Th22 response induced by Mycobacterium tuberculosis strains is closely related to severity of pulmonary lesions and bacillary load in patients with multi-drug-resistant tuberculosis.

The role of interleukin-22 (IL-22) in the pathogenesis or tissue repair in human tuberculosis (TB) remains to be established. Here, we aimed to explore the ex-vivo and in-vitro T helper 22 (Th22) response in TB patients and healthy donors (HD) induced by different local multi-drug-resistant (MDR) Mvcobacterium tuberculosis (Mtb) strains. For this purpose, peripheral blood mononuclear cells from drug-susceptible (S-TB) MDR-TB patients and HD were stimulated with local MDR strains and the laboratory strain H37Rv. IL-22 and IL-17 expression and senescent status were assessed in CD4+ and CD8+ cells by flow cytometry, while IL-22 amount was measured in plasma and culture supernatants by enzyme-linked immunosorbent assay (ELISA). We found lower IL-22 amounts in plasma from TB patients than HD, together with a decrease in the number of circulating T cells expressing IL-22. In a similar manner, all Mtb strains enhanced IL-22 secretion and expanded IL-22+ cells within CD4+ and CD8+ subsets, being the highest levels detected in S-TB patients. In MDR-TB, low systemic and Mtb-induced Th22 responses associated with high sputum bacillary load and bilateralism of lung lesions, suggesting that Th22 response could be influencing the ability of MDR-TB patients to control bacillary growth and tissue damage. In addition, in MDR-TB patients we observed that the higher the percentage of IL-22+ cells, the lower the proportion of programmed cell death 1 (PD-1)+ or CD57+ T cells. Furthermore, the highest proportion of senescent T cells was associated with severe lung lesions and bacillary load. Thus, T cell senescence would markedly influence Th22 response mounted by MDR-TB patients.

Coinfection of domestic felines by distinct Sporothrix brasiliensis in the Brazilian sporotrichosis hyperendemic area.

Microbial interactions may impact patient's diagnosis, prognosis and treatment. Sporotrichosis is a hyperendemic neglected zoonosis in Brazil, caused by Sporothrix brasiliensis. Four pairs of clinical isolates of Sporothrix were recovered from four diseased cats (CIM01-CIM04, two isolates per animal) raising the possibility of coinfection in a sporotrichosis hyperendemic area, Brazil. Each isolate of the pair had distinct pigmentation in mycological culture, and was designated as "Light" or "Dark", for low and high pigmentation, respectively. Dark isolates reacted strongly with monoclonal antibodies to melanin (p ≤ 0.05) by both ELISA and FACS quantitation, and displayed a ring pattern with some regions exhibiting higher punctuated labeling at cell wall by immunofluorescence. In turn, Light isolates reacted less intensely, with few and discrete punctuated labeling at the cell wall. PCR identified all isolates as S. brasiliensis, MAT1-2 idiomorph. Sequencing of ß-tubulin and calmodulin genes followed by phylogenetic analysis placed all eight isolates within the same cluster as others from the Brazilian hyperendemic area. The ability of these strains to stimulate cytokine production by human PBMCs (Peripheral blood mononuclear cells) was also analyzed. CIM01 and CIM03 Light and Dark isolates showed similar cytokine profiles to the control strain, while CIM02 and CIM04 behaved differently (p < 0.001), suggesting that differences in the surface of the isolates can influence host-fungus interaction. MICs for amphotericin B, terbinafine, caspofungin, micafungin, itraconazole, fluconazole, and voriconazole were obtained (CLSI M38-A2/M27-A3). Pairwise comparisons showed distinct MICs between Sporothrix Light and Dark isolates, higher than at least two-fold dilutions, to at least one of the antifungals tested. Isolates from the same pair displayed discrepancies in relation to fungistatic or fungicidal drug activity, notably after itraconazole exposure. Since S. brasiliensis Light and Dark isolates show disparate phenotypic parameters it is quite possible that coinfection represents a common occurrence in the hyperendemic area, with potential clinical implications on feline sporotrichosis dynamics. Alternatively, future studies will address if this specie may have, as reported for other fungi, broad phenotypic plasticity.

Apoptosis Transcriptional Profile Induced by Porphyromonas gingivalis HmuY.

This study aimed at evaluating the transcriptional profile of apoptosis-related genes after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) derived from individuals with periodontitis (P) and healthy nonperiodontitis (NP) control subjects with P. gingivalis HmuY protein. PBMCs from the P and NP groups were stimulated with HmuY P. gingivalis protein, and the expression of genes related to apoptosis was assessed by custom real-time polymerase chain reaction array (Custom RT2 PCR Array). Compared with the NP group, the P group showed low relative levels of apoptosis-related gene expression, downregulated for FAS, FAS ligand, TNFSF10 (TRAIL), BAK1, CASP9, and APAF1 after P. gingivalis HmuY protein stimulation. Furthermore, the P group exhibited low levels of relative gene expression, downregulated for CASP7 when the cells were not stimulated. Our data suggest that P. gingivalis HmuY protein might participate differently in the modulation of the intrinsic and extrinsic apoptosis pathways.

Diagnostic performance of GM-CSF and IL-2 in response to long-term specific-antigen cell stimulation in patients with active and latent tuberculosis infection.

BACKGROUND: A simple blood test for detecting active tuberculosis (TB) could be key to this epidemic containment, given that a large proportion of patients are unable to produce sputum for testing. Currently available interferon-γ release assays (IGRAs) are inadequate to diagnose active TB, with reported pooled sensitivity and specificity both under 81%. OBJECTIVE: To explore whether cytokines/chemokines other than interferon-γ in response to long-term cell stimulation could improve the ability to distinguish between different TB infection status. METHODS: We prospectively enrolled subjects with newly diagnosed pulmonary TB and their household contacts in Santiago. All contacts were tested with IGRA. Peripheral blood mononuclear cells were obtained and antigen-specific stimulated for 72 h before collecting their culture supernatants. RESULTS: Subjects with active TB displayed markedly low cytokines/chemokines secretion upon PBMC stimulation, with lower GM-CSF being the best differentiator from IGRA(+) contacts, with 71% (95% CI 53-85) sensitivity, 86% (95% CI 65-97) specificity and AUC = 0.79 (p = 0.0003). On the other hand, when compared to the uninfected IGRA(-) contacts, higher level of IL-2 secretion was the best indicator of active TB, with 73.5% (95% CI 56-87) sensitivity, 85% (95% CI 66-96) specificity and AUC = 0.79 (p = 0.0001). No single cytokine/chemokine released upon stimulation could accurately differentiate between active TB and all TB contacts grouped together. CONCLUSION: GM-CSF and IL-2 provided the best yield to differentiate active TB from latent TB and from TB uninfected, respectively, with higher specificities than that reported for IGRAs. However, none of both resulted sensitive enough to be used as a stand-alone biomarker for active TB.

Brucella abortus Traverses Brain Microvascular Endothelial Cells Using Infected Monocytes as a Trojan Horse.

Neurobrucellosis is an inflammatory disease caused by the invasion of Brucella spp. to the central nervous system (CNS). The pathogenesis of the disease is not well characterized; however, for Brucella to gain access to the brain parenchyma, traversing of the blood-brain barrier (BBB) must take place. To understand the CNS determinants of the pathogenesis of B. abortus, we have used the in vitro BBB model of human brain microvascular endothelial cells (HBMEC) to study the interactions between B. abortus and brain endothelial cells. In this study, we showed that B. abortus is able to adhere and invade HBMEC which was dependent on microtubules, microfilaments, endosome acidification and de novo protein synthesis. After infection, B. abortus rapidly escapes the endosomal compartment of HBMEC and forms a replicative Brucella-containing vacuole that involves interactions with the endoplasmic reticulum. Despite the ability of B. abortus to invade and replicate in HBMEC, the bacterium was unable by itself to traverse HBMEC, but could traverse polarized HBMEC monolayers within infected monocytes. Importantly, infected monocytes that traversed the HBMEC monolayer were a bacterial source for de novo infection of glial cells. This is the first demonstration of the mechanism whereby B. abortus is able to traverse the BBB and infect cells of the CNS. These results may have important implications in our understanding of the pathogenesis of neurobrucellosis.

Impaired TNF, IL-1ß, and IL-17 production and increased susceptibility to Mycobacterium tuberculosis infection in HTLV-1 infected individuals.

IFN-γ and TNF play critical roles in the control of Mycobacterium tuberculosis infection. Despite leading to an exaggerated production of inflammatory cytokines, HTLV-1 infection increases the risk of developing tuberculosis (TB). However, the immune mechanisms accounting for this phenomenon are still unclear. The aim of this study was to evaluate immunological aspects of the HTLV-1/M. tuberculosis co-infection. In this cross-sectional study, the levels of TNF, IL-1ß, and IL-17 were determined by ELISA in the supernatants of either unstimulated or tuberculin purified protein derivative (PPD) stimulated peripheral blood mononuclear cells. Cells from HTLV-1 infected individuals produced lower levels of TNF following PPD stimulation compared to unstimulated cells. IL-1ß and IL-17 production by cells from HTLV-1/M. tuberculosis co-infected individuals was lower than in cells from patients with TB. Impairment in TNF, IL-1ß, and IL-17 production upon stimulation with mycobacterial antigens may contribute to the increased susceptibility to M. tuberculosis infection observed in HTLV-1 infected individuals.

Differential In Vitro Cytokine Induction by the Species of Cryptococcus gattii Complex.

Cryptococcal species vary in capsule and cell size, thermotolerance, geographic distribution, and affected populations. Cryptococcus gattii sensu stricto and C. deuterogattii affect mainly immunocompetent hosts; however, C. bacillisporus, C. decagattii, and C. tetragattii cause infections mainly in immunocompromised hosts. This study aimed to compare the capacities of different species of the C. gattii species complex to induce cytokines and antimicrobial molecules in human peripheral blood mononuclear cells (PBMCs). Cryptococcus bacillisporus and C. deuterogattii induced the lowest levels of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and IL-6 among the five species of the C. gattii complex. Cryptococcus deuterogattii induced higher levels of IL-22 than those induced by C. tetragattii and the environmental species C. flavescens In addition, C. bacillisporus and C. gattii sensu stricto proliferated inside human monocyte-derived macrophages after 24 h of infection. All Cryptococcus species were able to generate reactive oxygen species (ROS) in human PBMCs, with C. bacillisporus and C. deuterogattii being more efficient than the other species. In conclusion, C. bacillisporus and C. deuterogattii induce lower levels of the proinflammatory cytokines TNF-α, IL-1ß, and IL-6 and higher ROS levels than those induced by the other species. Species of the Cryptococcus gattii complex have different abilities to induce cytokine and ROS production by human PBMCs.

Cryptococcus neoformans and gattii promote DNA damage in human peripheral blood mononuclear cells.

Cryptococcosis, a systemic mycosis capable of disseminating to the central nervous system with frequent lethal effects, is caused by the species Cryptococus neoformans and Cryptococcus gattii. Several infectious agents such as virus, bacteria, and parasites may be associated to DNA damage and carcinogenesis in humans. Products of the oxidative metabolism, such as NO, produced as a host defense mechanism to destroy these pathogens, have been implicated in this damage process, due to excessive production related to an established chronic inflammatory response. Here, we investigated whether C. neoformans and /or C. gattii can cause DNA damage in human peripheral blood mononuclear cells (PBMCs) and whether this process is related to NO levels produced by PBMCs. We found that both species are equally able to induce genotoxicity in PBMCs. However, an association between DNA damage and high NO levels was only detected in relation to C. gattii. The results point to the possibility that patients with cryptococcosis are more susceptible to the development of other diseases.

The clinical recovery of tuberculosis patients undergoing specific treatment is associated with changes in the immune and neuroendocrine responses.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a health problem worldwide. Patients with pulmonary TB show a neuro-immune-endocrine imbalance characterized by an impaired cellular immunity together with increased plasma levels of cortisol, pro- and anti-inflammatory cytokines and markedly decreased dehydroepiandrosterone (DHEA) levels. Extending these findings, we now investigated the immune-endocrine profile of TB patients undergoing specific treatment. Patients (n = 24) were bled at diagnosis (T0), 2, 4, 6 months after treatment initiation and 3 months following its completion. At T0, TB patients showed increased plasma levels of interleukin-6 (IL-6), C reactive protein, interferon-gamma (IFN-γ) and transforming growth factor beta (TGF-ß). These mediators decreased during treatment, reaching levels similar to those from healthy controls (n = 26). Specific treatment led to an increased lymphoproliferative response along with clinical improvement. Newly diagnosed patients had low levels of DHEA, with increased cortisol amounts and cortisol/DHEA ratio, which normalized upon specific treatment. As regards glucocorticoid receptors (GR), TB patients at diagnosis presented a reduced mRNA GRα/GRß ratio in their peripheral blood mononuclear cells. Furthermore, multivariate analysis showed that cortisol/DHEA ratio was positively associated with inflammatory mediators for which this ratio may constitute a disease biomarker. Anti-mycobacterial treatment results in a better immune-endocrine scenario for the control of physiopathological processes accompanying disease development and hence implied in clinical recovery.

Systemic Immunological changes in patients with distinct clinical outcomes during Mycobacterium tuberculosis infection.

BACKGROUND: The lung lesions in an individual infected with tuberculosis (TB) are surprisingly variable and independent of each other. However, there is no circulating biomarker yet able to segregate patients according to the extent of lung lesions. MATERIALS AND METHODS: In this study, the phenotypic and functional profile of leukocytes of patients with active pulmonary tuberculosis (TB) and controls (CO) were fully scrutinized by immunophenotyping assays and in vitro short-term whole blood culture. The TB group was subdivided according to the extent of lung lesions as unilateral (UNI) and bilateral (BI). RESULTS: The results show that TB group display an altered leukocyte profile in the peripheral blood with significant lower counts of NK-cells, CD3+CD56+CD16+/- NKT-cells, CD4+T-cells, CD19+B-cells when compared to CO. Increased CD4+T-cells and CD8+T-cell activation was observed by the upregulation of activation markers (HLA-DR) as well as of chemokine receptors (CCR2, CCR3, and CXCR4). In addition, TB group presented a significant decrease proportion of CD14LowCD16+ monocytes despite the increase in HLA-DR expression. Regarding the severity of the disease, in the BI group a reduction in frequency of CD19+CD5+ B-cells and expression of HLA-DR in CD14LowCD16+ monocytes was observed. Furthermore, the extent of lung lesions influences the production of molecules as observed by significantly larger production of IL-4 by neutrophils, total T-cells, CD4+T-cells, CD8+T-cells and CD19+B-cells in UNI as compared to BI. By contrast, in BI group the frequency of high producers of both IL-17+CD4+T-cells and IL-17+CD8+T-cells were significantly increased than UNI, suggesting the deleterious role of these subsets during active pulmonary Mtb infection. CONCLUSION: The immunophenotypic characterization of unilateral and bilateral active TB performed in the present study indicates that the extent of lung lesion could be associated with a fine-tuning between immunological responses during untreated Mtb infection.

Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host.

We have morphologically characterizedCandida tropicalisisolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of ß-1,3-glucan in the cell wall but not with detectable changes in chitin content. In line with this, AmB-resistant strains showed reduced susceptibility to Congo red. Moreover, mitogen-activated protein kinases (MAPKs) involved in cell integrity were already activated during regular growth in these strains. Finally, we investigated the response elicited by human blood cells and found that AmB-resistant strains induced a stronger proinflammatory response than susceptible strains. In agreement, AmB-resistant strains also induced stronger melanization ofGalleria mellonellalarvae, indicating that the effect of alterations of the cell wall on the immune response is conserved in different types of hosts. Our results suggest that resistance to AmB is associated with pleiotropic mechanisms that might have important consequences, not only for the efficacy of the treatment but also for the immune response elicited by the host.

Longitudinal immune profiles in type 1 leprosy reactions in Bangladesh, Brazil, Ethiopia and Nepal.

BACKGROUND: Acute inflammatory reactions are a frequently occurring, tissue destructing phenomenon in infectious- as well as autoimmune diseases, providing clinical challenges for early diagnosis. In leprosy, an infectious disease initiated by Mycobacterium leprae (M. leprae), these reactions represent the major cause of permanent neuropathy. However, laboratory tests for early diagnosis of reactional episodes which would significantly contribute to prevention of tissue damage are not yet available. Although classical diagnostics involve a variety of tests, current research utilizes limited approaches for biomarker identification. In this study, we therefore studied leprosy as a model to identify biomarkers specific for inflammatory reactional episodes. METHODS: To identify host biomarker profiles associated with early onset of type 1 leprosy reactions, prospective cohorts including leprosy patients with and without reactions were recruited in Bangladesh, Brazil, Ethiopia and Nepal. The presence of multiple cyto-/chemokines induced by M. leprae antigen stimulation of peripheral blood mononuclear cells as well as the levels of antibodies directed against M. leprae-specific antigens in sera, were measured longitudinally in patients. RESULTS: At all sites, longitudinal analyses showed that IFN-γ-, IP-10-, IL-17- and VEGF-production by M. leprae (antigen)-stimulated PBMC peaked at diagnosis of type 1 reactions, compared to when reactions were absent. In contrast, IL-10 production decreased during type 1 reaction while increasing after treatment. Thus, ratios of these pro-inflammatory cytokines versus IL-10 provide useful tools for early diagnosing type 1 reactions and evaluating treatment. Of further importance for rapid diagnosis, circulating IP-10 in sera were significantly increased during type 1 reactions. On the other hand, humoral immunity, characterized by M. leprae-specific antibody detection, did not identify onset of type 1 reactions, but allowed treatment monitoring instead. CONCLUSIONS: This study identifies immune-profiles as promising host biomarkers for detecting intra-individual changes during acute inflammation in leprosy, also providing an approach for other chronic (infectious) diseases to help early diagnose these episodes and contribute to timely treatment and prevention of tissue damage.

Behçet's disease heterogeneity: cytokine production and oxidative burst of phagocytes are altered in patients with severe manifestations.

OBJECTIVES: To test the hypothesis that classical phagocytic functions are constitutively stimulated in patients with Behçet's disease (BD). METHODS: Four study groups were analysed: active BD (aBD; n=30), inactive BD (iBD; n=31); septic patients (SP; n=25); healthy controls (HC; n=30). Microbicide activity against Streptococcus pneumoniae, Streptococcus sanguinis and Candida albicans was determined by means of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and absorbance read by ELISA. Flow cytometry analysis evaluated phagocytosis (zymosan particles and microrganisms) and oxidative burst by dihidrorhodamine oxidation before and after stimulation with phorbol myristate acetate (PMA). The supernatant of PBMC cultures under TLR or microbial stimuli and of neutrophil cultures under PMA, LPS or microbial stimuli were used for determination of cytokine production by ELISA. RESULTS: We found no significant differences between the BD patient groups and control groups with regard to oxidative burst, phagocytic activity, microbicide activity or cytokine production. However, the cells from patients with severe BD (based on clinical manifestation) exhibit significantly higher oxidative burst activity, both before and after PMA stimulation, compared to cells from patients with mild BD. Furthermore, we found significant correlations between the BD patients' scores on the simplified Behçet's Disease Current Activity Form adapted for Portuguese (BR-BDCAFs) and Streptococcus sanguinis-stimulated production of IL23 by PBMC and IL8 by neutrophils, and between BR-BDCAFs score and constitutive production of TNF-α, IFNγ, IL6 and IL23 by PBMC. CONCLUSIONS: Patients with severe active BD do exhibit phagocytic dysfunction and some evidence of constitutive activation regarding oxidative burst and cytokine production.

Genetically engineered immunomodulatory Streptococcus thermophilus strains producing antioxidant enzymes exhibit enhanced anti-inflammatory activities.

The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti-inflammatory activities.

Candida albicans delays HIV-1 replication in macrophages.

Macrophages are one of the most important HIV-1 target cells. Unlike CD4(+) T cells, macrophages are resistant to the cytophatic effect of HIV-1. They are able to produce and harbor the virus for long periods acting as a viral reservoir. Candida albicans (CA) is a commensal fungus that colonizes the portals of HIV-1 entry, such as the vagina and the rectum, and becomes an aggressive pathogen in AIDS patients. In this study, we analyzed the ability of CA to modulate the course of HIV-1 infection in human monocyte-derived macrophages. We found that CA abrogated HIV-1 replication in macrophages when it was evaluated 7 days after virus inoculation. A similar inhibitory effect was observed in monocyte-derived dendritic cells. The analysis of the mechanisms responsible for the inhibition of HIV-1 production in macrophages revealed that CA efficiently sequesters HIV-1 particles avoiding its infectivity. Moreover, by acting on macrophages themselves, CA diminishes their permissibility to HIV-1 infection by reducing the expression of CD4, enhancing the production of the CCR5-interacting chemokines CCL3/MIP-1α, CCL4/MIP-1ß, and CCL5/RANTES, and stimulating the production of interferon-α and the restriction factors APOBEC3G, APOBEC3F, and tetherin. Interestingly, abrogation of HIV-1 replication was overcome when the infection of macrophages was evaluated 2-3 weeks after virus inoculation. However, this reactivation of HIV-1 infection could be silenced by CA when added periodically to HIV-1-challenged macrophages. The induction of a silent HIV-1 infection in macrophages at the periphery, where cells are continuously confronted with CA, might help HIV-1 to evade the immune response and to promote resistance to antiretroviral therapy.

An assessment of whole blood and fractions by nested PCR as a DNA source for diagnosing canine ehrlichiosis and anaplasmosis.

Ehrlichiosis and anaplasmosis are tick-borne diseases. Ehrlichia canis and Anaplasma platys infect mainly white cells and platelets, respectively. The main DNA source for PCR is peripheral blood, but the potential of blood cell fractions has not been extensively investigated. This study aims at assessment of whole blood (WB) and blood fractions potential in nested PCR (nPCR) to diagnose canine ehrlichiosis and anaplasmosis. The 16S rRNA gene was amplified in 71.4, 17.8, 31.57, and 30% of the WB, granulocyte (G), mononuclear cells (M), and buffy coat (BC) samples. Compared to the WB, the sensitivity of the PCR was 42.86% for the M, and BC fractions, 21.43% for the G, and 33.33% for the blood clot (C). There was fair agreement between the WB and M, BC and C, and slight with the G. Fair agreement occurred between the nPCR and morulae in the blood smear. One animal was coinfected with A. platys and E. canis. This study provided the first evidence of A. platys infection in dogs in Paraíba, Brazil, and demonstrated that WB is a better DNA source than blood fractions to detect Ehrlichia and Anaplasma by nPCR, probably because of the plasma bacterial concentration following host cell lysis.

TGF-ß neutralization abrogates the inhibited DHEA production mediated by factors released from M. tuberculosis-stimulated PBMC.

Supernatants (SN) from cultures of peripheral blood mononuclear cells (PBMC) of tuberculosis (TB) patients inhibit dehydroepiandrosterone (DHEA) secretion by the adrenal cell line NCI-H295R. To analyze whether TGF-ß is involved in this effect, SN of PBMC from healthy controls or patients with severe TB infections, stimulated or not with Mycobacterium tuberculosis (Mtb SN), were added to adrenal cells under basal conditions or following stimulation with forskolin. Cortisol and DHEA concentrations were evaluated in supernatants of the adrenal cells cultured with or without the addition of anti-TGF-ß. Treatment with Mtb SN from TB inhibited DHEA production, and this effect was reversed when SN were treated with anti-TGF-ß. The increase in cortisol production induced by SN from TB patients was not affected by TGF-ß neutralization. Mediators released during the anti-TB immune response differentially modulate steroid production by adrenal cells, and TGF-ß is a cytokine implicated in the inhibition of DHEA production observed in TB.

Transcriptional response of peripheral blood mononuclear cells from cattle infected with Mycobacterium bovis.

Mycobacterium bovis is the causative agent of most cases of bovine tuberculosis. The identification of bTB biomarkers in specific stages of the disease will contribute to a better understanding of the immunopathology associated with tuberculosis and will enable their use in disease diagnosis and prognosis. The aim of this study was to evaluate the gene expression profile induced after specific stimulation of bovine peripheral blood mononuclear cells from cattle infected with M. bovis using the Affymetrix® GeneChip® Bovine Genome Array. A total of 172 genes showed differential expression profile that was statistically significant with log2-fold change >2.5 and <-2.5. Twenty-four out of these genes were upregulated and 148 were downregulated in bovine peripheral blood mononuclear cells of M. bovis-infected cattle. The highest differentially-expressed genes were related to immune and inflammatory responses, apoptosis, endocytosis, cellular trafficking and genes encoding proteins involved in cellular matrix degradation. Microarray results were confirmed in another group of infected cattle by RT-qPCR for the CD14, IL-1R, THBS1, MMP9 and FYVE genes. This study confirms previous findings that have shown that M. bovis infection in cattle results in the downregulation of immune response-related genes. Moreover, it validates the use of microarray platforms in combination with RT-qPCR to identify biomarkers of bovine tuberculosis. In addition, we propose CD14, IL-1R, THBS1, MMP9 and FYVE as potential biomarkers of bovine tuberculosis.

High expression of human monocyte iNOS mRNA induced by Paracoccidioides brasiliensis is not associated with increase in NO production.

In this study we investigated the role of nitric oxide (NO) in monocyte fungicidal activity against Paracoccidioides brasiliensis. We found that cells primed with IFN-γ, TNF-α or GM-CSF and challenged with a high-(Pb18) or low-virulence (Pb265) strain of the fungus increase their fungicidal activity. Expression of iNOS mRNA was increased after priming cells with each cytokine, and tended to be inhibited by Pb18. Despite up-regulation of iNOS mRNA expression by Pb265, an equivalent increase in NO production was not detected, as metabolite levels were similar in all cultures. The results indicated that high expression of human monocyte iNOS mRNA induced by P. brasiliensis is not correlated with NO concentrations produced.