The monocyte-derived dendritic cells (moDCs) are a subset of dendritic cells widely used in immunological studies as a convenient and easy approach after isolation of mononuclear cells directly from peripheral blood mononuclear cells (PBMC). Both the purification and cell culture of monocytes impact on the differentiation of monocytes into moDCs. The methodology to isolate and differentiate monocytes into moDCs is still controversial. We aimed to compare three different protocols for monocyte isolation from PBMC: 1) Cold-aggregation; 2) Percoll gradient; and 3) Magnetic beads cell-enrichment. Additionally we also compared four different monocyte differentiation and culture techniques: 1) Cell culture media; 2) Serum sources; 3) required GM-CSF and IL-4 concentrations; 4) Cell culture systems. We used flow cytometry analysis of light scattering and/or expression of pan surface markers, such as CD3, CD14 and CD209 to determine isolation/differentiation degree. Purified PBMC followed by two steps of cold aggregation, yielded cell viability around 95% with poor monocyte enrichment (monocytes increase vs. lymphocytes reduction was not statistically significant, p>0.05). Conversely, monocyte isolation from PBMC with discontinuous Percoll gradient generated around 50% cell viability. Albeit, we observed a significant reduction (p≤0.05) of lymphocytes contaminants. The magnetic beads cell-enrichment yield cell viability higher than 95%, as high as a significant lymphocyte depletion (p≤0.005) when compared to all other techniques employed. The moDCs showed better differentiation based on increased CD209 expression, but lower CD14 levels, when cells were cultured in RPMI medium plus 500IU/mL of both GM-CSF and IL-4 in a semi-adherent fashion. Serum sources showed no influence on the culture performance. In conclusion, the magnetic beads cell-enrichment showed superior cell viability, indicating that this approach is a better choice to isolate monocytes, and moDCs cultured afterwards in appropriate medium, serum, cytokines and culture system might influence the monocytes differentiation into moDC.

Using a short-term bulk culture protocol designed for an intracellular-staining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokine-producing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae- and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-alpha was the first (8 h) to be produced, followed by IFN-gamma (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-alpha than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-gamma. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations. The method can also be used to define immune status in non-infected and contact individuals.

Biomarkers or biosignature profiles have become accessible over time in population-based studies for Chagas disease. Thus, the identification of consistent and reliable indicators of the diagnosis and prognosis of patients with heart failure might facilitate the prioritization of therapeutic management to those with the highest chance contracting this disease. The purpose of this paper is to review the recent state and the upcoming trends in biomarkers for human Chagas disease. As an emerging concept, we propose a classification of biomarkers based on plasmatic-, phenotype-, antigenic-, genetic- and management-related candidates. The available data revisited here reveal the lessons learned thus far and the existing challenges that still lie ahead to enable biomarkers to be employed consistently in risk evaluation for this disease. There is a strong need for biomarker validation, particularly for biomarkers that are specific to the clinical forms of Chagas disease. The current failure to achieve the eradication of the transmission of this disease has produced determination to solve this validation issue. Finally, it would be strategic to develop a wide variety of biomarkers and to test them in both preclinical and clinical trials.

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

Tuberculosis (TB) remains among the world's leading cause of mortality. For its control, studies of TB vaccines are needed. Since live-attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only TB vaccine currently in use, studies on the protective role of BCG are required. In this study, we analyzed host cells purified directly from whole blood of human immunodeficiency virus (HIV)-negative volunteers, comprising adult healthy donors (HD) and neonates (umbilical cord bloods, UCB), with the aim to directly compare in vitro immune responses with distinct BCG strains in human mononuclear cells. The Moreau, Pasteur, and Danish BCG strains were used to infect mononuclear cells in vitro for 48 h; bacilli viability and cell-death were subsequently detected by flow cytometry. In addition, cell culture supernatants were used in cytokine detection assays. Overall, the Moreau BCG strain induced higher levels of apoptosis than the Pasteur and Danish BCG strains in both the HD and UCB groups (p-value < 0.05), and a human monocytic cell-line mirrored those cell-death patterns after BCG infection. The Moreau BCG strain, exclusively, induced Th1 cytokines at the highest levels in cells from adults (p-value < 0.05) when compared with both Pasteur and Danish BCG strains, whereas TGF-β1 levels were reduced significantly (p-value < 0.01) in the HD group when cells were infected with the Moreau BCG vaccine. As expected, eight out of 22 pro-inflammatory cytokines were secreted at significant levels (p-value < 0.05) above the baseline rates in all BCG-infected cell cultures, in the HD group only. When analyzing these results, we excluded confounding factors related to storage and viability of the BCG strains used. These findings suggest that Moreau BCG is a more potent immunostimulating agent than the Pasteur and Danish BCG strains. Clinical trials will be needed to confirm these findings.

The current tuberculosis (TB) vaccine Mycobacterium bovis BCG has been employed for some 70 years in Brazil and lessons from its use should be taken in account for the development or improvement of new TB vaccines. The vast majority of the current population has been vaccinated with BCG, with the possible requirement for a booster immunisation in adulthood for TB protection. BCG Moreau strain also protects against leprosy, meningitis and extrapulmonary forms of TB. Factors related to differences in strain, dosage and BCG administering protocol have been responsible for the variable efficacy of BCG. This vaccine is clearly affected by, as yet unclear, host and/or environmental variables. In this brief review, we describe some aspects of BCG immunisation observed in Brazil that may be of importance for improving or replacing BCG.