Mycobacterium bovis BCG as immunostimulating agent prevents the severe form of chronic experimental Chagas disease.

Introduction: There is currently no vaccine against Chagas disease (ChD), and the medications available confer multiple side effects. Mycobacterium bovis Bacillus Calmette-Guérin (BCG) produces balanced Th1, Th2, and Th17 modulatory immune responses and has improved efficacy in controlling chronic infections through nonspecific immunity. We aimed to improve the response to infection by inducing a stronger immune response and greater protection against the parasite by trained immunity. Methods: BALB/c mice were immunized with BCG subcutaneously, and 60 days later, they were infected with Trypanosoma cruzi intraperitoneally. An evaluation of the progression of the disease from the acute to the chronic stage, analyzing various aspects such as parasitemia, survival, clinical status, and humoral and cellular immune response, as well as the appearance of visceral megas and the histopathological description of target organs, was performed. Results: Vaccination reduced parasitemia by 70%, and 100% survival was achieved in the acute stage; although the presentation of clinical signs was reduced, there was no increase in the antibody titer or in the differential production of the isotypes. Conclusion: Serum cytokine production indicated a proinflammatory response in infected animals, while in those who received BCG, the response was balanced by inducing Th1/Th2-type cytokines, with a better prognosis of the disease in the chronic stage.

Diversity of midgut microbiota in ticks collected from white-tailed deer (Odocoileus virginianus) from northern Mexico.

Ticks host different pathogens as endosymbiont and nonpathogenic microorganisms and play an important role in reproductive fitness and nutrient provision. However, the bacterial microbiomes of white-tailed deer ticks have received minimal attention. This study aimed to examine the bacterial microbiome of ticks collected from Odocoileus virginianus on the Mexico-United States border to assess differences in microbiome diversity in ticks of different species, sexes, and localities. Five different tick species were collected: Rhipicephalus microplus, Dermacentor nitens, Otobius megnini, Amblyomma cajennense, and A. maculatum. The tick microbiomes were analyzed using next-generation sequencing. Among all tick species, the most predominant phylum was Proteobacteria, followed by Actinobacteria and Firmicutes. The ticks from Tamaulipas and Nuevo León presented the highest bacterial species diversity. Acinetobacter johnsonii and A. lwoffii were the common bacterial species in the microbiome of all ticks, Coxiella were present in R. microplus, and Dermacentor nitens also exhibited a Francisella-like endosymbiont. The microbiome of most females in D. nitens was less diverse than that of males, whereas R. microplus occurs in females, suggesting that microbiome diversity is influenced by sex. In the bacterial communities of A. maculatum and O. megnini, Candidatus Midichloria massiliensis, and Candidatus Endoecteinascidia fumentensis were the most predominant endosymbionts. These results constitute the initial report on these bacteria, and this is also the first study to characterize the microbiome of O. megnini.

In vitro anti-Trypanosoma cruzi activity of methanolic extract of Bidens pilosa and identification of active compounds by gas chromatography-mass spectrometry analysis.

Chagas disease, caused by Trypanosoma cruzi parasite, is a significant but neglected tropical public health issue in Latin America due to the diversity of its genotypes and pathogenic profiles. This complexity is compounded by the adverse effects of current treatments, underscoring the need for new therapeutic options that employ medicinal plant extracts without negative side effects. Our research aimed to evaluate the trypanocidal activity of Bidens pilosa fractions against epimastigote and trypomastigote stages of T. cruzi, specifically targeting the Brener and Nuevo León strains-the latter isolated from Triatoma gerstaeckeri in General Terán, Nuevo León, México. We processed the plant's aerial parts (stems, leaves, and flowers) to obtain a methanolic extract (Bp-mOH) and fractions with varying solvent polarities. These preparations inhibited more than 90% of growth at concentrations as low as 800 µg/ml for both parasite stages. The median lethal concentration (LC50) values for the Bp-mOH extract and its fractions were below 500 µg/ml. Tests for cytotoxicity using Artemia salina and Vero cells and hemolytic activity assays for the extract and its fractions yielded negative results. The methanol fraction (BPFC3MOH1) exhibited superior inhibitory activity. Its functional groups, identified as phenols, enols, alkaloids, carbohydrates, and proteins, include compounds such as 2-hydroxy-3-methylbenzaldehyde (50.9%), pentadecyl prop-2-enoate (22.1%), and linalool (15.4%). Eight compounds were identified, with a match confirmed by the National Institute of Standards and Technology (NIST-MS) software through mass spectrometry analysis.

The Enolase of the Haemophilus influenzae Mediates Binding to Collagens: An Extracellular Matrix Component.

Enolase proteins play a significant role as moonlighting proteins. In their role as surface-associated enolase, they have multiple functions as they interact with extracellular matrix proteins. Type I and III collagens are the major constituents of this extracellular matrix, and collagen is one of the targets of interaction with the enolase of many pathogens, thereby helping the colonization process and promoting the subsequent invasion of the host. This work aimed to determine the participation of non-typeable H. influenzae enolase as a collagen-binding protein. In this study, through the use of in vitro tests it was demonstrated that recombinant enolase of non-typeable H. influenzae (rNTHiENO) strongly binds to type I collagen. Using molecular docking, the residues that could take part in the interaction of non-typeable H. influenzae enolase-type I collagen (NTHiENO-Cln I) and non-typeable H. influenzae enolase-type III collagen (NTHiENO-Cln III) were identified. However, in vitro assays show that NTHiENO has a better affinity to interact with Cln I, concerning type Cln III. The interaction of NTHiENO with collagen could play a significant role in the colonization process; this would allow H. influenzae to increase its virulence factors and strengthen its pathogenesis.

Nitazoxanide: A Drug Repositioning Compound with Potential Use in Chagas Disease in a Murine Model.

Chagas disease (ChD), caused by Trypanosoma cruzi, is the most serious parasitosis in the western hemisphere. Benznidazole and nifurtimox, the only two trypanocidal drugs, are expensive, difficult to obtain, and have severe side effects. Nitazoxanide has shown to be effective against protozoa, bacteria, and viruses. This study aimed to evaluate the nitazoxanide efficacy against the Mexican T. cruzi Ninoa strain in mice. Infected animals were orally treated for 30 days with nitazoxanide (100 mg/kg) or benznidazole (10 mg/kg). The clinical, immunological, and histopathological conditions of the mice were evaluated. Nitazoxanide- or benznidazole-treated mice had longer survival and less parasitemia than those without treatment. Antibody production in the nitazoxanide-treated mice was of the IgG1-type and not of the IgG2-type as in the benznidazole-treated mice. Nitazoxanide-treated mice had significantly high IFN-γ levels compared to the other infected groups. Serious histological damage could be prevented with nitazoxanide treatment compared to without treatment. In conclusion, nitazoxanide decreased parasitemia levels, indirectly induced the production of IgG antibodies, and partially prevented histopathological damage; however, it did not show therapeutic superiority compared to benznidazole in any of the evaluated aspects. Therefore, the repositioning of nitazoxanide as an alternative treatment against ChD could be considered, since it did not trigger adverse effects that worsened the pathological condition of the infected mice.

External pH Variations Modify Proliferation, Erythrophagocytosis, Cytoskeleton Remodeling, and Cell Morphology of Entamoeba histolytica Trophozoites.

To infect the human host, Entamoeba histolytica carries out processes requiring cytoskeleton remodeling, which involves reorganizing the actin fibers. However, little is known about the external influence factors, e.g., the pH, on the parasite's cytoskeleton remodeling or cell morphology. Such influence becomes relevant given the pH gradient that the amoeba cope with when going through the human colonic mucus during infection. Therefore, we analyzed the proliferation, the reorganization of the actin fibers, and other actin structures and cell shape during adhesion to fibronectin and erythrophagocytosis in trophozoites at different external pH conditions (6.0, 6.5, 6.8, 7.5, 8.0). We found that the best condition of external pH to perform such functions was 6.8. At acid pH, the trophozoites presented better-defined actin fibers that formed a more compact network, while at alkaline pH, the fibers reorganized, forming a looser and less defined network. Similarly, the number of actin dots also changed from acid to alkaline pH. In conclusion, the external pH alters the proliferation of the amoebas and promotes the dynamic restructuration of their cytoskeleton, allowing them to carry out their functions.

Disordered glycemic control in women with type 2 diabetes is associated with increased TNF receptor-2 levels.

BACKGROUND: Evidence implicates tumor necrosis factor (TNF) in the pathophysiology of Type 2 Diabetes (T2D) through unclear mechanisms. We hypothesized that disordered glycemic control leads to TNF activation and increases in soluble-TNF (sTNF) and its receptors-1 (sTNFR1) and -2 (sTNFR2). METHODS: We characterized 265 T2D and non-diabetic Latin American subjects and assessed the relationship between the TNF system and fasting plasma glucose (FPG), hemoglobin-A1C (A1C), insulin (FPI), C-peptide and HOMA-Beta. RESULTS: sTNF and sTNFR2 but not sTNFR1 levels were higher in T2D than non-diabetics (P<0.0001). In T2D, sTNFR2 was associated with A1C and C-peptide (R2=0.354, b=0.504, P<0.0001; b=0.167, P=0.049). Also, T2D patients with disordered glycemic control had increased sTNFR2 levels that correlated with FPG (Rho:0.393, P<0.001), A1C (Rho:0.451, P<0.001) and HOMA-Beta (Rho:-0.308, P=0.005); events not observed in T2D patients with adequate glycemic control. Furthermore, sex-based comparative analyses of T2D patients showed that women compared to men had higher sTNFR2 levels (P=0.017) that correlated with FPG, A1C, FPI and HOMA-Beta. CONCLUSIONS: Disordered glycemic control is associated with sTNF and sTNFR2. sTNFR2 levels were higher in T2D women than men. Thus, increased sTNFR2 levels may be an important biomarker for disordered glucose and inflammatory complications in T2D patients and women in particular.

Effect of Prophylactic Vaccination with the Membrane-Bound Acid Phosphatase Gene of Leishmania mexicana in the Murine Model of Localized Cutaneous Leishmaniasis.

Leishmaniasis is a disease caused by an intracellular protozoan parasite of the genus Leishmania. Current treatments for leishmaniasis are long, toxic, and expensive and are not available in some endemic regions. Attempts to develop an effective vaccine are feasible, but no vaccine is in active clinical use. In this study, the LmxMBA gene of Leishmania mexicana was selected as a possible vaccine candidate using the reverse vaccinology approach, and the prophylactic effect generated by DNA vaccination with this gene in a murine model of cutaneous leishmaniasis was evaluated. The results showed that prophylactic vaccination with pVAX1::LmxMBA significantly reduced the size of the lesion and the parasitic load on the footpad, compared to the control groups. At a histological level, a smaller number of parasites were evident in the dermis, as well as the absence of connective tissue damage. Mice immunized with plasmid pVAX1::LmxMBA induced immunity characterized by an increase in the IgG2a/IgG1 > 1 ratio and a higher rate of lymphocyte proliferation. In this study, immunization with the plasmid promoted an improvement in the macroscopic and microscopic clinical manifestations of the experimental infection by L. mexicana, with a T helper 1 response characterized by an IgG2a/IgG1 > 1 ratio and high lymphoproliferative response. These findings support immunization with the plasmid pVAX1::LmxMBA as a preventive strategy against cutaneous infection of L. mexicana.

Effect of bile acids on the expression of MRP3 and MRP4: An In vitro study in HepG2 cell line.

INTRODUCTION AND OBJECTIVES: Free and conjugated bile acids (BA's) cannot cross cell membranes; therefore, a particular transport system is required by the cell. Members of the family of ABC (ATP-binding proteins) transporters transfer bile acids in and out of the cell, preventing their accumulation. High intracellular concentrations of bile acids, such as those observed in cholestasis, have been related to oxidative stress and apoptosis, which in many cases are the leading causes of hepatocyte damage. MRP3 and MRP4 (multidrug resistance-associated protein 3 and 4) proteins belong to the ABC subfamily C, and are transporters of the hepatocyte's basolateral membrane with a compensatory role. Both transporters' increased expression constitutes an essential role in the protective and adaptive responses of bile acid overload, such as cholestasis. This work aimed to analyze both transporters' mRNA and protein expression in an in vitro model of cholestasis using HepG2 cell line treated with main bile acids. METHODS: The expression of transporters was investigated through confocal microscopy immunofluorescence, Western Blot, and RT-qPCR after the main bile acids in HepG2 line cells. RESULTS: The results showed the relation between confluence and expression of both transporters in the plasma membrane. MRP3 showed atypical and heterogeneous distribution in this cell line. CDCA (chenodeoxycholic acid) at low concentrations induced the expression of mRNA of both transporters. In contrast, protein expression was induced by CA (cholic acid) at high concentrations. CONCLUSION: Primary bile acids (CDCA and CA) induce overexpression of the MRP4 and MRP3 transporters in the HepG2 cell line.

Low molecular weight protein tyrosine phosphatase (LMW-PTP2) protein can potentially modulate virulence of the parasite Entamoeba histolytica.

The Entamoeba histolytica parasite is the causative agent of amebiasis, infecting approximately 1% of the world population and causing 100,000 deaths per year. It binds to Fibronectin (FN), activating signaling pathways regulated by kinases and phosphatases. EhLMW-PTPs genes from E. histolytica encode for Low Molecular Weight Tyrosine Phosphatases expressed in trophozoites and amoebic cysts. The role of these phosphatases in the virulence of the parasite has not yet been well characterized. Our results showed a differential expression of the EhLMW-PTPs, at the mRNA and protein levels, in an asynchronous trophozoites culture. Furthermore, we observed that trophozoites transfected that overexpressed EhLMW-PTP2 phagocytized fewer erythrocytes, possibly due to decreased phagocytic cups, and showed deficiencies in adherence to FN and less cytopathic effect. These analyzes suggest that the parasite's EhLMW-PTPs have an essential role in the mechanisms of proliferation, adhesion, and phagocytosis, regulating its pathogenicity.

Characterization of low molecular weight protein tyrosine phosphatases of Entamoeba histolytica.

Entamoeba histolytica is an intestinal protozoan parasite of humans and is endemic in developing countries. E. histolytica has two low molecular weight protein tyrosine phosphatase (LMW-PTP) genes, EhLMW-PTP1 and EhLMW-PTP2, which are expressed in cultured trophozoites, clinical isolates, and cysts. The amino acid sequences of proteins EhLMW-PTP1 and EhLMW-PTP2 showed only one amino acid difference between them at position A85V, respectively. Both genes are expressed in cultured trophozoites, mainly EhLMW-PTP2, and in trophozoites recovered from amoebic liver abscess, the expression of EhLMW-PTP1 is downregulated. We cloned the two genes and purified the corresponding recombinant (rEhLMW-PTPs) proteins. Antibodies anti-rEhLMW-PTP2 showed that during red blood cells uptake by E. histolytica, the EhLMW-PTPs were found in the phagocytic cups based on analysis of fluorescence signals. On the other hand, rEhLMW-PTPs showed an optimum phosphatase activity at pH 6.0 with p-nitrophenyl phosphate as the substrate. They dephosphorylate phosphotyrosine and 3-O-methylfluorescein phosphate, but not phosphoserine or phosphothreonine, and the enzymatic activity is inhibited by orthovanadate. rEhLMW-PTP1 and rEhLMW-PTP2 exhibited optimum temperatures of activities at 60 °C and 58 °C, respectively, with high thermal stability at 50 °C. Also, the rEhLMW-PTPs showed high specific activities and specific km value with pNPP or OMFP as the substrates at the physiological temperature (37 °C).

Electrolyzed Oxidizing Water Modulates the Immune Response in BALB/c Mice Experimentally Infected with Trypanosoma cruzi.

Chagas disease is a major public health problem in Latin America. The mixed Th1/Th2 immune response is required against Trypanosoma cruzi. Electrolyzed oxidizing water (EOW) has been shown to have germicidal efficacy. The objective of this study was to evaluate the EOW effectiveness in T. cruzi-infected BALB/c mice clinically, immunologically, and histologically. The severity of the infection was assessed by parasitaemia, general health condition, mortality, mega syndromes, and histological lesions. IgG, TNF-alpha, IFN-gamma, and IL-1 beta levels were quantified. The EOW administration showed a beneficial effect on parasitaemia, general physical condition, and mortality. High levels of IgG1 at 50 days postinfection were observed. Prophylactic EOW treatment was able to induce a predominantly TH1 immune response based on an IgG2a levels increase at the late acute phase, and a 10-fold increase of INF-gamma in whole acute phase. EOW was able to control the acute phase infection as effectively as benznidazole. Splenomegaly was caused by EOW treatment and lymphadenopathy was stimulated by T. cruzi infection in all groups. Severe tissue damage was not prevented by EOW treatments. Moderate efficacy may be due to immunomodulatory properties and not to a direct toxic effect on the parasite.

DNA Vaccine Treatment in Dogs Experimentally Infected with Trypanosoma cruzi.

Chagas disease is a chronic and potentially lethal disorder caused by the parasite Trypanosoma cruzi, and an effective treatment has not been developed for chronic Chagas disease. The objective of this study was to determine the effectiveness of a therapeutic DNA vaccine containing T. cruzi genes in dogs with experimentally induced Chagas disease through clinical, pathological, and immunological analyses. Infection of Beagle dogs with the H8 T. cruzi strain was performed intraperitoneally with 3500 metacyclic trypomastigotes/kg body weight. Two weeks after infection, plasmid DNA immunotherapy was administered thrice at 15-day intervals. The clinical (physical and cabinet studies), immunological (antibody and cytokine profiles and lymphoproliferation), and macro- and microscopic pathological findings were described. A significant increase in IgG and cell proliferation was recorded after immunotherapy, and the highest stimulation index (3.02) was observed in dogs treated with the pBCSSP4 plasmid. The second treatment with both plasmids induced an increase in IL-1, and the third treatment with the pBCSSP4 plasmid induced an increase in IL-6. The pBCSP plasmid had a good Th1 response regulated by high levels of IFN-gamma and TNF-alpha, whereas the combination of the two plasmids did not have a synergistic effect. Electrocardiographic studies registered lower abnormalities and the lowest number of individuals with abnormalities in each group treated with the therapeutic vaccine. Echocardiograms showed that the pBCSSP4 plasmid immunotherapy preserved cardiac structure and function to a greater extent and prevented cardiomegaly. The two plasmids alone controlled the infection moderately by a reduction in the inflammatory infiltrates in heart tissue. The immunotherapy was able to reduce the magnitude of cardiac lesions and modulate the cellular immune response; the pBCSP treatment showed a clear Th1 response; and pBCSSP4 induced a balanced Th1/Th2 immune response that prevented severe cardiac involvement. The pBCSSP4 plasmid had a better effect on most of the parameters evaluated in this study; therefore, this plasmid can be considered an optional treatment against Chagas disease in naturally infected dogs.

Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin-Binding Proteins.

Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha-actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.

Leishmania mexicana gp63 is the enzyme responsible for cyclooxygenase (COX) activity in this parasitic protozoa.

Cyclooxygenase-2 (COX-2) is an enzyme responsible of prostaglandins production, such as prostaglandin E2 (PGE2), an immune response modulator that regulates the immune system to inhibit Th1 and to promote Th2 cytokines production. Many parasites modulate their host immune response through PGE2 effects; however, in parasites, only one protein with COX activity has been described, the α-actinin of Entamoeba histolytica. Prostanoids production has been reported in some species of Leishmania but not the enzymes responsible of their production. To identify the protein responsible for COX activity in Leishmania mexicana, we examined total extracts of promastigotes and samples with COX activity were subjected to ion exchange column purification and precipitation with ammonium sulphate; fractions with activity were analyzed by SDS-PAGE and Western blot using an anti-mouse COX-2 polyclonal antibody. Results showed that in those samples with enzymatic activity, the anti-mouse COX-2 polyclonal antibody recognized a protein with an approximate molecular weight of 66 KDa. Bands recognized by the antibody were subjected to mass spectrometry analysis and the results showed that several peptides from the bands purified by two different methods, and that were recognized by the anti-mouse COX-2 polyclonal antibody corresponded to the Leishmania mexicana gp63 surface protease. L. mexicana gp63 was purified by a Concanavalin A (Con-A) affinity column and subjected to immunoprecipitation with a commercial anti-Leishmania gp63 polyclonal antibody; the immunoprecipitated sample was analyzed for COX activity showing that the anti-gp63 antibody did immunoprecipitate the COX activity. The presence of COX activity was further confirmed in amastigotes extracts of L. mexicana. Moreover, a recombinant gp63 protein was produced and its COX activity tested, confirming that gp63 is the molecule responsible for COX activity.

Recombinant Enolase of Trypanosoma cruzi as a Novel Vaccine Candidate against Chagas Disease in a Mouse Model of Acute Infection.

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease, which is considered by the World Health Organization to be a neglected tropical disease. Two drugs exist for the treatment of Chagas disease, nifurtimox and benznidazole; they are only effective in the acute phase, and a vaccine is currently not available. In this study, we used the recombinant enolase from T. cruzi H8 strain (MHOM/MX/1992/H8 Yucatán) (rTcENO) and its encoding DNA (pBKTcENO) to immunize mice and evaluate their protective effects in an experimental murine model of acute phase infection. Our results showed that mice vaccinated with rTcENO or its encoding DNA were able to generate typical specific antibodies (IgG1, IgG2a, and IgG2b), suggesting that a mixed Th1/Th2 immune response was induced. The parasite burden in the blood was reduced to 69.8% and 71% in mice vaccinated with rTcENO and pBKTcENO, respectively. The group vaccinated with rTcENO achieved 75% survival, in contrast to the group vaccinated with pBKTcENO that showed no survival in comparison to the control groups. Moreover, rTcENO immunization elevated the production of IFN-γ and IL-2 after the parasite challenge, suggesting that the Th1-type immune response was polarized. These results indicated that rTcENO could be used as a vaccine against Chagas disease.

Characterization of the protein tyrosine phosphatase PRL from Entamoeba histolytica.

Protein tyrosine phosphatase of regenerating liver (PRL) is a group of phosphatases that has not been broadly studied in protozoan parasites. In humans, PRLs are involved in metastatic cancer, the promotion of cell migration and invasion. PTPs have been increasingly recognized as important effectors of host-pathogen interactions. We characterized the only putative protein tyrosine phosphatase PRL (PTP EhPRL) in the eukaryotic human intestinal parasite Entamoeba histolytica. Here, we reported that the EhPRL protein possessed the classical HCX5R catalytic motif of PTPs and the CAAX box characteristic of the PRL family and exhibited 31-32% homology with the three human PRL isoforms. In amebae, the protein was expressed at low but detectable levels. The recombinant protein (rEhPRL) had enzymatic activity with the 3-o-methyl fluorescein phosphate (OMFP) substrate; this enzymatic activity was inhibited by the PTP inhibitor o-vanadate. Using immunofluorescence we showed that native EhPRL was localized to the cytoplasm and plasma membrane. When the trophozoites interacted with collagen, EhPRL relocalized over time to vesicle-like structures. Interaction with fibronectin increased the presence of the enzyme in the cytoplasm. Using RT-PCR, we demonstrated that EhPRL mRNA expression was upregulated when the trophozoites interacted with collagen but not with fibronectin. Trophozoites recovered from amoebic liver abscesses showed higher EhPRL mRNA expression levels than normal trophozoites. These results strongly suggest that EhPRL may play an important role in the biology and adaptive response of the parasite to the host environment during amoebic liver abscess development, thereby participating in the pathogenic mechanism.

Tritrichomonas foetus: characterisation of ecto-phosphatase activities in the endoflagelar form and their possible participation on the parasite's transformation and cytotoxicity.

The protist parasite Tritrichomonas foetus displays a pear-shaped (PS) and a pseudocystic or endoflagellar form (EFF). Here, we characterised the ecto-phosphatase activity on the surface of EFF and compare its biochemical properties to that of the PS regarding rate of substrate hydrolysis, pH activation profile and sensitivity to well-known phosphatases inhibitors. Two strains exhibiting low- and high-cytotoxicity were used. The enzyme activities of PS and EFF exhibited similar characteristics of protein tyrosine phosphatases (PTP). However, the ecto-phosphatase activities for both forms presented distinct kinetic parameters and different inhibition patterns by PTP inhibitors, suggesting the presence of distinct ecto-enzyme activities between PS and EFF, as well, between both strains. Ultrastructural cytochemistry confirmed the differential distribution of the ecto-phosphatase activity during the EFF transformation. An increase in the percentage of the EFF resulted in a proportional increase in the ecto-phosphatase activity. During EFF reversion, ecto-phosphatase activity decreased and was restored to the level found in the parasites before EFF induction. PS and EFF from the high-cytotoxic strain exhibited higher ecto-phosphatase activities than PS and EFF from the low-cytotoxic strain, respectively. In both strains, the EFF was more cytotoxic and exhibited higher ecto-phosphatase activity when compared to the PS. A large part of the ecto-phosphatase activities of EFF from both strains and PS from the high-cytotoxic strain was irreversibly inhibited when the parasites were pre-treated with a specific antibody against amoebic PTP (anti-EhPRL). Immunoreaction assays revealed that the anti-EhPRL antibody cross-reacted with a 24-kDa protein differentially expressed on the cell surface of PS and EFF T. foetus. A positive correlation was observed between the surface expression of 24-kDa protein and ecto-phosphatase activity. Irreversible inhibition of a part of the ecto-phosphatase activities partially blocked the EFF induction and the cytotoxic effects exerted by both forms. These results suggest that the ecto-phosphatase activities could play a role on the EFF transformation and cytotoxicity of T. foetus.

Association of Trypanosoma cruzi infection with risk factors and electrocardiographic abnormalities in northeast Mexico.

BACKGROUND: American trypanosomiasis is a major disease and public health issue, caused by the protozoan parasite Trypanosoma cruzi. The prevalence of T. cruzi has not been fully documented, and there are few reports of this issue in Nuevo Leon. The aim of this study was to update the seroprevalence rate of T. cruzi infection, including an epidemiological analysis of the risk factors associated with this infection and an electrocardiographic (ECG) evaluation of those infected. METHODS: Sera from 2,688 individuals from 10 municipalities in the state of Nuevo Leon, Mexico, were evaluated using an enzyme-linked immunosorbent assay and an indirect hemagglutination assay. An ECG case-control study was performed in subjects seropositive for T. cruzi and the results were matched by sex and age to seronegative residents of the same localities. A univariate analysis with χ2 and Fisher's exact tests was used to determine the association between seropositivity and age (years), sex, and ECG changes. A multivariate analysis was then performed to calculate the odd ratios between T. cruzi seropositivity and the risk factors. RESULTS: The seropositive rate was 1.93% (52/2,688). In the ECG study, 22.85% (8/35) of the infected individuals exhibited ECG abnormalities. Triatoma gerstaeckeri was the only vector reported. The main risk factors were ceiling construction material (P ≤ 0.0024), domestic animals (P ≤ 0.0001), and living in rural municipalities (P ≤ 0.0025). CONCLUSIONS: These findings demonstrate a 10-fold higher prevalence of Chagas disease than previously reported (0.2%), which implies a serious public health threat in northeastern Mexico. The epidemiological profile established in this study differs from that found in the rest of Mexico, where human populations live in close proximity to domiciliary triatomines.

In silico approach for the identification of immunological properties of enolase from Trypanosoma cruzi and its possible usefulness as vaccine in Chagas disease.

Nowadays, Chagas disease is a major health problem in Latin America that has been disseminated also into non-endemic countries. Currently, a vaccine against Chagas disease does not exist. In the present study, the gene encoding Trypanosoma cruzi enolase (TcENO) was amplified, cloned, and sequenced and the recombinant protein was purified. We used in silico and an experimental assay to investigate the immunological role of TcENO. The in silico assays showed that TcENO sequence contains characteristic motifs of enolase; additionally, a transmembranal region was identified, and this could indicate the potential membrane localization of TcENO. Moreover, both B lymphocyte and cytotoxic T lymphocytes (CTL) predicted epitopes were localized; these results suggest the possibility that TcENO can develop both humoral and cellular immune responses. Furthermore, the presence of antibodies was verified by western blot assays, showing that the purified recombinant protein was detected by sera from experimentally infected mice and sera of patients with Chagas disease. These results indicate that TcENO is immunogenic and could be used as a vaccine candidate.