Humoral Immune Response Kinetics in Philander opossum and Didelphis marsupialis Infected and Immunized by Trypanosoma cruzi Employing an Immunofluorescence Antibody Test

Philander opossum and Didelphis marsupialis considered the most ancient mammals and an evolutionary success, maintain parasitism by Trypanosoma cruzi without developing any apparent disease or important tissue lesion. In order to elucidate this well-balanced interaction, we decided to compare the humoral immune response kinetics of the two didelphids naturally and experimentally infected with T. cruzi and immunized by different schedules of parasite antigens, employing an indirect fluorescence antibody test (IFAT). Both didelphids responded with high serological titers to different immunization routes, while the earliest response occurred with the intradermic route. Serological titers of naturally infected P. opossum showed a significant individual variation, while those of D. marsupialis remained stable during the entire follow-up period. The serological titers of the experimentally infected animals varied according to the inoculated strain. Our data suggest that (1) IFAT was sensitive for follow-up of P. opossum in natural and experimental T. cruzi infections; (2) both P. opossum and D. marsupialis are able to mount an efficient humoral immune response as compared to placental mammals; (3) experimentally infected P. opossum and D. marsupialis present distinct patterns of infection, depending on the subpopulation of T. cruzi, (4) the differences observed in the humoral immune responses between P. opossum and D. marsupialis, probably, reflect distinct strategies selected by these animals during their coevolution with T. cruzi.

A Preliminary Investigation on the Chemical Composition of the Cell Surface of Five Enteropathogenic Escherichia coli Serotypes

The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy. Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins. Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues. The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100oC for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon. Phosphate, total sugar and protein contents were determined. Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose. Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction. Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction.

Kinetics of T cell-activation molecules in response to Mycobacterium tuberculosis antigens

The phenotypic features acquired subsequent to antigen-specific stimulation in vitro were evaluated by means of the kinetic expressions of CD69 and CD25 activation molecules on T lymphocytes and assayed by flow cytometry in response to PPD, Ag85B, and ferritin in PPD-positive healthy control individuals. In response to PHA, CD69 staining on both CD4+ and CD8+ T cells became initially marked after 4 h, peaked at 24 h, and quickly decreased after 120 h. For CD25, a latter expression was detected around 8 h, having increased after 96 h. As expected, the response rate to the mycobacterial antigens was much lower than that to the mitogen. Positive staining was high after 96 h for CD25 and after 24 h for CD69. CD69 expression was significantly enhanced (p < 0.05) on CD8+ as compared to CD4+ T cells. High levels were also found between 96-120 h. Regarding Ag85B, CD25+ cells were mostly CD4+ instead of CD8+ T cells. Moreover, in response to ferritin, a lower CD25 expression was noted. The present data will allow further characterization of the immune response to new mycobacterial-specific antigens and their evaluation for possible inclusion in developing new diagnostic techniques for tuberculosis as well in a new vaccine to prevent the disease.

Experimental kinetics of infection induced by Yersinia pseudotuberculosis isolated from stock animals

The course of in vivo infection of five isolates of Yersinia pseudotuberculosis was followed for three weeks in Swiss mice. The strains were isolated from diarrheic and normal feces and mesenteric lymph nodes of healthy and sick stock animals. Four strains of serogroup O:3 and one of serogroup O:1a, with and without the virulence plasmid, were inoculated intragastrically and intravenously in the mice. Groups of five animals were sacrificed at 6 h and 3, 6, 10, 15, and 21 days after inoculation, and organs and tissues were checked for possible macroscopic alterations. Development of infection was monitored at these times by performing viable bacterial counts in homogenates of selected tissues. The animals were cheked daily for clinical alterations. The results of the study showed that strains with the virulence plasmid infected organs and tissues at various times and at varying intensity by both routes of infection, the strain of type O:1a being the most invasive. Moreover, clinical and pathological alterations occurred only in animals inoculated with bacteria carrying the virulence plasmid, regardless of the route of infection.

Antimicrobial activity and chemical investigation of Brazilian Drosera

The antimicrobial activity of three different extracts (hexanic, ethyl acetate, methanol) obtained from Brazilian Drosera species (D. communis, D. montana var. montana, D. brevifolia, D. villosa var. graomogolensis, D. villosa var. villosa, Drosera sp. 1, and Drosera sp. 2 ) were tested against Staphylococcus aureus (ATCC 25923), Enterococcus faecium (ATCC23212), Pseudomonas aeruginosa (ATCC27853), Escherichia coli (ATCC11229), Salmonella choleraesuis (ATCC10708), Klebsiella pneumoniae (ATCC13883), and Candida albicans (a human isolate). Better antimicrobial activity was observed with D. communis and D. montana var. montana ethyl acetate extracts. Phytochemical analyses from D. communis, D. montana var. montana and D. brevifolia yielded 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin); long chain aliphatic hydrocarbons were isolated from D. communis and from D. villosa var. villosa, a mixture of long chain aliphatic alcohols and carboxylic acids, was isolated from D. communis and 3b-O-acetylaleuritolic acid from D. villosa var. villosa.

Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.

Infection kinetics of human adenovirus serotype 41 in HEK 293 cells

The purpose of this work was to acquire an overview of the infectious cycle of HAdV-41 in permissive HEK 293 cells and compare it to that observed with the prototype of the genus, Human adenovirus C HAdV-2. HEK 293 cells were infected with each virus separately and were harvested every 12 h for seven days. Infection kinetics were analysed using confocal and electronic microscopy. The results show that, when properly cultivated, HAdV-41 was not fastidious. It had a longer multiplication cycle, which resulted in the release of complete viral particles and viral stocks reached high titres. After 60 h of infection, the export of viral proteins from the infected cell to the extracellular milieu was observed, with a pattern similar to that previously described for HAdV-2 penton-base trafficking after 30 h of infection. HAdV-41 had a non-lytic cycle and the infection spread from the first infected cell to its neighbours. The release process of the viral particles is unknown. The results observed for HAdV-41 infection in HEK 293 cells show how different this virus is from the prototype HAdV-2 and provides information for the development of this vector for use in gene therapy.

Chemical composition and antiprotozoal activities of Colombian Lippia spp essential oils and their major components

The chemical composition and biological activities of 19 essential oils and seven of their major components were tested against free and intracellular forms of Leishmania chagasi and Trypanosoma cruzi parasites as well as Vero and THP-1 mammalian cell lines. The essential oils were obtained from different species of Lippia, a widely distributed genus of Colombian plants. They were extracted by microwave radiation-assisted hydro-distillation and characterised by GC-FID and GC-MS. The major components were geranial, neral, limonene, nerol, carvacrol, p-cymene, γ-terpinene, carvone and thymol. The essential oil of Lippia alba exhibited the highest activity against T. cruzi epimastigotes and intracellular amastigotes with an IC50 of 5.5 μg/mL and 12.2 μg/mL, respectively. The essential oil of Lippia origanoides had an IC50 of 4.4 μg/mL in L. chagasi promastigotes and exhibited no toxicity in mammalian cells. Thymol (IC50 3.2 ± 0.4 μg/mL) and S-carvone (IC50 6.1 ± 2.2 μg/mL), two of the major components of the active essential oils, were active on intracellular amastigotes of T. cruziinfected Vero cells, with a selective index greater than 10. None of the essential oils or major components tested in this study was active on amastigotes of L. chagasi infected THP-1 cells.

Growth, cysts and kinetics of Borrelia garinii (Spirochaetales: Spirochaetacea) in different culture media

The aim of the present paper was to evaluate cyst formation and growth parameters of Borrelia garinii in a range of media differing in formulation and cost. A qualitative assessment of morphology and motility of B. garinii was conducted. All media were prepared aseptically and used in test tubes or Petri dishes. For each medium, the initial spirochete concentration was standardized to 10³ spirochets/mL. The following culture media were suitable to grow B. garinii: Barbour-Stoenner-Kelly, brain heart infusion and PMR. Growth was minimal at six weeks post-inoculation and maximum spirochete density was observed between 9-12 weeks. Often, the cultures developed cysts of different sizes, isolated or in groups, with a spiraled portion of variable sizes, mainly in unfavorable culture media. Brazilian Lyme disease-like illness, also known as Baggio-Yoshinari syndrome (BYS), is a new and interesting emerging tick-borne disease, caused by Borrelia burgdorferi sensu lato spirochetes, only during its cystic forms. It has been assumed that the peculiar clinical and laboratory features of BYS are consequential to the absence of a human sucker Ixodes ricinus complex tick at risk areas in Brazil, supporting the concept that the borrelia phenotypic expression pattern is modified as it is transmitted through the host.

Modulation of expression and activity of cytochrome P450s and alteration of praziquantel kinetics during murine schistosomiasis

In this study, we investigated the expression and activity of liver cytochrome P450s (CYPs) and praziquantel (PZQ) kinetics in mice infected with Schistosoma mansoni. Swiss Webster (SW) mice of both genders were infected (100 cercariae) on postnatal day 10 and killed on post-infection days (PIDs) 30 or 55. Non-infected mice of the same age and sex served as controls. Regardless of mouse sex, infection depressed the activities of CYP1A [ethoxy/methoxy-resorufin-O-dealkylases (EROD/MROD)], 2B9/10 [pentoxy/benzyloxy-resorufin-O-dealkylases (PROD, BROD)], 2E1 [p-nitrophenol-hydroxylase (PNPH)] and 3A11 [erythromycin N-demethylase (END)] on PID 55 but not on PID 30. On PID 55, infection decreased liver CYP mRNA levels (real-time reverse transcription-polymerase chain reaction). On PID 30, whereas mRNA levels remained unaltered in males, they were depressed in females. Plasma PZQ (200 and 400 mg/kg body weight intraperitoneally) levels were measured (high-performance liquid chromatography) at different post-treatment intervals. In males and females, infection delayed the PZQ clearance on PID 55, but not on PID 30. Therefore, it can be concluded that schistosomiasis down-modulated CYP expression and activity and delayed PZQ clearance on PID 55, when a great number of parasite eggs were lodged in the liver. On PID 30, when egg-laying was initiated by the worms, no change of CYP expression and activity was found, except for a depression of CYP1A2 and 3A11 mRNAs in female mice.

A single nucleotide polymorphism, rs129679860, in the IL28B locus is associated with the viral kinetics and a sustained virological response in a chronic, monoinfected hepatitis C virus genotype-1 Brazilian population treated with pegylated interferon-ribavirin

Single nucleotide polymorphisms (SNPs) in the interleukin (IL)28B locus have been associated with a sustained virological response (SVR) in interferon-ribavirin (IFN-RBV)-treated chronic hepatitis C virus (HCV)-infected patients in European and African populations. In this study, the genotype frequency of two IL28B SNPs (rs129679860 and rs8099917) in a cohort of chronic HCV-monoinfected patients in Brazil was evaluated and the SNP sufficient to predict the treatment response outcome was determined. A total of 66 naïve genotype-1 chronic HCV-infected patients were genotyped and the associated viral kinetics and SVR were assessed. The overall SVR was 38%. Both the viral kinetics and SVR were associated with rs129679860 genotypes (CC = 62% vs. CT = 33% vs. TT = 18%, p = 0.016). However, rs8099917 genotypes were only associated with SVR (TT = 53% vs. TG = 33% vs. GG = 18%; p = 0.032). In this population, the analysis of a single SNP, rs12979860, successfully predicts SVR in the IFN-RBV treatment of HCV.

Superoxide dismutases and glutaredoxins have a distinct role in the response of Candida albicans to oxidative stress generated by the chemical compounds menadione and diamide

To cope with oxidative stress, Candida albicans possesses several enzymes involved in a number of biological processes, including superoxide dismutases (Sods) and glutaredoxins (Grxs). The resistance of C. albicans to reactive oxygen species is thought to act as a virulence factor. Genes such as SOD1 and GRX2, which encode for a Sod and Grx, respectively, in C. albicans are widely recognised to be important for pathogenesis. We generated a double mutant, Δgrx2/sod1, for both genes. This strain is very defective in hyphae formation and is susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, the double null mutant was susceptible to menadione and resistant to diamide. The reintegration of the SOD1 gene in the null mutant led to recovery in resistance to menadione, whereas reintegration of the GRX2 gene made the null mutant sensitive to diamide. Despite having two different roles in the responses to oxidative stress generated by chemical compounds, GRX2 and SOD1 are important for C. albicans pathogenesis because the double mutant Δgrx2/sod1 was very susceptible to neutrophil killing and was defective in hyphae formation in addition to having a lower virulence in an animal model of systemic infection.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

Morphological, chemical and developmental aspects of the Dufour gland in some eusocial bees (Hymenoptera, Apidae): a review

Morphological, chemical and developmental aspects of the Dufour gland in some eusocial bees (Hymenoptera, Apidae): a review. The present revision focused on the more recent data about the Dufour gland in some eusocial bees, taking in account general aspects of its morphology, secretion chemical nature, bio-synthetic pathway and development. Many functions have been attributed to this gland in eusocial bees, but none are convincing. With the new data about this gland, not only the secretion chemical pathway of the Dufour gland may be reasonable understood, as its function in some eusocial bees, especially Apis mellifera Linné, 1758, which has been extensively studied in the last years.

Influence of tree species on the herbaceous understory and soil chemical characteristics in a silvopastoral system in semi-arid northeastern Brazil

Studies from some semi-arid regions of the world have shown the beneficial effect of trees in silvopastoral systems, by promoting the formation of resource islands and increasing the sustainability of the system. No data are available in this respect for tree species of common occurrence in semi-arid Northeastern Brazil. In the present study, conducted in the summer of 1996, three tree species (Zyziphus joazeiro, Spondias tuberosa and Prosopis juliflora: ) found within Cenchrus ciliaris pastures were selected to evaluate differences on herbaceous understory and soil chemical characteristics between samples taken under the tree canopy and in open grass areas. Transects extending from the tree trunk to open grass areas were established, and soil (0-15 cm) and herbaceous understory (standing live biomass in 1 m² plots) samples were taken at 0, 25, 50, 100, 150 and 200% of the average canopy radius (average radius was 6.6 ± 0.5, 4.5 ± 0.5, and 5.3 ± 0.8 m for Z. joazeiro, P. juliflora, and S. tuberosa , respectively). Higher levels of soil C, N, P, Ca, Mg, K, and Na were found under the canopies of Z. joazeiro and P. juliflora: trees, as compared to open grass areas. Only soil Mg organic P were higher under the canopies of S. tuberosa trees, as compared to open grass areas. Herbaceous understory biomass was significantly lower under the canopy of S. tuberosa and P. juliflora trees (107 and 96 g m-2, respectively) relatively to open grass areas (145 and 194 g m-2). No herbaceous biomass differences were found between Z. joazeiro canopies and open grass areas (107 and 87 g m-2, respectively). Among the three tree species studied, Z. joazeiro was the one that presented the greatest potential for use in a silvopastoral system at the study site, since it had a larger nutrient stock in the soil without negatively affecting herbaceous understory biomass, relatively to open grass areas.