A comparative approach on the activation of the three complement system pathways in different hosts of Visceral Leishmaniasis after stimulation with Leishmania infantum.

Visceral Leishmaniasis is an infectious disease that affects mainly humans and dogs, with the latter being important reservoirs of the parasite. Conversely, cats are naturally resistant. The immune system can offer important explanation to this problematic as there is no evidence on the role that the complement system plays in cats. In this context, effect of the complement system from human, dog and cat sera on Leishmania infantum was evaluated. Activation of the classical, alternative and lectin pathways was assessed through hemolytic and ELISA assays. Lytic activity of the complement on the parasite's viability was investigated by Transmission Electron Microscopy and Flow Cytometry. Complement proteins were more consumed in dog serum on the classical and alternative pathways, leading to less hemolytic activity, and only in cat serum they were consumed on the lectin pathway when incubated with L. infantum. Lytic activity on the parasite's surface was more accentuated in human serum, and varied throughout the parasite's developmental stages.

Pneumococcal Predictive Proteins Selected by Microbial Genomic Approach Are Serotype Cross-Reactive and Bind to Host Extracellular Matrix Proteins.

Streptococcus pneumoniae is a colonizer of the human nasopharynx, which accounts for most of the community-acquired pneumonia cases and can cause non-invasive and invasive diseases. Current available vaccines are serotype-specific and the use of recombinant proteins associated with virulence is an alternative to compose vaccines and to overcome these problems. In a previous work, we describe the identification of proteins in S. pneumoniae by reverse vaccinology and the genetic diversity of these proteins in clinical isolates. It was possible to purify a half of 20 selected proteins in soluble form. The expression of these proteins on the pneumococcal cells surface was confirmed by flow cytometry. We demonstrated that some of these proteins were able to bind to extracellular matrix proteins and were recognized by sera from patients with pneumococcal meningitis infection caused by several pneumococcal serotypes. In this context, our results suggest that these proteins may play a role in pneumococcal pathogenesis and might be considered as potential vaccine candidates.

Flow Cytometry as a Tool for Quality Control of Fluorescent Conjugates Used in Immunoassays.

The use of antibodies in immunodiagnostic kits generally implies the conjugation of these proteins with other molecules such as chromophores or fluorochromes. The development of more sensitive quality control procedures than spectrophotometry is essential to assure the use of better fluorescent conjugates since the fluorescent conjugates are critical reagents for a variety of immunodiagnostic kits. In this article, we demonstrate a new flow cytometric protocol to evaluate conjugates by molecules of equivalent soluble fluorochromes (MESF) and by traditional flow cytometric analysis. We have coupled microspheres with anti-IgG-PE and anti-HBSAg-PE conjugates from distinct manufactures and/or different lots and evaluated by flow cytometry. Their fluorescence intensities were followed for a period of 18 months. Our results showed that there was a great difference in the fluorescence intensities between the conjugates studied. The differences were observed between manufactures and lots from both anti-IgG-PE and anti-HBSAg-PE conjugates. Coefficients of variation (CVs) showed that this parameter can be used to determine better coupling conditions, such as homogenous coupling. The MESF analysis, as well as geometric mean evaluation by traditional flow cytometry, showed a decrease in the values for all conjugates during the study and were indispensable tools to validate the results of stability tests. Our data demonstrated the feasibility of the flow cytometric method as a standard quality control of immunoassay kits.