Tânia Mara Pinto Dabés Guimarães

CD4-CD8-αβ and γδ T cells display inflammatory and regulatory potentials during human tuberculosis

T-cells play an important role controlling immunity against pathogens and therefore influence the outcome of human diseases. Although most T-lymphocytes co-express either CD4 or CD8, a smaller T-cell subset found the in the human peripheral blood that expresses the αβ or γδ T-cell-receptor (TCR) lacks the CD4 and CD8 co-receptors. These double negative (DN) T-cells have been shown to display important immunological functions in human diseases. To better understand the role of DN T-cells in human Mycobacterium tuberculosis, we have characterized their frequency, activation and cytokine profile in a well-defined group of tuberculosis patients, categorized as severe and non-severe based on their clinical status. Our data showed that whereas high frequency of αβ DN T-cells observed in M. tuberculosis-infected patients are associated with disease severity, decreased proportion of γδ DN T-cells are found in patients with severe tuberculosis. Together with activation of CD4+ and CD8+ T-cells, higher frequencies of both αβ and γδ DN T-cells from tuberculosis patients also express the chronic activation marker HLA-DR. However, the expression of CD69, an early activation marker, is selectively observed in DN T-cells. Interestingly, while αβ and γδ DN T-cells from patients with non-severe tuberculosis display a pro-inflammatory cytokine profile, characterized by enhanced IFN-γ, the γδ DN T-cells from patients with severe disease express a modulatory profile exemplified by enhanced interleukin-10 production. Overall, our findings suggest that αβ and γδ DN T-cell present disparate immunoregulatory potentials and seems to contribute to the development/maintenance of distinct clinical aspects of TB, as part of the complex immunological network triggered by the TB infection.

Peripheral blood mononuclear cells immunophenotyping in pulmonary tuberculosis patients before and after treatment.

Tuberculosis (TB) is a lung disease caused by Mycobacterium tuberculosis. The interaction between the bacillus and the host may lead to a protective cellular immune response. In the present study, we propose the “in vitro” evaluation of this cellular immune response in patients with tuberculosis before and after chemotherapic treatment. Eleven patients with TB and 9 asymptomatic subjects with tuberculin skin test negative (TST–) (purified protein derivative (PPD) ≤10 mm) were evaluated. The peripheral lymphocytes of the subjects were analyzed utilizing the following surface markers: CD3+, CD4+, CD8+, CD19+, CD25+, CD56+, CD14+, CD16+ and HLA‐DR+. At the end of the treatment, symptomatic patients presented a significant predominance (P<0.05) of CD4+ lymphocytes, a significant decrease (P<0.05) in activated CD8+ T cells and a significant increase (P<0.05) in the marker CD19+. A predominance of mature NK cells and a significant decrease (P<0.05) in NKT cells were observed. Also observed was a trend toward decrease in immunoregulatory T cells and a predominance of pro‐inflammatory macrophages. TST– subjects presented a predominance of CD4+ over CD8+ and predominance of CD19+ and of mature NK cells in comparison to the group of patients, both before and after treatment. Thus, several cell types, such as CD4+ and CD8+ T cells, NK cells and their subpopulations, NKT cells and pro‐inflammatory macrophages could act in a synergic way to control the growth and multiplication of M. tuberculosis.

Pulmonary tuberculosis: evaluation of interferon-gamma levels as an immunological healing marker based on the response to the Bacillus Calmette-Guerin

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis whose interaction with the host may lead to a cell-mediated protective immune response. The presence of interferon gamma (IFN-gamma) is related to this response. With the purpose of understanding the immunological mechanisms involved in this protection, the lymphoproliferative response, IFN-gamma and other cytokines like interleukin (IL-5, IL-10), and tumor necrosis factor alpha (TNF-alpha) were evaluated before and after the use of anti-TB drugs on 30 patients with active TB disease, 24 healthy household contacts of active TB patients, with positive purified protein derivative (PPD) skin tests (induration > 10 mm), and 34 asymptomatic individuals with negative PPD skin test results (induration < 5 mm). The positive lymphoproliferative response among peripheral blood mononuclear cells of patients showed high levels of IFN-gamma, TNF-alpha, and IL-10. No significant levels of IL-5 were detected. After treatment with rifampicina, isoniazida, and pirazinamida, only the levels of IFN-gamma increased significantly (p < 0.01). These results highlight the need for further evaluation of IFN-gamma production as a healing prognostic of patients treated.

Natural killer cell subpopulations in putative resistant individuals and patients with active Mycobacterium tuberculosis infection.

Herein, we intended to perform flow‐cytometric analyses of peripheral blood NK‐cell subsets in patients with active tuberculosis (TB) and those putative resistant subjects displaying positive tuberculin skin test (TST+) and compared with TST− healthy controls. Our findings demonstrated distinct phenotypic features in TST+ as compared with TB. While lower values of NK‐cells with increased frequency of CD3−CD16+ CD56− and CD3−CD16−CD56+ subsets besides lower frequency of CD3−CD16+ CD56+ NK‐cells was observed in TST+, unaltered levels of NK‐cells with increased levels of CD3−CD16+ CD56− NK‐cells with lower frequency of CD3−CD16+ CD56+ NK‐cells was found in TB. Additional analysis highlighted a shift towards increased levels of CD3−CD16−/+CD56bright NK‐cells as the hallmark of TST+, whereas unaltered frequency was observed in TB. Increased levels of CD3+CD56+ cells were observed in both TST+ and TB. Further focusing on the monocyte/NK‐cell network, we have reported that enhanced frequency of CD14+ CD16+ monocytes particularly observed in TST+. Outstanding were the distinct correlation profiles observed between CD3−CD16−CD56+ NK‐cells and CD3+ CD56+ cells CD14+ CD16+ monocytes for TST+ and TB. These data suggested that high levels of CD3−CD16−CD56+ NK‐cells aside CD14+ CD16+ monocytes as well as non‐concurrent increment of CD3+ CD56+ cells, may be involved in protective mechanisms in putative tuberculosis‐resistant individuals. On the other hand, the basal levels of macrophage‐like monocytes despite its positive correlation with increased levels of CD3+ CD56+ cells may count for the lack of the protective immunity in patients with active tuberculosis. Further studies focusing on the cytokine profiling of peripheral blood innate immunity cells before and after chemotherapic treatment are currently under evaluation.

Vaccination of C57BL/10 mice against cutaneous leishmaniasis using killed promastigotes of different strains and species of Leishmania

Antigenic extracts from five Leishmania stocks were used to vaccinate C57BL/10 mice. The Leishvacin(R) and PH8 monovalent vaccine yielded the highest IFN-gamma levels in the supernatants of spleen cell culture from vaccinated animals. Each single strain immunized group showed evidence of protective immunity six months after the challenge with promastigotes of Leishmania (Leishmania) amazonensis. No differences were detected between the vaccinated groups. It can be concluded that vaccines composed of single Leishmania stocks can provide protection to C57BL/10 mice against L. (L.) amazonensis infection.

Assessment of immunity induced in mice by glycoproteins derived from different strains and species of Leishmania

A comparative study was undertaken on the immunogenic properties of 63kDa glycoproteins obtained from five different strains/species of Leishmania and assessed in C57BL/10 mice. The humoral immune response was assessed by ELISA against the five different antigens of the immunized animals. The cellular immune response was derived from Leishmania. The response was found to be species-specific in all of determined by means of the cytokine profiles secreted by the spleen cells of immunized animals. The presence of gamma-IFN and IL-2, and the absence of IL-4 in the supernatants of cells stimulated by L. amazonensis antigen established that the cellular response is of Th1 type. The five glycoproteins tested were equally effective in protecting C57BL/10 mice against challenge by L. amazonensis. About 50% of the immunized animals were protected for six months.

Detection of a Giardia lamblia coproantigen by using a commercially available immunoenzymatic assay, in Belo Horizonte, Brazil

It is known that fecal examination to detect Giardia lamblia cysts or trophozoites produces a high percentage of false-negative results. A commercially available immunoenzymatic assay (ProSpecT Giardia Microplate Assay, Alexon, Inc., BIOBRAS) to detect G. lamblia specific coproantigen was evaluated for the first time in Brazil. A total of 90 specimens were tested. Each specimen was first tested as unpreserved stool, and then it was preserved in 10% Formalin to be tested 2 months later. The assay was able to identify all the 30 positive patients (sensitivity = 100.0%) by visual or spectrophotometric examination in the unpreserved specimens and was negative in 57 of the 60 patients without G. lamblia (specificity = 95.0%). The assay identified 27 of the 30 positive patients (sensitivity = 90.0%) and was negative in 59 of the 60 negatives (specificity = 98.3%) in the preserved stools according to both readings. A marked difference was observed in the optical densities in both groups, preserved and unpreserved stools, when the G. lamblia-positive specimens were compared to the negative or positive for other intestinal parasites than G. lamblia. The assay seems a good alternative for giardiasis diagnosis, especially when the fecal examination was repeatedly negative and the patient presents giardiasislike symptoms.

Physicochemical Characterization and Study of in vitro Interactions of pH-sensitive Liposomes with the Complement System

Complement activation is an important step in the acceleration of liposome clearance. The anaphylatoxins released following complement activation may motivate a wide variety of physiologic changes. We performed physicochemical characterization and in vitro studies of the interaction of complement system with both noncirculating and long-circulating pH-sensitive and nonpH-sensitive liposomes. The liposomes were characterized by diameter, zeta potential, and atomic force microscopy (AFM). The study of liposome interactions with complement system was conducted using hemolytic assay in rat serum. All liposomes presented a similar mean diameter (between 99.8 and 124.3 nm). The zeta potential was negative in all liposome preparations, except in liposomes modified with aminopoly (ethyleneglycol) 2000-distearoylphosphatidylethanolamine (aPEG2000-DSPE), which presented positive zeta potential. Atomic force microscopy images showed that non–long-circulating pH-sensitive liposomes are prone to vesicles aggregation. Non–pH-sensitive liposomes complement system activates, while pH-sensitive liposomes showed to be poor complement activators in rat serum.

Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis

For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegros skin test conversion rates induced by MtVac and by PhVac was 68.06% and 85.9%, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

Comparison of cytokine levels and metabolic parameters of stored platelet concentrates of the Fundação Hemominas, Belo Horizonte, Brazil

Background Prolonged storage of platelets could improve availability and logistical management and decrease wastage. Immunobiochemical methods can be used to guarantee the quality of platelets after prolonged storage. Objective The aim of this study was to compare storage-related changes in buffy coat-derived platelet concentrations versus platelet-rich plasmal. Methods Units of whole blood were drawn using a quadruple-bag blood container system. Platelet-rich plasma and buffy coat prepared from whole blood following standard methods were stored for 9 days. During this period test samples were aseptically collected for analysis on Days 1, 2, 3, 5, 7 and 9. Results The highest CD42b expression was greater than 95%. The percentage of CD62p was significantly lower than the CD42b expression. The pH remained fairly stable during storage. Measurement of pO2 and pCO2 showed that oxygen levels were significantly higher than carbon dioxide levels. There were no significant differences in bicarbonate levels, glucose consumption and lactate production between the groups. The swirling effect with platelet-rich plasma samples decreased after 5 days of storage and after 7 days of storage for buffy coat samples. There was a significant twenty-fold increase in the mean IL-1β after 5 days of storage for both groups. Slight increases in IL-6 and IL-8 levels were seen at 5 days. Conclusion The quality of platelet concentrates remained acceptable during 7 days of storage in respect to the swirling effect, pH and platelet activation. There were no significant differences between buffy coat-derived platelets and platelet-rich plasma in this study.