Morphological and functional characterizations of Schwann cells stimulated with Mycobacterium leprae

Nerve damage, a characteristic of leprosy, is the cause of patient deformities and a consequence of Schwann cells (SC) infection by Mycobacterium leprae. Although function/dysfunction of SC in human diseases like leprosy is difficult to study, many in vitro models, including SC lines derived from rat and/or human Schwannomas, have been employed. ST88-14 is one of the cell lineages used by many researchers as a model for M. leprae/SC interaction. However, it is necessary to establish the values and limitations of the generated data on the effects of M. leprae in these SC. After evaluating the cell line phenotype in the present study, it is close to non-myelinating SC, making this lineage an ideal model for M. leprae/SC interaction. It was also observed that both M. leprae and PGL-1, a mycobacterial cell-wall component, induced low levels of apoptosis in ST88-14 by a mechanism independent of Bcl-2 family members.

The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria

Malaria remains an important health problem in tropical countries like Brazil. Thrombocytopenia is the most common hematological disturbance seen in malarial infection. Oxidative stress (OS) has been implicated as a possible mediator of thrombocytopenia in patients with malaria. This study aimed to investigate the role of OS in the thrombocytopenia of Plasmodium vivax malaria through the measurement of oxidant and antioxidant biochemical markers in plasma and in isolated platelets. Eighty-six patients with P. vivax malaria were enrolled. Blood samples were analyzed for total antioxidant and oxidant status, albumin, total protein, uric acid, zinc, magnesium, bilirubin, total thiols, glutathione peroxidase (GPx), malondialdehyde (MDA), antibodies against mildly oxidized low-density lipoproteins (LDL-/nLDL ratio) and nitrite/nitrate levels in blood plasma and GPx and MDA in isolated platelets. Plasma MDA levels were higher in thrombocytopenic (TCP) (median 3.47; range 1.55-12.90 µmol/L) compared with the non-thrombocytopenic (NTCP) patients (median 2.57; range 1.95-8.60 µmol/L). Moreover, the LDL-/nLDL autoantibody ratio was lower in TCP (median 3.0; range 1.5-14.8) than in NTCP patients (median 4.0; range 1.9-35.5). Finally, GPx and MDA were higher in the platelets of TPC patients. These results suggest that oxidative damage of platelets might be important in the pathogenesis of thrombocytopenia found in P. vivax malaria as indicated by alterations of GPx and MDA.

Experimental reinfection of BALB/c mice with different recombinant type I/III strains of Toxoplasma gondii: involvement of IFN-³ and IL-10

To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-³ and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-³ were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates

Plasmodium vivax control is now being hampered by drug resistance. Orthologous Plasmodium falciparum genes linked to chloroquine or sulfadoxine-pyrimethamine chemoresistance have been identified in P. vivax parasites, but few studies have been performed. The goal of the present work is to characterise pvmdr1 and pvdhfr genes in parasite isolates from a Brazilian endemic area where no molecular investigation had been previously conducted. The pvmdr1 analysis revealed the existence of single (85.7%) and double (14.3%) mutant haplotypes, while the pvdhfr examination showed the presence of double (57.2%) and triple (42.8%) mutant haplotypes. The implications of these findings are discussed.

Sequencing of E2 and NS5A regions of HCV genotype 3a in Brazilian patients with chronic hepatitis

Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.

Comparative randomised trial of high and conventional doses of praziquantel in the treatment of schistosomiasis mansoni

The efficacy of oral praziquantel in the treatment of schistosomiasis has been considered low by most public health institutions. In this paper, we compared the efficacy of two dosages of praziquantel (80 mg/kg vs. 50 mg/kg) in patients with chronic schistosomiasis mansoni. Two hundred eighty-eight patients with schistosomiasis from a community in Brazil were randomly divided into two groups: 145 patients (Group 1) received 80 mg/kg body weight of oral praziquantel divided in two equal doses with 1 h interval and 143 patients (Group 2) received 50 mg/kg body weight of oral praziquantel. To keep the study masked, patients in Group 2 received placebo 1 h after the first dose. All patients were subjected to clinical and ultrasonographic examination. Cure assessment was performed by repeating two stool examinations, by a quantitative method, at 30, 90 and 180 days after treatment. The morbidity of schistosomiasis was low, with a few cases of light periportal thickening and 16 cases of mild splenomegaly. The cure rates were 89.7% for Group 1 and 83.9% for Group 2. There was no difference in the efficacy of both therapeutic dosages of praziquantel assayed. The adverse reactions were more frequent with higher dosage.

Correlation of biological serum markers with the degree of hepatic fibrosis and necroinflammatory activity in hepatitis C and schistosomiasis patients

Liver biopsy is the gold-standard method to stage fibrosis; however, it is an invasive procedure and is potentially dangerous. The main objective of this study was to evaluate biological markers, such as cytokines IL-13, IFN-γ, TNF-α and TGF-β, platelets, bilirubins (Bil), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total proteins, γ-glutamil transferase (γ-GT) and alkaline phosphatase (AP), that could be used to predict the severity of hepatic fibrosis in schistosomiasis and hepatitis C (HC) as isolated diseases or co-infections. The following patient groups were selected: HC (n = 39), HC/hepatosplenic schistosomiasis (HSS) (n = 19), HSS (n = 22) and a control group (n = 13). ANOVA and ROC curves were used for statistical analysis. P < 0.05 was considered significant. With HC patients we showed that TNF-α (p = 0.020) and AP (p = 0.005) could differentiate mild and severe fibrosis. With regard to necroinflammatory activity, AST (p = 0.002), γ-GT (p = 0.034) and AP (p = 0.001) were the best markers to differentiate mild and severe activity. In HC + HSS patients, total Bil (p = 0.008) was capable of differentiating between mild and severe fibrosis. In conclusion, our study was able to suggest biological markers that are non-invasive candidates to evaluate fibrosis and necroinflammatory activity in HC and HC + HSS.

Developmental and reproductive patterns of Triatoma brasiliensis infected with Trypanosoma cruzi under laboratory conditions

The aim of this work was to study the interaction between Trypanosoma cruzi-1 and Triatoma brasiliensis. A group of 1st instar nymphs was initially fed on T. cruzi-infected mice and a control group was fed on uninfected mice. From the second feeding onwards, both groups were otherwise fed on non-infected mice. The resulting adults were grouped in pairs: infected male/uninfected female, uninfected male/infected female, infected male and female and uninfected male/uninfected female. The infection affected only the 1st instar nymphs, which took significantly more time to reach the 2nd instar than uninfected nymphs. The differences in the molting time between the infected and uninfected nymphs from the 2nd to the 5th instars were not statistically significant. Both groups presented similar rates of nymphal mortality and reproductive performance was not significantly affected by infection in any of the treatments.

Vaccination with Trypanosoma rangeli modulates the profiles of immunoglobulins and IL-6 at local and systemic levels in the early phase of Trypanosoma cruzi experimental infection

In America, there are two species of Trypanosoma that can infect humans: Trypanosoma cruzi, which is responsible for Chagas disease and Trypanosoma rangeli, which is not pathogenic. We have developed a model of vaccination in mice with T. rangeli epimastigotes that protects against T. cruzi infection. The goal of this work was to study the pattern of specific immunoglobulins in the peritoneum (the site of infection) and in the sera of mice immunized with T. rangeli before and after challenge with T. cruzi. Additionally, we studied the effects triggered by antigen-antibodies binding and the levels of key cytokines involved in the humoral response, such as IL-4, IL-5 and IL-6. The immunization triggered the production of antibodies reactive with T. cruzi in peritoneal fluid (PF) and in serum, mainly IgG1 and, to a lesser magnitude, IgG2. Only immunized mice developed specific IgG3 antibodies in their peritoneal cavities. Antibodies were able to bind to the surface of the parasites and agglutinate them. Among the cytokines studied, IL-6 was elevated in PF during early infection, with higher levels in non-immunized-infected mice. The results indicate that T. rangeli vaccination against T. cruzi infection triggers a high production of specific IgG isotypes in PF and sera before infection and modulates the levels of IL-6 in PF in the early periods of infection.

Correlation of meta 1 expression with culture stage, cell morphology and infectivity in Leishmania (Leishmania) amazonensis promastigotes

The parasitic protozoan Leishmania (Leishmania) amazonensis alternates between mammalian and insect hosts. In the insect host, the parasites proliferate as procyclic promastigotes andthen differentiate into metacyclic infective forms. The meta 1 gene is preferentially expressed during metacyclogenesis. Meta 1 expression profile determination along parasite growth curves revealed that the meta 1 mRNA level peaked at the early stationary phase then decreased to an intermediate level. No correlation was observed between meta 1 expression and infectivity. Conversely, infectivity correlated with the increase of apoptotic cells in the late stationary phase.

A comparative evaluation of endemic and non-endemic region of visceral leishmaniasis (Kala-azar) in India with ground survey and space technology

In visceral leishmaniasis, phlebotomine vectors are targets for control measures. Understanding the ecosystem of the vectors is a prerequisite for creating these control measures. This study endeavours to delineate the suitable locations of Phlebotomus argentipes with relation to environmental characteristics between endemic and non-endemic districts in India. A cross-sectional survey was conducted on 25 villages in each district. Environmental data were obtained through remote sensing images and vector density was measured using a CDC light trap. Simple linear regression analysis was used to measure the association between climatic parameters and vector density. Using factor analysis, the relationship between land cover classes and P. argentipes density among the villages in both districts was investigated. The results of the regression analysis indicated that indoor temperature and relative humidity are the best predictors for P. argentipes distribution. Factor analysis confirmed breeding preferences for P. argentipes by landscape element. Minimum Normalised Difference Vegetation Index, marshy land and orchard/settlement produced high loading in an endemic region, whereas water bodies and dense forest were preferred in non-endemic sites. Soil properties between the two districts were studied and indicated that soil pH and moisture content is higher in endemic sites compared to non-endemic sites. The present study should be utilised to make critical decisions for vector surveillance and controlling Kala-azar disease vectors.

Evaluation of rapid techniques for the detection of mycobacteria in sputum with scanty bacilli or clinically evident, smear negative cases of pulmonary and extra-pulmonary tuberculosis

The objective of the current study was to compare two rapid methods, the BBL Mycobacteria Growth Indicator Tube (MGIT TM) and Biotec FASTPlaque TB TM (FPTB) assays, with the conventional Löwenstein-Jensen (LJ) media assay to diagnose mycobacterial infections from paucibacillary clinical specimens. For evaluation of the clinical utility of the BBL MGIT TM and FPTB assays, respiratory tract specimens (n = 208), with scanty bacilli or clinically evident, smear negative cases and non-respiratory tract specimens (n = 119) were analyzed and the performance of each assay was compared with LJ media. MGIT and FPTB demonstrated a greater sensitivity (95.92% and 87.68%), specificity (94.59% and 98.78%), positive predictive value (94.91% and 99.16%) and negative predictive value (96.56% and 90.92%), respectively, compared to LJ culture for both respiratory tract and non-respiratory tract specimens. However, the FPTB assay was unable to detect nontuberculous mycobacteria and few Mycobacterium tuberculosis complex cases from paucibacillary clinical specimens. It is likely that the analytical sensitivity of FPTB is moderately low and may not be useful for the direct detection of tuberculosis in paucibacillary specimens. The current study concluded that MGIT was a dependable, highly efficient system for recovery of M. tuberculosis complexes and nontuberculous mycobacteria from both respiratory and non-respiratory tract specimens in combination with LJ media.

Monoclonal auto-antibodies and sera of autoimmune patients react with Plasmodium falciparum and inhibit its in vitro growth

The relationship between autoimmunity and malaria is not well understood. To determine whether autoimmune responses have a protective role during malaria, we studied the pattern of reactivity to plasmodial antigens of sera from 93 patients with 14 different autoimmune diseases (AID) who were not previously exposed to malaria. Sera from patients with 13 different AID reacted against Plasmodium falciparum by indirect fluorescent antibody test with frequencies varying from 33-100%. In addition, sera from 37 AID patients were tested for reactivity against Plasmodium yoelii 17XNL and the asexual blood stage forms of three different P. falciparum strains. In general, the frequency of reactive sera was higher against young trophozoites than schizonts (p < 0.05 for 2 strains), indicating that the antigenic determinants targeted by the tested AID sera might be more highly expressed by the former stage. The ability of monoclonal auto-antibodies (auto-Ab) to inhibit P. falciparum growth in vitro was also tested. Thirteen of the 18 monoclonal auto-Ab tested (72%), but none of the control monoclonal antibodies, inhibited parasite growth, in some cases by greater than 40%. We conclude that autoimmune responses mediated by auto-Ab may present anti-plasmodial activity.

The infection of microvascular endothelial cells with ExoU-producing Pseudomonas aeruginosa triggers the release of von Willebrand factor and platelet adhesion

An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.

The in vivo antimalarial activity of methylene blue combined with pyrimethamine, chloroquine and quinine

The effectiveness of methylene blue (MB) combined with pyrimethamine (PYR), chloroquine (CQ) or quinine (Q) was examined in a classical four-day suppressive test against a causative agent of rodent malaria, Plasmodium berghei. A marked potentiation was observed when MB was administered at a non-curative dose of 15 mg/kg/day in combination with PYR (0.19 mg/kg/day) or Q (25 mg/kg/day). No synergy was found between MB (15 mg/Kg) and CQ (0.75 mg/Kg). Our results suggest that the combination of MB with PYR or Q may improve the efficacy of these currently used antimalarial drugs.