Evaluation of the allergenicity potential of TcPR-10 protein from Theobroma cacao.

BACKGROUND: The pathogenesis related protein PR10 (TcPR-10), obtained from the Theobroma cacao-Moniliophthora perniciosa interaction library, presents antifungal activity against M. perniciosa and acts in vitro as a ribonuclease. However, despite its biotechnological potential, the TcPR-10 has the P-loop motif similar to those of some allergenic proteins such as Bet v 1 (Betula verrucosa) and Pru av 1 (Prunus avium). The insertion of mutations in this motif can produce proteins with reduced allergenic power. The objective of the present work was to evaluate the allergenic potential of the wild type and mutant recombinant TcPR-10 using bioinformatics tools and immunological assays. METHODOLOGY/PRINCIPAL FINDINGS: Mutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by site-directed mutagenesis, cloned into pET28a and expressed in Escherichia coli BL21(DE3) cells. Changes in molecular surface caused by the mutant substitutions was evaluated by comparative protein modeling using the three-dimensional structure of the major cherry allergen, Pru av 1 as a template. The immunological assays were carried out in 8-12 week old female BALB/c mice. The mice were sensitized with the proteins (wild type and mutants) via subcutaneous and challenged intranasal for induction of allergic airway inflammation. CONCLUSIONS/SIGNIFICANCE: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of IgE production and cellular infiltration in the lungs. On the other hand, in vitro assays show that the TcPR-10 mutants still present antifungal and ribonuclease activity against M. perniciosa RNA. In conclusion, the mutant proteins present less allergenic potential than the wild TcPR-10, without the loss of interesting biotechnological properties.

Reactive oxygen species and nitric oxide in cutaneous leishmaniasis.

Cutaneous leishmaniasis affects millions of people around the world. Several species of Leishmania infect mouse strains, and murine models closely reproduce the cutaneous lesions caused by the parasite in humans. Mouse models have enabled studies on the pathogenesis and effector mechanisms of host resistance to infection. Here, we review the role of nitric oxide (NO), reactive oxygen species (ROS), and peroxynitrite (ONOO(-)) in the control of parasites by macrophages, which are both the host cells and the effector cells. We also discuss the role of neutrophil-derived oxygen and nitrogen reactive species during infection with Leishmania. We emphasize the role of these cells in the outcome of leishmaniasis early after infection, before the adaptive T(h)-cell immune response.

Glycoinositolphospholipids from Leishmania braziliensis and L. infantum: modulation of innate immune system and variations in carbohydrate structure.

The essential role of the lipophosphoglycan (LPG) of Leishmania in innate immune response has been extensively reported. However, information about the role of the LPG-related glycoinositolphospholipids (GIPLs) is limited, especially with respect to the New World species of Leishmania. GIPLs are low molecular weight molecules covering the parasite surface and are similar to LPG in sharing a common lipid backbone and a glycan motif containing up to 7 sugars. Critical aspects of their structure and functions are still obscure in the interaction with the vertebrate host. In this study, we evaluated the role of those molecules in two medically important South American species Leishmania infantum and L. braziliensis, causative agents of visceral (VL) and cutaneous Leishmaniasis (CL), respectively. GIPLs derived from both species did not induce NO or TNF-α production by non-primed murine macrophages. Additionally, primed macrophages from mice (BALB/c, C57BL/6, TLR2-/- and TLR4-/-) exposed to GIPLs from both species, with exception to TNF-α, did not produce any of the cytokines analyzed (IL1-ß, IL-2, IL-4, IL-5, IL-10, IL-12p40, IFN-γ) or p38 activation. GIPLs induced the production of TNF-α and NO by C57BL/6 mice, primarily via TLR4. Pre incubation of macrophages with GIPLs reduced significantly the amount of NO and IL-12 in the presence of IFN-γ or lipopolysaccharide (LPS), which was more pronounced with L. braziliensis GIPLs. This inhibition was reversed after PI-specific phospholipase C treatment. A structural analysis of the GIPLs showed that L. infantum has manose rich GIPLs, suggestive of type I and Hybrid GIPLs while L. braziliensis has galactose rich GIPLs, suggestive of Type II GIPLs. In conclusion, there are major differences in the structure and composition of GIPLs from L. braziliensis and L. infantum. Also, GIPLs are important inhibitory molecules during the interaction with macrophages.

Infecções genitais por Chlamydia trachomatis em mulheres / Genital infections for Chlamydia trachomatis in women

Os autores apresentam uma revisão da literatura sobre cervicovaginites causadas por Chlamydia trachomatis e discorrem sobre a epidemiologia, a teraia e o seguimento das pacientes. Atualmente mulheres que após o tratamento apresentam resultados positivos para C. trachomatis são consideradas como reinfectadas. Contudo, discutem estudos que mostram evidências de reemergência de infecções latentes persistentes em mulheres tratadas, ressaltando a importância do seguimento dessas paciente.

The authors present a literature review about cervicovaginitis caused by Chlamydia trachomatis and discuss the epidemiology, therapy and follow-up of the patients. Nowadays, women previously treated for chlamydial infection and presenting positive tests are considered reinfected. However, the authors discuss studies showing evidence of latent infection that persists after treatment, and therefore emphasize the importance of treatment follow-up.

Genotyping of Chlamydia trachomatis from endocervical specimens in Brazil.

BACKGROUND: There is no data concerning genotyping of Chlamydia trachomatis from Brazilian samples. GOAL: To characterize the genotype of C. trachomatis detected in women assisted at a STD public clinic and establish the prevalence of this infection in that population. STUDY DESIGN: Endocervical samples of a group of 100 women were tested for chlamydial infection with PCR directed to C. trachomatis cryptic plasmid. Genotyping of positive samples were done after omp1 amplification and sequencing. RESULTS: The overall prevalence of C. trachomatis infection was 19%, with the highest prevalence in women between 15 and 25 years old (68.4%). Four genotypes were found associated with endocervical infections: D, E, F, and K. Sequence analysis revealed a coinfection of genotypes D and E in 1 woman. CONCLUSIONS: To our knowledge this is the first study to characterize Brazilian C. trachomatis endocervical samples and Brazilian C. trachomatis genotype coinfection. Our results also emphasize the importance of routine diagnosis of C. trachomatis for the control of this STD.

In vitro activation of the hemolysin in Prevotella nigrescens ATCC 33563 and Prevotella intermedia ATCC 25611.

Hemolytic activity was evaluated in the putative periodontopathogens Prevotella intermedia and Prevotella nigrescens. Whole cells of both species present weak hemolytic activity evidenced only by solid media assays after 48 h of bacterial growth or after 5 h of interaction with erythrocytes at 37 degrees C in liquid assays. In this work we show that the use of crude extract allowed the detection of a higher hemolytic activity for P. intermedia, but surprisingly not for P. nigrescens. Incubation at 37 degrees C for 9 h, or treatment with trypsin or proteinase K, increased or exposed the hemolytic activity of P. intermedia and P. nigrescens crude extract, respectively. The activation process was inhibited by TLCK and PMSF but not by EDTA, E-64 or pepstatin A, indicating the serino-protease nature of the factor involved in activation of P. intermedia and P. nigrescens hemolysins. Both the buffer and the pH employed for cell fractionation influenced the activation of hemolysin, and the best results were obtained with Universal buffer at pH 8.0. The activated hemolysins acted optimally at pH 6.5 at 37 degrees C and the maximum hemolytic activity was detected at the early log phase of growth. The results of this study show for the first time a strong hemolytic activity for P. nigrescens and evidence of proteolytic activation of hemolysins produced by periodontopathogens.

Hemolytic activity of Prevotella intermedia and Prevotella nigrescens strains: influence of abiotic factors in solid and liquid assays.

The influence of growth medium, hemin and menadione, blood source and atmosphere of incubation on the expression of hemolytic activity of 25 strains of Prevotella intermedia and Prevotella nigrescens was evaluated. The best hemolytic activity was observed for samples of both species growing in brain heart infusion agar and incubated in Brewer-like anaerobic jars for 48 h. Hemolysis was less intense and occurred later in the presence of hemin and menadione in solid media. beta-Hemolysis was detected for medium supplemented with horse or human blood and alpha-hemolysis was observed when sheep blood was used. These results suggesting some specificity for the hemolytic activity were also observed in liquid assays in which sheep erythrocytes were found to be resistant to hemolysis while horse and human cells where lysed. In liquid assays, the hemolytic activity of all studied strains remained stable in the pH range of 6.0 to 8.5 and was not altered by iron-scavenging compounds or atmosphere of incubation. The phenomenon of hot/cold hemolysis was ruled out as the mechanism of action of P. intermedia and P. nigrescens hemolysin.