Effects of cyclooxygenase inhibitors on parasite burden, anemia and oxidative stress in murine Trypanosoma cruzi infection

Prostaglandins are known to be produced by macrophages when challenged with Trypanosoma cruzi, the etiological agent of Chagas' disease. It is not known whether these lipid mediators play a role in oxidative stress in host defenses against this important protozoan parasite. In this study, we demonstrated that inducible cyclooxygenase-mediated prostaglandin production is a key chemical mediator in the control of parasite burden and erythrocyte oxidative stress during T. cruzi infection in C57BL/6 and BALB/c mice, prototype hosts for the study of resistance and susceptibility in murine Chagas' disease. The results suggested the existence of at least two mechanisms of oxidative stress, dependent or independent with regard to the nitric oxide and cyclooxygenase pathway, where one or the other is more evident depending on the mouse strain.     

Prevalence of anti-R-13 antibodies in human Trypanosoma cruzi infection

Abstract Infection with Trypanosoma cruzi develops in three phases: acute, indeterminate or asymptomatic, and chronic phase (with cardiac or digestive manifestations). Moreover, transmission may occur from infected mothers to newborn, the so-called congenital form. In the present study, humoral responses against T. cruzi total extract and against the 13 amino acid peptide named R-13 derived from the parasite ribosomal P protein, previously described as a possible marker of chronic Chagas heart disease, were determined pateints and in blood bank donors from endemic areas. While in sera from acute phase, only IgM anti- T.cruzi response was observed, both IgM and IgG anti- T. cruzi antibodies were detected in sera from congenitally infected newborns. The percentage of positive response in sera from blood bank donors was relatively high in endemic regions. Antibodies against the R-13 peptide were present in a large proportion of cardiac chagasic patients but were totally lacking in patients with digestive form of Chagas disease. Furthermore, anti-R-13 positive responses were detected in congenitally infected newborns.     

Involvement of nitric oxide (NO) and TNF-alpha in the oxidative stress associated with anemia in experimental Trypanosoma cruzi infection

Trypanosoma cruzi infection in mice is associated with severe hematological changes, including anemia, which may contribute to mortality. TNF-alpha and nitric oxide (NO) play a critical role in establishing host resistance to this pathogen. We hypothesized that phagocyte-derived NO damages erythrocytes and contributes to the anemia observed during T. cruzi infection. To test this hypothesis, two strains of mice that differed in susceptibility and NO response to T. cruzi infection were used in these studies. We also blocked endogenous NO production by aminoguanidine (AG) treatment or blocked TNF-alpha with a neutralizing antibody and used mice that cannot produce phagocyte-derived NO (C57BL/6 iNOS(-/-)). Following infection with T. cruzi, resistant (C57BL/6) and susceptible (Swiss) mice displayed a parasitemia that peaked at the same time (i.e., day 9), yet parasitemia was 3-fold higher in Swiss mice (P < 0.05). All Swiss mice were dead by day 23 post-infection, while no C57BL/6 mice died during the study. At 14 days post-infection anemia in C57BL/6 mice was more severe than in Swiss mice. Treatment of both strains with the NO inhibitor, AG (50 mg/kg), and the use of iNOS(-/-) mice, revealed that the anemia in T. cruzi-infected mice is not caused by NO. However, the reticulocytosis that occurs during infection was significantly reduced after treatment with AG in both Swiss and C57BL/6 mice (P < 0.05). In addition, we showed that neutralization of TNF-alpha in vivo induced a significant increase in circulating reticulocytes in T. cruzi-infected C57BL/6 mice (P < 0.05), but did not modify other hematologic parameters in these mice. The evaluation of the oxidative stress after induction by t-butyl hydroperoxide (t-BHT) revealed that the treatment with AG completely protected against NO-mediated haemoglobin oxidation. Further, treatment with AG, but not with anti-TNF-alpha, protected against the infection-induced reduction of antioxidant capacity of erythrocytes as assessed by oxygen uptake and induction time. In summary, this is the first report showing the participation of NO and TNF-alpha in the oxidative stress to erythrocytes in acute T. cruzi infection. Further, our data suggest that NO does not play a direct role in development of the anemia. However, NO may contribute to other hematological changes noted during T. cruzi infection, such as the elevation of circulating reticulocytes and the reduction in circulating leukocytes and neutrophils.     

The problem of interlab variation in methods for mitochondrial disease diagnosis: enzymatic measurement of respiratory chain complexes

Due to the large importance of mitochondrial function for numerous diseases the detection of respiratory chain defects for diagnostic purposes is an important task of mitochondrial medicine. For comparing the methods, standard mitochondrial homogenate prepared from bovine skeletal muscle was sent on dry ice to 14 labs of 8 countries. Activities of complexes I, I + III, II, II + III, IV (cytochrome c oxidase) and V (F0F1ATPase) as well as of citrate synthase were measured. For all enzymes the results differed at more than one order of magnitude. From eight labs we were able to compare the results with their control values for human skeletal muscle. Four labs found normal activity of cytochrome-c-oxidase whereas three labs found higher and one lab found lower activities compared to the own controls. Since all labs used different temperatures (25, 30 and 37 degrees C) in one lab the temperature dependencies were measured experimentally. The temperature correction did not much reduce the divergence of the results. It is concluded that differences in the lab protocols are the reason for the large variation of results. Since the experimental results strongly depend on the used method a strict standardization is necessary.     

Oxidative stress induces mitochondrial dysfunction and a protective unfolded protein response in RPE cells

How cells degenerate from oxidative stress in aging-related disease is incompletely understood. This study’s intent was to identify key cytoprotective pathways activated by oxidative stress and determine the extent of their protection. Using an unbiased strategy with microarray analysis, we found that retinal pigmented epithelial (RPE) cells treated with cigarette smoke extract (CSE) had overrepresented genes involved in the antioxidant and unfolded protein response (UPR). Differentially expressed antioxidant genes were predominantly located in the cytoplasm, with no induction of genes that neutralize superoxide and H₂O₂ in the mitochondria, resulting in accumulation of superoxide and decreased ATP production. Simultaneously, CSE induced the UPR sensors IRE1α, p-PERK, and ATP6, including CHOP, which was cytoprotective because CHOP knockdown decreased cell viability. In mice given intravitreal CSE, the RPE had increased IRE1α and decreased ATP and developed epithelial–mesenchymal transition, as suggested by decreased LRAT abundance, altered ZO-1 immunolabeling, and dysmorphic cell shape. Mildly degenerated RPE from early age-related macular degeneration (AMD) samples had prominent IRE1α, but minimal mitochondrial TOM20 immunolabeling. Although oxidative stress is thought to induce an antioxidant response with cooperation between the mitochondria and the ER, herein we show that mitochondria become impaired sufficiently to induce epithelial–mesenchymal transition despite a protective UPR. With similar responses in early AMD samples, these results suggest that mitochondria are vulnerable to oxidative stress despite a protective UPR during the early phases of aging-related disease.     

Anomalous electrophoretic behaviour of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi in relation to its apparent molecular mass

The molecular mass of cruzipain, the major cysteine proteinase from Trypanosoma cruzi epimastigotes, is 36.3 kDa as calculated from its sequence; this value can increase to about 41 kDa if the three potential N-glycosylation sites are glycosylated in vivo. Yet the apparent molecular mass of the enzyme, as determined by SDS-polyacrylamide gel electrophoresis, has been reported in a range of values from 60 to 40 kDa. We show that the purified enzyme had apparent molecular masses ranging from 51 to 33 kDa, depending on the experimental conditions. This variation is likely to be due to both N-glycosylation and the presence of several disulfide bridges, which make electrophoretic mobility dependent on acrylamide concentration, and reduction and/or boiling of the sample.     

Trypanosoma cruzi: Cardiac mitochondrial alterations produced by different strains in the acute phase of the infection

The parasite Trypanosoma cruzi is the causative agent of Chagas disease. T. cruzi invasion and replication in cardiomyocytes induce cellular injuries and cytotoxic reactions, with the production of inflammatory cytokines and nitric oxide, both source of reactive oxygen species. The myocyte response to oxidative stress involves the progression of cellular changes primarily targeting mitochondria. We studied the cardiac mitochondrial structure and the enzymatic activity of citrate synthase and respiratory chain CI–CIV complexes, in Albino Swiss mice infected with T. cruzi, Tulahuen strain and SGO Z12 isolate, in two periods of the acute infection. Changes in the mitochondrial structure were detected in both infected groups, reaching values of 71% for Tulahuen and 88% for SGO Z12 infected mice, 30 days post infection. The citrate synthase activity was different according to the evolution of the infection and the parasite strain, but the respiratory chain alterations were similar with either strain.     

Inhibition of xenograft rejection reaction in the bug Triatoma infestans during infection with a protozoan, Trypanosoma cruzi

Autografts and allografts implanted into the body cavity of the bug Triatoma infestans provoked a poor hemocyte encapsulation, while xenografts stimulated significantly stronger reaction. This reaction of xenograft rejection, however, appeared distinctly inhibited in Trypanosoma cruzi-infected bugs. Similar effect was produced in triatomas by injecting them with the fluid from culture of the parasites. The results of these studies suggest that under natural conditions some parasites may avoid destruction and develop within the insect's body cavity following their suppression of host's defense reactions.     

Ecology of Triatoma brasiliensis in northeastern Brazil: seasonal distribution,feeding resources,and Trypanosoma cruzi infection in a sylvatic population

We assessed some ecological parameters of Triatoma brasiliensis in rock piles in the state of Ceará during the rainy and dry seasons. The greatest density was in April (median = 12.5 triatomines/site). The greatest abundance was in December, when the insects were more dispersed and the density per site was lower (6 triatomines/site). The nutritional status of females and 5^th instar nymphs was increased in July. The rate of T. cruzi infection reached its highest peak in July (10.9%). ELISA revealed that the principal food sources were birds (33.1%), followed by armadillos (18.8%). Food sources were more frequently identified during the rainy season. T. brasiliensis specimens collected in the drought tended to: i) present lower rates of T. cruzi infection and gut content reactivity to tested antisera, ii) have a poorer nutritional status, iii) exhibit lower fecundity, iv) be more dispersed among the studied collection sites, and v) be more abundant and easily collected in the surface of the rocks, possibly reflecting an increased searching for blood meals. Such findings underscore epidemiological concerns and allow inferences about the season when triatomines can more frequently invade the peridomestic environment in search of food and recolonize artificial structures.     

DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells

Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction. In this study, mitochondrial and nuclear DNA DSBs were generated in the A549 human lung adenocarcinoma cell line by infecting with retroviruses expressing the restriction endonuclease PstI fused to a mitochondrial targeting sequence (MTS) or nuclear localization sequence (NLS) and a hemagglutinin antigen epitope tag (HA). Expression of MTS-PstI-HA or NLS-PstI-HA activated the DNA damage response defined by phosphorylation of ATM, the tumor suppressor protein p53 (TP53), KRAB-associated protein (KAP)-1, and structural maintenance of chromosomes (SMC)-1. Phosphorylated ATM and SMC1 were detected in nuclear fractions, whereas phosphorylated TP53 and KAP1 were detected in both mitochondrial and nuclear fractions. PstI also enhanced expression of the cyclin-dependent kinase inhibitor p21 and inhibited cell growth. This response to DNA damage occurred in the absence of detectable mitochondrial dysfunction and excess production of ROS. These findings reveal that DNA DSBs are sufficient to activate ATM independent of mitochondrial dysfunction and suggest that the activated form of ATM and some of its substrates are restricted to the nuclear compartment, regardless of the site of DNA damage.     

Effects of mammal host diversity and density on the infection level of Trypanosoma cruzi in sylvatic kissing bugs

Several reports have described host species diversity and identity as the most important factors influencing disease risk, producing either dilution or amplification of the pathogen in a host community. Triatomine vectors, mammals and the protozoan Trypanosoma cruzi (Trypanosomatida: Trypanosomatidae) Chagas are involved in the wild cycle of Chagas disease, in which infection of mammals occurs by contamination of mucous membranes or skin abrasions with insect‐infected faeces. We examined the extent to which host diversity and identity determine the infection level observed in vector populations (i.e. disease risk in humans). We recorded infection in triatomine colonies and on the coexisting host mammalian species in semi‐arid Chile. Host diversity, and total and infected host species densities are used as predictor variables for disease risk. Disease risk did not correlate with host diversity changes. However, the densities of each infected rodent species were positively associated with disease risk. We suggest that the infected host density surrounding the vector colonies is a relevant variable for disease risk and should be considered to understand disease dynamics. It is crucial to pay attention on the spatial scale of analysis, considering the pattern of vector dispersal, when the relationship between host diversity and disease risk is studied.     

Cardioprotective effects of verapamil on myocardial structure and function in a murine model of chronic Trypanosoma cruzi infection (Brazil Strain): an echocardiographic study.

Verapamil has been shown to attenuate the extent of myocardial injury in murine models of chronic Trypanosoma cruzi infection. Infected mice treated with verapamil have significantly lower myocardial expression of inducible nitric oxide synthase and cytokines and substantially less inflammatory infiltrate and myocyte necrosis at necropsy. In the present study, we examined the cardiac structural and functional correlates of verapamil treatment in CD1 mice infected with the Brazil strain of T. cruzi using serial transthoracic echocardiography. There were four groups: uninfected- untreated control, uninfected-verapamil-treated, infected-untreated control, and infected-verapamil-treated. Verapamil was given in drinking water (1 gm/l) continuously from the day of infection for a total of 120 days. Mice were evaluated at baseline, 40 and 150 days p.i. Mice in the untreated-infected group compared with the mice in the infected-verapamil-treated group showed thinning of the left ventricular wall (0.84 +/- 0.02-vs-0.92 +/- 0.04, P<0.05 mm), increase in the left ventricular end-diastolic diameter (3.27 +/- 0.15-vs-2.74 +/- 0.05 mm, P<0.05) and reduction in percent fractional shortening (37 +/- 2-vs-53 +/- 4%, P<0.05). No differences in these parameters were noted among mice in the uninfected-untreated and uninfected-verapamil-treated groups. Furthermore, right ventricular dilation was more severe in mice from the infected-untreated group as compared with those in the infected- verapamil-treated group (visual grade 1.9 +/- 0.4-vs-1.0 +/- 0.2, P<0.05). At necropsy, the extent of myocardial injury, as determined histologically, was significantly greater in the infected-untreated mice. These data provide cardiac structural and functional correlates for the previously observed cardioprotective effects of verapamil in chronic chagasic cardiomyopathy.     

Trypanosoma cruzi: effects of repetitive stress during the development of experimental infection

Activation of the hypothalamus-pituitary-adrenal axis plays a major role in the suppression of the immune system. We have investigated the effects of repetitive stress on Wistar rats infected with the Y strain of Trypanosoma cruzi and a control group that underwent stressor stimuli by exposure to ether vapor for one minute twice a day. Repetitive stress resulted in an elevated number of circulating parasites accompanies by deep tissue disorganization, and cardiac histopathological alterations. The infected and stressed group displayed a decrease in body weight, and an increased parasite burden in heart tissue, and adrenal glands. Histological analysis of the heart also showed a moderate to severe diffused mononuclear inflammatory process. These results suggest that repetitive stress could be considered an important factor during development of experimental Chagas' disease, enhancing pathogenesis through disturbance of the host's immune system.     

Congenital transmission of Trypanosoma cruzi in central Brazil. A study of 1,211 individuals born to infected mothers

Transmission of Trypanosoma cruzi during pregnancy is estimated tooccur in less than 20% of infected mothers; however, the etiopathogenesis is notcompletely understood. The Centre for Studies on Chagas Disease provides confirmationof T. cruzi infection for individuals living in central Brazil.In this retrospective hospital-based study, all requests for diagnosis of T.cruzi infection in individuals less than 21 years old from 1994-2014 weresearched. We end with 1,211 individuals and their respective infected mothers.Congenital transmission of infection was confirmed in 24 individuals (2%) in centralBrazil, an area where the main T. cruzi lineage circulating inhumans is TcII. This low prevalence of congenital Chagas disease is discussed inrelation to recent findings in the south region of Brazil, where TcV is the mainlineage and congenital transmission has a higher prevalence (approximately 5%),similar to frequencies reported in Argentina, Paraguay and Bolivia. This is the firstreport to show geographical differences in the rates of congenital transmissionof T. cruzi and the relationship between the prevalence ofcongenital transmission and the type of Tc prevalent in each region.     

Oxidative stress-mediated activation of extracellular signal-regulated kinase contributes to mild cognitive impairment-related mitochondrial dysfunction

Mild cognitive impairment (MCI) occurs during the predementia stage of Alzheimer disease (AD) and is characterized by a decline in cognitive abilities that frequently represents a transition between normal cognition and AD dementia. Its pathogenesis is not well understood. Here, we demonstrate the direct consequences and potential mechanisms of oxidative stress and mitochondrial dynamic and functional defects in MCI-derived mitochondria. Using a cytoplasmic hybrid (cybrid) cell model in which mitochondria from MCI or age-matched non-MCI subjects were incorporated into a human neuronal cell line depleted of endogenous mitochondrial DNA, we evaluated the mitochondrial dynamics and functions, as well as the role of oxidative stress in the resultant cybrid lines. We demonstrated that increased expression levels of mitofusin 2 (Mfn2) are markedly induced by oxidative stress in MCI-derived mitochondria along with aberrant mitochondrial functions. Inhibition of oxidative stress rescues MCI-impaired mitochondrial fusion/fission balance as shown by the suppression of Mfn2 expression, attenuation of abnormal mitochondrial morphology and distribution, and improvement in mitochondrial function. Furthermore, blockade of MCI-related stress-mediated activation of extracellular signal-regulated kinase (ERK) signaling not only attenuates aberrant mitochondrial morphology and function but also restores mitochondrial fission and fusion balance, in particular inhibition of overexpressed Mfn2. Our results provide new insights into the role of the oxidative stress–ERK–Mfn2 signal axis in MCI-related mitochondrial abnormalities, indicating that the MCI phase may be targetable for the development of new therapeutic approaches that improve mitochondrial function in age-related neurodegeneration.     

Production of hydrogen peroxide by peripheral blood monocytes and specific macrophages during experimental infection with Trypanosoma cruzi in vivo

Acute Chagas' disease triggers potent inflammatory reaction characterized by great increase of peripheral blood monocyte (PBM) and macrophage numbers. We studied the respiratory burst responses of PBM and peritoneal and splenic macrophages to in vivo infection (rats). The ultrastructure of heart inflammatory macrophages was also investigated. The infection increased the hydrogen peroxide (H2O2) production by PBM and splenic macrophages but not by peritoneal macrophages. Accordingly, the PBM and spleen cell numbers increased but the total number of peritoneal cells was similar to controls. Heart macrophages of infected rats exhibited increase (number and size) and activated morphology in parallel to high cardiomyocyte parasitism. Our data highlight the importance of innate immunity and H2O2production to host resistance during acute phase of T. cruzi infection. A novel finding is that H2O2production seems related to specific types of monocytes/macrophages that are able to release this agent when in presence of high parasite load.     

Relationships between altitude,triatomine (Triatoma dimidiata) immune response and virulence of Trypanosoma cruzi,the causal agent of Chagas' disease

Little is known about how the virulence of a human pathogen varies in the environment it shares with its vector. This study focused on whether the virulence of Trypanosoma cruzi (Trypanosomatida: Trypanosomatidae), the causal agent of Chagas' disease, is related to altitude. Accordingly, Triatoma dimidiata (Hemiptera: Reduviidae) specimens were collected at three different altitudes (300, 700 and 1400 m a.s.l.) in Chiapas, Mexico. The parasite was then isolated to infect uninfected T. dimidiata from the same altitudes, as well as female CD‐1 mice. The response variables were phenoloxidase (PO) activity, a key insect immune response, parasitaemia in mice, and amastigote numbers in the heart, oesophagus, gastrocnemius and brain of the rodents. The highest levels of PO activity, parasitaemia and amastigotes were found for Tryp. cruzi isolates sourced from 700 m a.s.l., particularly in the mouse brain. A polymerase chain reaction‐based analysis indicated that all Tryp. cruzi isolates belonged to a Tryp. cruzi I lineage. Thus, Tryp. cruzi from 700 m a.s.l. may be more dangerous than sources at other altitudes. At this altitude, T. dimidiata is more common, apparently because the conditions are more beneficial to its development. Control strategies should focus activity at altitudes around 700 m a.s.l., at least in relation to the region of the present study sites.     

Oxidative stress-related mechanisms affecting response to aspirin in diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a major cardiovascular risk factor. Persistent platelet activation plays a key role in atherothrombosis in T2DM. However, current antiplatelet treatments appear less effective in T2DM patients vs nondiabetics at similar risk. A large body of evidence supports the contention that oxidative stress, which characterizes DM, may be responsible, at least in part, for less-than-expected response to aspirin, with multiple mechanisms acting at several levels. This review discusses the pathophysiological mechanisms related to oxidative stress and contributing to suboptimal aspirin action or responsiveness. These include: (1) mechanisms counteracting the antiplatelet effect of aspirin, such as reduced platelet sensitivity to the antiaggregating effects of NO, due to high-glucose-mediated oxidative stress; (2) mechanisms interfering with COX acetylation especially at the platelet level, e.g., lipid hydroperoxide-dependent impaired acetylating effects of aspirin; (3) mechanisms favoring platelet priming (lipid hydroperoxides) or activation (F₂-isoprostanes, acting as partial agonists of thromboxane receptor), or aldose-reductase pathway-mediated oxidative stress, leading to enhanced platelet thromboxane A₂ generation or thromboxane receptor activation; (4) mechanisms favoring platelet recruitment, such as aspirin-induced platelet isoprostane formation; (5) modulation of megakaryocyte generation and thrombopoiesis by oxidative HO-1 inhibition; and (6) aspirin–iron interactions, eventually resulting in impaired pharmacological activity of aspirin, lipoperoxide burden, and enhanced generation of hydroxyl radicals capable of promoting protein kinase C activation and platelet aggregation. Acknowledgment of oxidative stress as a major contributor, not only of vascular complications, but also of suboptimal response to antiplatelet agents in T2DM, may open the way to designing and testing novel antithrombotic strategies, specifically targeting oxidative stress-mediated mechanisms of less-than-expected response to aspirin.