The BCG Moreau Vaccine Upregulates In Vitro the Expression of TLR4, B7-1, Dectin-1 and EP2 on Human Monocytes.

Background: Tuberculosis (TB) is currently the second greatest killer worldwide and is caused by a single infectious agent. Since Bacillus Calmette−Guérin (BCG) is the only vaccine currently in use against TB, studies addressing the protective role of BCG in the context of inducible surface biomarkers are urgently required for TB control. Methods: In this study, groups of HIV-negative adult healthy donors (HD; n = 22) and neonate samples (UCB; n = 48) were voluntarily enrolled. The BCG Moreau strain was used for the in vitro mononuclear cell infections. Subsequently, phenotyping tools were used for surface biomarker detection. Monocytes were assayed for TLR4, B7-1, Dectin-1, EP2, and TIM-3 expression levels. Results: At 48 h, the BCG Moreau induced the highest TLR4, B7-1, and Dectin-1 levels in the HD group only (p-value < 0.05). TIM-3 expression failed to be modulated after BCG infection. At 72 h, BCG Moreau equally induced the highest EP2 levels in the HD group (p-value < 0.005), and higher levels were also found in HD when compared with the UCB group (p-value < 0.05). Conclusions: This study uncovers critical roles for biomarkers after the instruction of host monocyte activation patterns. Understanding the regulation of human innate immune responses is critical for vaccine development and for treating infectious diseases.

The in vitro Mycobacterium bovis BCG Moreau infection of human monocytes that induces Caspase-1 expression, release and dependent cell death is mostly reliant upon cell integrity.

BACKGROUND: Caused by Mycobacterium tuberculosis, tuberculosis (TB) is an extremely contagious disease predominantly affecting the lungs. TB is found worldwide and has a major impact on public health safety primarily due to its high mortality rate. Applied for over a hundred years as a preventive measure, Mycobacterium bovis BCG remains the only available TB vaccine. Only one seminal study about the apoptotic pathways induced by this vaccine in the monocytic lineage of the host cell has found the effects of BCG on regulation of apoptosis. The aim of this study was to explore beyond that pioneer study the pathway related to the in vitro cell-death pattern and the inflammatory response to the BCG vaccine in human monocytes. METHODS: Cohorts of HIV-negative volunteers were enrolled: adult Healthy Donors (HD) and neonates' Umbilical Cord Blood (UCB) individuals. Host mononuclear cells were infected with the M. bovis Moreau strain of BCG vaccine at 16, 24, 48, and 72 h. The Real-Time RT-PCR for TRADD, Bcl-2, and Caspases-1 and -3 were performed, and supernatants were assayed in parallel for Caspase-1, NLRP3, HO-1, and IL-1ß levels whereas caspases were assessed intracellularly. The effect of a BCG infection in monocytes was characterized via a metabolic activity assay by LDH release profiles. RESULTS: Overall, the BCG vaccine induced significantly higher Caspase-1 and Bcl-2 mRNA levels in both the HD and UCB groups (p-value ≤0.05). In addition, a significant increase solely in Caspase-1 protein levels was also noted in both HD and UCB (p-value ≤0.05) notwithstanding the absence of any damaged cell membranes. CONCLUSIONS: Our data directly corroborate other findings showing that BCG Moreau led to an increased secretion of IL-1ß but not IL-18, two Caspase-1-activated cytokines, and are also in support of the model that the BCG Moreau infection of human mononuclear cells may induce a cell-death pattern involving Caspase-1 activation.

Human neonates display altered ex vivo monokine production related to healthy adults.

The inflammatory response plays an important role during the induction of several neonatal diseases. Previous studies have shown that during newborn infections, the natural imbalance between pro- and anti-inflammatory responses shifts toward the production of pro-inflammatory cytokines. In this study, we employed an array system to detect 9 pro- and anti-inflammatory cytokines, and performed ELISA for 6 other cytokines. We then compared the immune response profiling in umbilical cord blood (UV) plasma samples with circulating levels in otherwise healthy donors (HD). Concentrations of ex vivo monokine levels, such as interleukins (IL)-18, IL-23 and IL-27, were profoundly reduced in the UV in relation to the HD group (p-values of 0.003, 0.009 and <0.0001, respectively). Conversely, UV-plasmatic TGF-ß1 levels displayed marked enhancement (p-value=0.005) in relation to HD. Several factors may be implicated in these neonatal alterations, and additional characterization of a broader cytokine panel is warranted to reveal other possible candidates.

A whole blood assay as a simple, broad assessment of cytokines and chemokines to evaluate human immune responses to Mycobacterium tuberculosis antigens.

In vitro stimulation of whole blood or isolated peripheral blood cells with specific antigens is used for several purposes. We sought to identify a reliable, reproducible, fast and feasible in vitro method to assess human cellular immune responses to Mycobacterium tuberculosis. In contrast to peripheral blood mononuclear cell (PBMC) culture, a whole blood assay (WBA) provides a more physiological environment, which may provide a broader assessment of serum biomarker, biosignature profiles. Twenty-three asymptomatic individuals with M. tuberculosis infection were recruited. Total cells from the WBA (diluted 1:3 in completed RPMI) and PBMC (2×10(5)cells/ml) plus M. tuberculosis Ag85A, Ag85B, ESAT-6 and Mycobacterium bovis 65kDa were characterized by flow cytometry, then added in 96-well plates and on day 5 plasma and supernatants were harvested for detection of 17 cytokines by a Luminex array system. There was agreement between PBMC and WBA for IL-2, IL-5, IL-6, IL-7, IL-13, IFN-γ, TNF-α, MCP-1 and MIP-1ß. There was evidence toward higher IL-10 (p≤0.049) and G-CSF (p≤0.012) plasma production, and higher IL-1ß (p≤0.048), IL-4 (p≤0.044), IL-12p70 (p≤0.006), IL-17 (p≤0.002) and GM-CSF (p≤0.049) production for PBMC vs. WBA. Both methods provided virtually no reaction to the internal, negative control. Due to technical issues linked to data out of range, IL-8 data were not considered. These results suggest that, depending on the method employed, PBMC and/or WBA techniques provide fine conditions for the model proposed and thus whole blood cultures are well-suited low-cost proxy-measures during search for serum biomarkers.

Neglected and emerging fungal infections: review of hyalohyphomycosis by Paecilomyces lilacinus focusing in disease burden, in vitro antifungal susceptibility and management.

Paecilomyces lilacinus is an emerging pathogenic fungus that can cause different clinical manifestations ranging from cutaneous and sub-cutaneous infections to severe oculomycosis. This review discusses infections caused by P. lilacinus, as well as their symptoms and correlates of immune responses, morphological characteristics of the fungus, therapies, in vitro susceptibility tests, laboratory diagnosis and the experimental models available.