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.

Experimental Hyalohyphomycosis by Purpureocillium lilacinum: Outcome of the Infection in C57BL/6 Murine Models.

Purpureocillium lilacinum is a filamentous, hyaline fungus considered an emerging pathogen in humans. The aim of our study was to evaluate the outcome of hyalohyphomycosis in C57BL/6 murine models inoculated with two clinical P. lilacinum isolates (S1 and S2). Each isolate was inoculated in mice randomly distributed in immunocompetent (CPT) and immunosuppressed (SPS) groups. Mice were evaluated at day 7, 21, and 45 after inoculation for histopathological analysis, recovery of fungal cells, and immunological studies. Histological analysis showed scarce conidia-like structures in lung tissue from CPT mice and a lot of fungal cells in SPS mice inoculated with S2 compared to mice inoculated with S1. The maximum recovery of fungal cells was seen in CPT mice inoculated with both isolates at day 7, but with mean significantly higher in those inoculated with S2 isolate. Phenotypical characterization of T cells showed TCD8+ lymphocytes predominance over TCD4+ in immunosuppressed mice infected and control groups. We also observed higher percentages of the central and effector memory/effector phenotype in CPT mice infected with S2 strain, especially in TCD8+ in the initial period of infection. Regulatory T cells showed higher percentages in immunosuppressed, predominantly after the acute phase. Our results showed that the P. lilacinum is a fungus capable to cause damages in competent and immunosuppressed experimental hosts. Furthermore, S2 isolate seems to cause more damage to the experimental host and it was possible to identify different cellular subsets involved in the mice immune response.

Scrutinizing the Biomarkers for the Neglected Chagas Disease: How Remarkable!

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 of 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.

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.

The role of host soluble inflammatory mediators induced by the BCG vaccine for the initiation of in vitro monocyte apoptosis in healthy Brazilian volunteers.

BACKGROUND: Tuberculosis (TB) is the second greatest killer worldwide that is caused by a single infectious agent. For its control, studies of TB vaccines are needed. Since Bacillus Calmette-Guerin (BCG) is the only vaccine against TB currently in use, studies addressing the protective role of BCG in the context of inducible inflammatory mediators are urgently required. METHODS: In this study, groups of HIV-negative adult healthy donors (HD; n = 42) and neonates (UV; n = 18) have been voluntarily enrolled, and BCG Moreau strain was used for the in vitro mononuclear cell infections for an initial period of 48 h. Subsequently, harvested conditioned medium (CM) was added to autologous resting cells for an additional 24, 48, and 120 h, and Annexin V, in conjunction with a vital dye, was then used for apoptosis detection. CM was also assayed for nitric oxide (NO), prostaglandin E2 (PGE2), leukotriene B4 (LTB4), interferon (IFN)-ß, and transforming growth factor (TGF)-ß1 levels. The p values were set up for any differences between two groups of individuals using Student's t-test and considered significant when ≤ 0.05. RESULTS: At 120 h, CM induced the highest apoptosis levels in both group studied, but necrosis was high in UV group only (p-value < 0.05). NO was released equally during BCG infection in both groups, but higher levels were found in HD when compared with UV group (p-value < 0.05). Overall, BCG Moreau triggered high PGE2, LTB4 and IFN-ß productions in macrophages from the UV group (p-value ≤ 0.05), whereas the prostanoid PGE2 and TGF-ß1 had an opposite pattern in the HD group. CONCLUSIONS: This study uncovers critical roles for endogenous compounds in the instruction of host macrophage cell death patterns. Understanding the regulation of human immune responses is critical for vaccine development and the treatment of infectious diseases. These findings shed new light on the potential condition for a booster immunization in individuals already vaccinated with BCG for TB protection, and further studies are warranted.

Detection of antibody to Purpureocillium lilacinum by immunofluorescent assay and flow cytometry in serum of infected C57BL/6 mice.

Purpureocillium lilacinum is an emerging pathogenic fungus that can cause different clinical manifestations ranging from cutaneous and sub-cutaneous infections to severe oculomycosis. In this study, using both conventional indirect immunofluorescence and non-conventional flow cytometry approaches, IgG antibodies were readily detected in both C57BL/6 immunocompetent and immunosuppressed mice after i.v. infection with P. lilacinum. The humoral immune response was specific, since virtually no antibodies were detected in the serum of control mice. Flow cytometry assays also showed both quantitative and qualitative differences in total IgG and its isotypes in sera of immunocompetent and immunosupressed infected mice. Although a good starting point, it is clear that the effectiveness of serological assays for P. lilacinum hyalohyphomycosis identification in clinical studies still requires further standardization. Upon further validation in humans, these techniques have the potential to be suitable to detect P. lilacinum infection in patients, thereby avoiding current laborious and time-consuming culture techniques.

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.

Polymorphisms in IL-1beta, vitamin D receptor Fok1, and Toll-like receptor 2 are associated with extrapulmonary tuberculosis.

BACKGROUND: Human genetic variants may affect tuberculosis susceptibility, but the immunologic correlates of the genetic variants identified are often unclear. METHODS: We conducted a pilot case-control study to identify genetic variants associated with extrapulmonary tuberculosis in patients with previously characterized immune defects: low CD4+ lymphocytes and low unstimulated cytokine production. Two genetic association approaches were used: 1) variants previously associated with tuberculosis risk; 2) single nucleotide polymorphisms (SNPs) in candidate genes involved in tuberculosis pathogenesis. Single locus association tests and multifactor dimensionality reduction (MDR) assessed main effects and multi-locus interactions. RESULTS: There were 24 extrapulmonary tuberculosis cases (18 black), 24 pulmonary tuberculosis controls (19 black) and 57 PPD+ controls (49 black). In approach 1, 22 SNPs and 3 microsatellites were assessed. In single locus association tests, interleukin (IL)-1beta +3953 C/T was associated with extrapulmonary tuberculosis compared to PPD+ controls (P = 0.049). Among the sub-set of patients who were black, genotype frequencies of the vitamin D receptor (VDR) Fok1 A/G SNP were significantly different in extrapulmonary vs. pulmonary TB patients (P = 0.018). In MDR analysis, the toll-like receptor (TLR) 2 microsatellite had 76% prediction accuracy for extrapulmonary tuberculosis in blacks (P = 0.002). In approach 2, 613 SNPs in 26 genes were assessed. None were associated with extrapulmonary tuberculosis. CONCLUSIONS: In this pilot study among extrapulmonary tuberculosis patients with well-characterized immune defects, genetic variants in IL-1beta, VDR Fok1, and TLR2 were associated with an increased risk of extrapulmonary disease. Additional studies of the underlying mechanism of these genetic variants are warranted.

Decreased CD4+ lymphocytes and innate immune responses in adults with previous extrapulmonary tuberculosis.

BACKGROUND: CD4+ lymphocytes control Mycobacterium tuberculosis infection through cytokine-mediated macrophage activation. Extrapulmonary tuberculosis is presumably a marker of immunodeficiency, but cytokine responses have not been well studied in such patients. OBJECTIVE: Assess immune defects in persons with previous extrapulmonary tuberculosis. METHODS: In vitro cytokine responses of PBMCs from HIV-seronegative adults with previous extrapulmonary tuberculosis (n = 10) were compared with responses from persons with previous pulmonary tuberculosis (n = 24) and latent M tuberculosis infection (n = 30) in a case-control study. RESULTS: Patients and controls did not differ according to age, sex, race, or monocytes. The median time between tuberculosis diagnosis and study entry was 72 and 122 weeks in extrapulmonary and pulmonary patients, respectively (P = .2). Median CD4+ counts were 660, 814, and 974 lymphocytes/mm3 in extrapulmonary, pulmonary, and latently infected patients, respectively (P = .03). At 48 hours, median unstimulated cytokine levels were uniformly lower in extrapulmonary patients than both sets of controls. These differences persisted after controlling for CD4+ count by linear regression analysis. Despite lower unstimulated levels, median TNF-alpha response was higher in patients with extrapulmonary and pulmonary tuberculosis than latently infected persons after stimulation with PHA 1% (P = .006) and PHA+IL-12 (1 ng/mL; P = .02); IL-10 remained low in patients with extrapulmonary tuberculosis after the same stimuli (P = .04 and .06, respectively). There was no primary immunodeficiency in the IL-12/23-IFN-gamma axis. CONCLUSION: HIV-seronegative adults with previous extrapulmonary tuberculosis had lower CD4+ lymphocytes and unstimulated cytokine production. This suggests a subtle abnormality in innate immune function. CLINICAL IMPLICATIONS: These characteristics could identify persons at risk for severe tuberculosis manifestations.

Cytokines and Mycobacterium leprae induce apoptosis in human Schwann cells.

The development of deformities during the course of leprosy disease is a major public health concern worldwide. It is possible that cytokine production and apoptosis of Schwann cells (SCs) directly affect nerve degeneration and regeneration leading to injury of the myelin sheath and axon. In the present study, the expression of TNFalpha, TGFbeta, and their receptors, in addition to cell death triggered by cytokines or whole Mycobacterium leprae were investigated in a human SC line. The results showed the presence of TNF-Rs and TGF-RII on the SC membrane and the shedding of TNF-Rs during the culture period. Evaluation of cell death was performed through TUNEL and flow cytometry techniques. TNFalpha/TGFbeta combination as well as M. leprae infection triggered an increase in the apoptosis rate in the cultured SC. Moreover, reverse transcriptase-polymerase chain reaction assay revealed that M. leprae upregulated the expression of such cytokines and their receptors on the SC line. Despite the detection of TNFalpha mRNA, no protein was found in the culture supernatants. The data indicate that induction of SC death after cell interaction with M. leprae may, in fact, be implicated in the pathogenesis of nerve damage, which can most likely be modulated by in vivo cytokine production.