosoma cruzi glucose-6-phosphate dehydrogenase (G6PDH) is encoded by several genes located in three of the parasite chromosomes. All the sequences present two possible start codons, 111bp apart, also present in its Trypanosoma brucei counterpart. As the 37 residues comprised between the two candidate initiator methionines of T. brucei and T. cruzi G6PDHs constitute an unusual N-terminal extension only present in trypanosomatids, two forms of the T. cruzi G6PDH were expressed in Escherichia coli: a long one (Tc-G6PDH-L) translated from the first ATG codon, and a short one (Tc-G6PDH-S) translated from the second. Both were purified and their kinetic constants determined. The apparent K(m) for glucose-6-phosphate was 189.9, 98.4, and 288microM, for Tc-G6PDH-L, Tc-G6PDH-S and native Tc-G6PDH, respectively. The apparent K(m) for NADP was similar for both recombinant proteins. The Tc-G6PDH-L as well as the native enzyme, was inactivated by DTT while the Tc-G6PDH-S was unaffected by the reducing agent. This behavior could be related to the presence of two Cys groups in the N-terminal extension of the Tc-G6PDH-L similarly to the redox regulated G6PDHs from chloroplasts and cyanobacteria. This property, together with a remarkable induction (up to 46-fold) of the T. cruzi G6PDH in metacyclic trypomastigotes under oxidative stress conditions, suggests that the enzyme may play a prominent role in the defense mechanisms of the parasite against oxidative stress becoming an important target for chemotherapy. Western blots using antibodies against the N-terminal extension in Tc-G6PDH-L show that this form is expressed in the parasite.     

A cardiac myosin-specific autoimmune response is induced by immunization with Trypanosoma cruzi proteins

Trypanosoma cruzi is the protozoan parasite that causes Chagas' heart disease, a potentially fatal cardiomyopathy prevalent in Central and South America. Infection with T. cruzi induces cardiac myosin autoimmunity in susceptible humans and mice, and this autoimmunity has been suggested to contribute to cardiac inflammation. To address how T. cruzi induces cardiac myosin autoimmunity, we investigated whether immunity to T. cruzi antigens could induce cardiac myosin-specific autoimmunity in the absence of live parasites. We immunized A/J mice with a T. cruzi Brazil-derived protein extract emulsified in complete Freund's adjuvant and found that these mice developed cardiac myosin-specific delayed-type hypersensitivity (DTH) and autoantibodies in the absence of detectable cardiac damage. The induction of autoimmunity was specific since immunization with extracts of the related protozoan parasite Leishmania amazonensis did not induce myosin autoimmunity. The immunogenetic makeup of the host was important for this response, since C57BL/6 mice did not develop cardiac myosin DTH upon immunization with T. cruzi extract. Perhaps more interesting, mice immunized with cardiac myosin developed T. cruzi-specific DTH and antibodies. This DTH was also antigen specific, since immunization with skeletal myosin and myoglobin did not induce T. cruzi-specific immunity. These results suggest that immunization with cardiac myosin or T. cruzi antigen can induce specific, bidirectionally cross-reactive immune responses in the absence of detectable cardiac damage.     

Pregnancy and humoral immune response in mice chronically infected by Trypanosoma cruzi.

The effect of pregnancy on the humoral immune response induced by Trypanosoma cruzi was studied in groups of chronically infected and pregnant mice (IP) or chronically infected and nonpregnant mice (INP) of strain BALB/c. Groups of noninfected and nonpregnant mice (NINP) or noninfected and pregnant mice (NIP) served as controls. The pregnant mice were killed on day 17 of pregnancy. Anti-T. cruzi immunoglobulin G (IgG) and IgM antibodies, detected by immunofluorescence or enzyme-linked immunosorbent assay or both, underwent a pregnancy-associated decrease of 20 to 40%, whereas complement-mediated lytic antibodies were unaffected by pregnancy. Immunoblotting analysis indicated identical specificities of the anti-T. cruzi antibodies in IP and INP groups. The levels of all the immunoglobulin isotypes (particularly IgG2a and IgG3), circulating immune complexes, rheumatoid-like factor, and anti-DNA antibodies were considerably increased during chronic infection (NINP versus INP), which could be related to the high degree of polyclonal B-cell activation occurring in T. cruzi infection. However, pregnancy significantly decreased (by 20 to 60%) such parameters. IgG levels were particularly affected (by 40 to 60%), and the decreases could be ordered as follows: IgG3 greater than IgG2a greater than IgG1 greater than IgG2b for IP versus INP. Comparisons between the noninfected groups indicated differences only in IgG levels. These results indicate the following. (i) The specific humoral anti-T. cruzi immune response is weakly affected by pregnancy, which is not sufficient to modify the course of the mother's infection. (ii) Pregnancy does not modify the expression of the anti-T. cruzi antibody repertory. (iii) Pregnancy reduces the polyclonal B-cell activation, particularly the levels of the IgG isotypes undergoing the greatest activation.     

枸杞多糖减轻过氧化氢诱导的人内皮样细胞EA.hy926氧化损伤

目的:研究枸杞多糖(LBP)对过氧化氢(H_2O_2)诱导人内皮样细胞EA.hy926氧化损伤的作用及机制。方法:用H_2O_2建立EA.hy926细胞氧化损伤模型,细胞共分为5个组:正常对照(control)组、模型(damage)组(H_2O_2,50 mmol/L)、LBP(100 mg/L)组、抗损伤(anti-damage)组(50 mg/L、100 mg/L和200 mg/L LBP+50 mmol/L H_2O_2)和LY294002(20μmol/L)组。采用CCK-8法检测细胞的活力;用Annexin V/PI双染法流式细胞术检测不同处理后EA.hy926细胞的凋亡;吖啶橙(AO)/溴乙啶(EB)染色法观察细胞凋亡的形态特征;划痕法测定细胞的迁移能力;一氧化氮(NO)试剂盒检测细胞培养上清中NO的水平;用ELISA法测定血管内皮生长因子(VEGF)的水平;Western blot检测cleaved caspase-3、Bax、Bcl-2、内皮型NO合酶(eNOS)、p-eNOS和p-Akt的蛋白水平。结果:量效结果显示浓度为100 mg/L的LBP预处理EA.hy926细胞1 h,然后加入H_2O_2(50 mmol/L)处理24 h对减轻H_2O_2诱导的EA.hy926细胞损伤的作用最显著。与damage组比较,LBP预处理可提高EA.hy926细胞的活力(P0.05),减少细胞凋亡(P0.05),并促进细胞迁移;上清液中VEGF和NO水平升高;Bcl-2/Bax比率明显升高,下调cleaved caspase-3蛋白水平,并上调eNOS和p-eNOS蛋白水平。此外,加入PI3K抑制剂LY294002后,LBP对H_2O_2损伤的EA.hy926细胞的保护作用减弱,表现为NO水平和p-Akt蛋白水平降低。结论:LBP能减轻H_2O_2对EA.hy926细胞的损伤作用,缓解H_2O_2诱导的凋亡,其机制与激活PI3K/Akt/eNOS信号通路密切相关。     

Adaptation to hydrogen peroxide enhances PC12 cell tolerance against oxidative damage

The adaptive responses to H2O2 and the resulting protective effect against oxidative stress have been investigated using PC12 cells. Pretreatment of sublethal doses of H2O2 significantly protected PC12 cells against the cytotoxicity induced by lethal H2O2. The endogenous antioxidant defense systems, catalase and glutathione peroxidase, were enhanced quickly by the pretreatment of low doses of H2O2. This pretreatment also exerted protective effect against the oxidative insults induced by 6-hydroxydopamine and paraquat, but not against alkyl peroxyl radicals. Our results, taken together, suggest that the stimulation by low dose of H2O2 enriches the cellular antioxidant defense systems, thereby enhancing cell tolerance against the forthcoming oxidative insults induced by H2O2 and related hydroxyl radicals.     

IgE dependent autoimmune response. Effect of Trypanosoma cruzi infection

The autoimmune response to mouse accessory glands (MAG) was investigated in male BALB/c mice immunized with different doses of chemically modified mouse accessory glands (MMAG) and complete Freund's adjuvant (CFA). This autoimmune response was studied at several time intervals using the skin test with MAG. It was found that 5 mg of MMAG induced on the day 15 an autoimmune response detected by specific skin test at 20 min., 3 h and 24 h. The results of the immediate type hypersensitivity (ITH) were higher than those with the other skin tests. In order to study the type of immunoglobulin involved, the ITH was also analyzed by passive cutaneous anaphylaxis (PCA) at different time intervals with treated and untreated sera at 56 degrees C. The findings suggest the presence of reaginic antibodies, IgE being the major antibody as detected by enzime linked immunosorbent assay (ELISA). The MAG was subsequently fractionated using Sephandex G-100 and the fractions thus obtained (FI,FII and FIII) were used to challenge mice immunized with MMAG. It was found that FI was the only fraction which revealed an ITH similar to that revealed by MAG. The effect of infection with Trypanosoma cruzi on the autoimmune response to MAG was analyzed with different mouse groups intraperitoneally treated with 2 x 10(3) blood trypomastigotes/animal at several time intervals: namely, on days -5, 0, +5 and +10 with respect to the immunization with MMAG. The autoimmune response to MAG showed suppression when the animals received the parasites on the same day as the autoantigen.     

Role of hydrogen peroxide and peroxidase in the cytotoxicity of Trypanosoma dionisii by human granulocytes.

The mechanism of the cytotoxic reaction of leukocytes to Trypanosoma dionisii was investigated. Cytotoxicity was measured by release of [99mTc]pertechnetate from labeled protozoa. Both granulocytes and lymphocytes were found to be cytotoxic to antibody-coated T. dionisii. The reaction was inhibited by diethyldithiocarbamate and by potassium cyanide, both of which inhibit myeloperoxidase. Myeloperoxidase from azurophil granules was toxic to T. dionisii, provided that hydrogen peroxide was also present. Hydrogen peroxide formation was induced in granulocytes and, to a lesser extent, in lymphocytes by antibody-coated T. dionisii. Inhibition of this hydrogen peroxide formation by treatment of the effector cell surface with p-diazobenzenesulfonic acid inhibited cytotoxicity. It is therefore concluded that granulocytes, and probably also lymphocytes, kill T. dionisii with hydrogen peroxide by a peroxidase-mediated reaction. Although hydrogen peroxide and myeloperoxidase alone were also cytotoxic to the lymphoblastoid cell line CLA4, it seems unlikely that this is the cytotoxic mechanism for this process because these cells were unable to induce hydrogen peroxide formation.     

Chemotactic activity generated in human serum from the fifth component of complement by hydrogen peroxide.

Exposure of normal human serum to various concentrations of hydrogen peroxide as low as 68.8 microM resulted in the generation of chemotactic activity for human neutrophils, which was inhibited by adding catalase prior to the exposure. Maximum chemotactic activity was obtained by hydrogen peroxide at a concentration of 1.1 mM, and the concentration greater than 1.1 mM expressed decreased activity. On the contrary, hydrogen peroxide less than 1.1 mM produced dose-dependent chemotactic activity. The generation of chemotactic activity is initiated at an incubation time of 5 minutes, and subsequently increases with up to 90 minutes. The suppression of chemotactic activity was minimum even after 180 minutes. The maximum activity was obtained by 1024-fold dilution of serum treated with 13.8 mM hydrogen peroxide. These results suggest that the disappearance of chemotactic activity at the high-dose range of hydrogen peroxide is due to neutrophil deactivation rather than inactivation of the chemotactic activity after it was generated. Similarly, purified human C5 exposed to hydrogen peroxide generated chemotactic activity. The chemotactic activity was inhibitable by antiserum to human C5. The molecular weight of chemotactically active substance was approximately 15,000. The generation of chemotactic activity was not inhibited by addition of EGTA or EDTA. These results imply that hydrogen peroxide generates C5a-like chemotactic factor through hydrolysis of C5.     

Denervation and the immune response in mice infected with Trypanosoma cruzi.

C57B1 mice were infected with Trypanosoma cruzi Y strain trypomastigotes and showed a peak of parasitaemia 9 days after infection. Virtually all mice survived the acute stage of infection and were aparasitaemic thereafter. Coincident with the peak of parasitaemia, there was a progressive loss of cardiac neurones (up to the 20th day after infection) and an appearance of T. cruzi antigen on myofibres. Anti-parasite immunity, evidenced by a significant inhibition of macrophage migration in the presence of T. cruzi antigens (MIF test) and the deposition of complement and immunoglobulin in vivo around the nests of parasites, developed between days 7-10 after infection. Immunity to "self' components (MIF) test using neurone and heart antigens) was not detected until 15-20 days after infection. Although the MIF test detected a progressive increase in anti-neurone immunity between 20-90 days after infection, there was no additional loss of cardiac neurones during this period. In contrast to current hypotheses, these data suggest that the immunity to heart and neuronal antigens commonly detected in animals infected with T. cruzi is the result rather than the cause, of host cell destruction.     

Proteomic analysis of oxidative modification in endothelial colony-forming cells treated by hydrogen peroxide

Endothelial progenitor cells (EPCs) which circulate in the peripheral blood and reside in blood vessels are proven to promote the repair of damaged endothelium and improve the function of endothelial cells after vascular injury. Recently, EPCs have been extensively studied as risk biomarkers and a potential therapeutic tool for cardiovascular disease. It is known that oxidative stress is one of the most important pathogenetic factors impairing endothelial function. During the repair process after endothelial injury, EPCs are exposed to oxidative stress. In this study, we treated endothelial colony-forming cells (ECFCs) with hydrogen peroxide (H?O?) as an oxidative stress model and observed the changes in cytology and morphology of ECFCs. In addition, we investigated the alterations in oxidative levels of proteins associated with H?O?-induced morphological and cytological changes in ECFCs by proteomic analysis of oxidative modification. The results showed that H?O? treatment led to a decreased proliferation, increased apoptosis and impaired tube-forming ability of ECFCs in a dose-dependent manner. Five proteins with upregulated oxidative levels were identified successfully. The upregulated oxidative levels of these five proteins may be responsible for the dysfunction of ECFCs under oxidative stress. Our results may provide some novel insights into the molecular mechanisms of oxidative stress action on ECFCs.     

Insights into IL-33 on inflammatory response during in vitro infection by Trypanosoma cruzi

Inflammatory and regulatory cytokines play an important role in the immunopathogenesis of Trypanosoma cruzi infection. Interleukin (IL)-33 is a member of the IL-1 superfamily of cytokines whose expression/production is upregulated following pro-inflammatory stimulation to alert the immune system in response to tissue stress or damage. The aim of this study was to evaluate the inflammatory profile induced in cultured J774 cells stimulated or not with IL-33 (10 ng/mL), with live parasites (1 × 10⁶ metacyclic trypomastigote forms) and/or total antigen, TcAg (100 µg/mL) and with both, IL-33 and TcAg/T. cruzi. The cultures were evaluated at 24 h and 48 h after addition of the stimuli. For this, the supernatants were collected for the measurement of TNF, IL-17, CCL2, and IL-10 by ELISA and of nitrite by the Griess method. TNF, IL-17, and CCL2 concentrations were elevated in the presence of TcAg or live T. cruzi parasites at 24 h, and the addition of IL-33 potentiated these effects at 48 h. In addition, the T. cruzi-amastigote forms reduced in those infected J774 cells stimulated with IL-33 at 48 h. In conclusion, the IL-33 elevated the production of the TNF, IL-17, and CCL2 in cultured J774 cells stimulated with T. cruzi and/or its antigen and reduced the intracellular parasites, providing impetus to new investigations on its potential actions on the parasite-induced inflammation.     

Chronic Chagas' disease cardiomyopathy patients display an increased IFN-gamma response to Trypanosoma cruzi infection

One-third of all Trypanosoma cruzi -infected patients eventually develop chronic Chagas' disease cardiomyopathy (CCC), a particularly lethal inflammatory dilated cardiomyopathy, where parasites are scarce and heart-infiltrating mononuclear cells seem to be the effectors of tissue damage. Since T. cruzi is a major inducer of interleukin-12 production, the role of inflammatory cytokines in the pathogenesis of CCC was investigated. We assayed cytokine production by peripheral blood mononuclear cells (PBMC) from CCC and asymptomatic T. cruzi -infected (ASY) individuals, as well as by T cell lines from endomyocardial biopsies from CCC patients. PBMC from CCC and ASY patients produced higher IFN-gamma levels than normal (N) individuals in response to B13 protein and phytohaemagglutinin PHA; IFN-gamma high responders (> or =1 ng/ml) were 2-3 fold more frequent among CCC patients than ASY individuals. Conversely, IL-4 production in response to the same stimuli was suppressed among T. cruzi -infected patients. The frequency of PHA-induced IFN gammaproducing cells on PBMC was significantly higher among CCC than ASY and N individuals. IFN-gamma and TNF-alpha were produced by ten out of ten PHAstimulated T cell lines from CCC patients; IL-2 and IL-10 were produced by four out of ten and one out of ten lines, respectively; IL-4, IL-1alpha, IL-1beta, IL-6 and IL-12 were undetectable. Our results suggest that CCC and ASY patients may respond differentially to the IFN-gamma-inducing stimulus provided by T. cruzi infection. Given the T(1)-type cytokine profile of heart-infiltrating T cell lines from CCC patients, the ability to mount a vigorous IFN-gamma response may play a role on the differential susceptibility to CCC development.     

Trypanosoma cruzi infection induces a global host cell response in cardiomyocytes

Chagas' disease, caused by the hemoflagellate protozoan Trypanosoma cruzi, affects millions of people in South and Central America. Chronic chagasic cardiomyopathy, the most devastating manifestation of this disease, occurs in approximately one-third of infected individuals. Events associated with the parasite's tropism for and invasion of cardiomyocytes have been the focus of intense investigation in recent years. In the present study, we use murine microarrays to investigate the cellular response caused by invasion of primary murine cardiomyocytes by T. cruzi trypomastigotes. These studies identified 353 murine genes that were differentially expressed during the early stages of invasion and infection of these cells. Genes associated with the immune response, inflammation, cytoskeleton organization, cell-cell and cell-matrix interactions, apoptosis, cell cycle, and oxidative stress are among those affected during the infection. Our data indicate that T. cruzi induces broad modulations of the host cell machinery in ways that provide insight into how the parasite survives, replicates, and persists in the infected host and ultimately defines the clinical outcome of the infection.     

Effect of hydroxyurea on the concanavalin-A proliferative response of Trypanosoma cruzi infected mice

Mice infected with Trypanosoma cruzi develop immunosuppression with a deficient production of interleukin-2 (IL-2). In this situation the deficient concanavalin A (Con A) T-cell response is not corrected by addition of exogenous IL-2. Here we show that elimination of cycling cells by treatment of infected mice with hydroxyurea (HU) fully restored the ability of spleen cells to respond to IL-2. Further, capacity for IL-2 production was restored to HU treated infected mice, but not as completely as the response to IL-2.