Antagonistic activity of Lactiplantibacillus plantarum 6.2 extracted from cocoa fermentation and its supernatant on Gardnerella vaginalis.

Search for alternative methods for the treatment of bacterial vaginosis has been growing, and probiotics being among them. The most well-known probiotic microorganisms are lactobacilli, which are naturally present in the vaginal microenvironment. Cocoa fermentation is a source of lactic acid bacteria, with lactobacilli being the most prominent. The aim of this study was to evaluate the antagonistic activity of Lactiplantibacillus plantarum 6.2 a strain of lactobacilli isolated from cocoa fermentation, and its cell-free supernatant on Gardnerella vaginalis. It was shown that Lpb. plantarum 6.2 and its supernatant, used at three concentrations, i.e., 40, 20 and 10 mg/mL, have a strong antagonistic activity against G. vaginalis, with a probable action of proteinaceous bacteriocins; the activity was lost after heat treatment. The ability to exclude and displace G. vaginalis from the adhesion site to vaginal HMVII epithelial cells was also demonstrated by the lactobacilli and the supernatant, with the latter showing a bactericidal effect. Thus, the Lpb. plantarum 6.2 strain presents itself as a good probiotic with potential to be used not only as a therapeutic alternative for vaginosis but also as a complement to existing therapies.

Metagenomic alkaline protease from mangrove sediment.

Functional screening of metagenomic libraries is an important tool for the discovery of new molecules. The metabolic diversity of microorganisms enables survival in harsh environments and is related to the production of enzymes. In this study, we identified a protease-producing clone from a metagenomic library derived from mangrove sediment. The protease was purified by ammonium sulphate precipitation and gel filtration chromatography, with a yield of 77.27% and a specific activity of 8.57 U µg-1 . It had a molecular weight of approximately 70 kDa. MS/MS in ESI-Q-TOF revealed nine peptides similar to a peptidase of Bacillus safensis. The aligned partial sequence showed 47.48% identity and 82.74% similarity to the conserved domains of a glutamyl aminopeptidase from the human gut metagenome and 32.12% total coverage. The protease had an optimal pH of 8.5 and optimal activity at 60°C. At pH 9-12, its activity was greater than 80%. It had moderate thermotolerance and thermostability at temperatures of 40 and 50 °C. The KM and Vmax values were estimated to be 0.92 mg ml-1 , and 13.15 mmol min-1 for azocasein. Substrate specificity analysis showed that PR4A3 was active on gelatin, blood, egg yolk, and milk. These results support the potential use of PR4A3 in biotechnological applications.

Application of PCR in serum samples for diagnosis of paracoccidioidomycosis in the southern Bahia-Brazil.

Paracoccidioidomycosis (PCM) cannot always be diagnosed by conventional means such as direct examination of histopathology or clinical samples, and serological methods, used as an alternative, still have many cases of cross-reactivity. In this scenario, molecular techniques seem to arise as a rapid approach, specific and direct that could be used in the diagnosis of this mycosis. In this study we analyzed 76 serum samples from patients in southern Bahia suspected of having paracoccidioidomycosis using a conventional PCR with primers for the ITS1 ribosomal DNA of P. brasiliensis. Of these 76 patients, 5 were positive for PCM by double immunodiffusion and/or direct examination and histopathology. To test specificity of PCR, we used human DNA and three isolates of P. lutzii (1578, 01 and ED01). Additionally, we analyzed by serial dilutions of DNA the limit of detection of the assay. The test of PCR proved specific, as only a 144 bp fragment of the three isolates of P. lutzii and no human DNA was amplified. Detection limit was 1.1 pg/µL of DNA. Despite the high detection limit and specificity of PCR none of the 76 serum samples were found positive by PCR, but a biopsy specimen obtained from one of the patients with PCM was positive. These results, albeit limited, show that PCR is not effective in detecting DNA of P. brasiliensis or P. lutzii in serum, but could perhaps be used with other types of clinical samples, especially in those instances in which conventional methods fail.

Interleukin-6 activation in ischemic stroke caused by Neisseria meningitidis serogroup C.

We report an unusual phenomenon in a patient with meningococcal meningitis who developed a brain infarct. Concentrations of interleukin-6 were notably elevated in the cerebrospinal fluid. We also discuss some clinical and pathophysiological features of cerebrovascular injury in meningococcal disease.

Severe myalgia of the lower extremities as the first clinical feature of meningococcal purpura fulminans.

In patients with meningococcal infection, devastating presentations, such as purpura fulminans, which can progress to extensive tissue necrosis of the limbs and digits, have a significant social impact. The case presented herein illustrates such a phenomenon in a patient who developed bilateral necrosis of the lower extremities as a result of infection with Neisseria meningitis. We emphasize that severe myalgia was the first clinical manifestation of meningococcal purpura fulminans in our case. However, myalgia has typically been overlooked and undervalued as an early clinical feature of meningococcal sepsis. Early recognition and prompt initial antibiotic therapy continue to be the cornerstones of the successful management of this dramatic disease, reducing morbidity and mortality.

Compartmentalization of interleukin-6 response in a patient with septic meningococcal peritonitis.

We report the first case of Neisseria meningitidis-induced septic peritonitis diagnosed by PCR assay of peritoneal fluid. Concentrations of interleukin-6 were notably higher in the peritoneal fluid than in the blood. PCR diagnosis of septic meningococcal peritonitis and the pathogenesis of the disease are discussed.

Cytokine activation in purulent pericarditis caused by Neisseria meningitidis serogroup C.

Herein, we report a case of purulent pericarditis caused by Neisseria meningitidis serogroup C in a previously healthy 5-year-old boy, which was detected in pericardial fluid by polymerase chain reaction. The concentrations of interleukin-6, interferon-gamma and interleukin-10 in pericardial fluid were notably increased compared with serum. The role of polymerase chain reaction, counterimmunoelectrophoresis and latex agglutination test in the diagnosis, as well as the participation of cytokines in the pathogenesis of meningococcal pericarditis, are discussed.

The role of interleukin-10 in the differential expression of interleukin-12p70 and its beta2 receptor on patients with active or treated paracoccidioidomycosis and healthy infected subjects.

Paracoccidioidomycosis patients present an antigen-specific Th1 immunosuppression. To better understand this phenomenon, we evaluated the interleukin (IL)-12 pathway by measuring IL-12p70 production and CD3+ T cell expression of the IL-12 receptor (IL-12R)beta1/beta2 chains, induced with the main fungus antigen (gp43) and a control antigen, from Candida albicans (CMA). We showed that gp43-induced IL-12p70 production and IL-12Rbeta2 expression were significantly decreased in acute and chronic patients as compared to healthy subjects cured from PCM or healthy infected subjects from endemic areas. Interestingly, the healthy infected subjects had higher gp43-induced IL-12p70 production and beta2 expression than the cured subjects. The addition of a neutralizing anti-IL-10 antibody to the cultures increased IL-12p70 levels and beta2 expression in acute and chronic patients to levels observed in cured subjects. Conversely, addition of the cytokine IL-10 strongly inhibited both parameters in the latter group. In conclusion, we have shown that paracoccidioidomycosis-related Th1 immunosuppression is associated with down-modulation of the IL-12 pathway, that IL-10 may participate in this process, and that patients cured from paracoccidioidomycosis may not fully recover their immune responsiveness.

The role of apoptosis in the antigen-specific T cell hyporesponsiveness of paracoccidioidomycosis patients.

Paracoccidioidomycosis is a deep endemic mycosis associated with an antigen-specific immunodeficiency. To examine the role of apoptosis in this immunodeficiency, peripheral blood mononuclear cells (PBMC) of patients with paracoccidioidomycosis and controls were stimulated with the main antigen of Paracoccidioides brasiliensis (gp43) and an unrelated fungal antigen (from Candida albicans, CMA) and analyzed for annexin V and propidium iodide staining by flow cytometry. Control PBMC proliferated well with both antigens. Patients' PBMC proliferated only with CMA, but presented higher levels of apoptosis with gp43 and CMA than in their own unstimulated cultures. Moreover, gp43-triggered apoptosis in control PBMC was lower than in those of the patients. Thus, patient but not control gp43-stimulated T cells apparently remained anergized and subsequently underwent apoptosis. While CMA-induced apoptosis is likely triggered by activation-induced cell death, this is apparently not the case in gp43-induced apoptosis because of the lack of cell cycling and IL-2 in the gp43-stimulated cultures. However, higher IL-10 levels were found in gp43-stimulated patient PBMC cultures. Addition of a neutralizing anti-IL-10 antibody to the cultures resulted in increased apoptosis levels only in gp43-stimulated patient PBMC cultures. Our results suggest that apoptosis plays a role in the patients' antigen-specific hyporesponsiveness and that IL-10 may have an antiapoptotic role.

IL-12 and neutralization of endogenous IL-10 revert the in vitro antigen-specific cellular immunosuppression of paracoccidioidomycosis patients.

Treatment of patients with paracoccidioidomycosis is still a challenge. Patients present defective lymphoproliferation and IFN-gamma responses to the main Paracoccidioides brasiliensis antigen (gp43), which correlates with disease severity. Here, we demonstrated that the patients show also a defective synthesis of interleukin (IL)-12. Therefore, we attempted to revert this immune disfunction by adding IL-12 and neutralizing anti-IL-10 antibody to gp-43-stimulated peripheral blood mononuclear cell cultures. Both treatments increased IFN-gamma secretion to levels observed with healthy sensitized individuals, but affected proliferation only modestly. When combined, the treatments further increased IFN-gamma synthesis and cell proliferation. The addition of suboptimal concentrations of IL-2 also further increased the IL-12-mediated secretion of IFN-gamma. Interestingly, the immune modulation was mostly antigen-specific, since the responses to Candida albicans' antigen were not affected. These results suggest that appropriate immune intervention with cytokines and/or anti-cytokines may help in the treatment of PCM.