Neonatal nasal necrosis: Case series and brief review.

There is limited information available on pressure-related neonatal nasal injuries. We present three neonates born with erythema and purpura of the nasal tip that subsequently ulcerated, then evolved into a thick eschar. Each healed well with conservative management but left behind significant scarring. The sharp demarcation and location of the lesions were suggestive of hypoxic tissue damage akin to halo scalp ring alopecia. Further investigation is necessary to elucidate the etiology and optimal management of this condition.

The relationship between host defense peptides and adrenal steroids. An account of reciprocal influences.

AIM: ß-defensins 2 and -3 (HBD-2 and HBD-3) and cathelicidin LL-37 are host defense peptides (HDPs) that play a crucial role in the immune response against mycobacteria. Given our former studies in tuberculosis patients wherein their plasma levels of such peptides correlated with steroid hormone concentrations, we now studied the reciprocal influence of cortisol and/or dehydroepiandrosterone (DHEA) on HDPs biosynthesis and LL-37 on adrenal steroidogenesis. MAIN METHODS: Cultures of macrophages derived from the THP-1 line were treated with cortisol (10-6M) and/or DHEA (10-6M and 10-7M) and stimulated with irradiated M. tuberculosis (Mi) or infected M. tuberculosis strain H37Rv to assess cytokine production, HDPs, reactive oxygen species (ROS) and colony forming units. Cultures of NCI-H295-R adrenal line were treated with LL37 (5, 10, and 15 µg/ml) for 24 h to further measure cortisol and DHEA levels together with steroidogenic enzyme transcripts. KEY FINDINGS: In macrophages, M. tuberculosis produced an increase of IL-1ß, TNFα, IL-6, IL-10, LL-37, HBD-2, and HBD-3 levels, irrespective of DHEA treatment. Adding cortisol to M. tuberculosis-stimulated cultures (with or without DHEA) decreased the amounts of these mediators, compared to only stimulated cultures. Although M. tuberculosis reduced ROS levels, DHEA increased these values in addition to diminishing intracellular mycobacterial growth (no matter cortisol treatment). In turn, studies on adrenal cells showed that LL-37 reduced the production of cortisol and DHEA besides modifying transcripts for some steroidogenic enzymes. SIGNIFICANCE: while adrenal steroids seem to influence the production of HDPs, the former compounds are also likely to modulate adrenal biogenesis.

Evidence that changes in antimicrobial peptides during tuberculosis are related to disease severity, clinical presentation, specific therapy and levels of immune-endocrine mediators.

Given the role of host defense peptides (HDPs) in the defensive response against mycobacteria, we analyzed the circulating levels of LL-37, ß-defensin-2 and -3 in newly diagnosed patients with pulmonary (PTB) or pleural tuberculosis (PLTB) in whom measurements of pleural fluids were also performed. Severe PTB patients displayed higher circulating amounts of ß-defensin-3, statistically different from controls, further decreasing upon antimycobacterial treatment. LL-37 concentrations appeared within the normal range at diagnosis, but tended to increase during treatment, becoming statistically upon its completion in moderate cases. PLTB patients revealed decreased levels of ß-defensin-2 in presence of increased amounts of ß-defensin-3 and LL-37; in their plasma or pleural fluids. Considering the immune-endocrine dysregulation of tuberculosis, we also performed correlation analysis detecting positive associations between levels of cortisol, IL-6 and ß-defensin-3 in plasma from untreated severe patients as did their dehydroepiandrosterone and LL-37 values. Increased presence of ß-defensins, may represent an attempt to improve defensive mechanisms; which also take part in the inflammatory reaction accompanying TB, reinforced by the association with immune-endocrine mediators. The divergent profile of PLTB patients, decreased ß-defensin-2 but increased ß-defensin-3 and LL-37 levels, suggests a differential role of these HDPs in a situation characterized for its better protective response.

Simultaneous determination of opiates, methadone, amphetamines, cocaine, and metabolites in human placenta and umbilical cord by LC-MS/MS.

LC-MS/MS methods for the quantification of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, 6-acetylmorphine, cocaine, benzoylecgonine, ecgonine methyl ester, hydroxybenzoylecgonine, cocaethylene, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), methadone, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in human placenta and umbilical cord were developed and validated. Specimens (1 ± 0.02 g) were homogenized with the Ultra-Turrax T8 disperser and centrifuged, and the supernatant was submitted to solid-phase extraction with Oasis MCX cartridges. Chromatographic separation was performed using an Atlantis T3 analytical column (100 × 2.1 mm, 3 µm) and a gradient of 0.1 % formic acid and acetonitrile. Selectivity was verified in 10 different blank specimens. The method was linear from 1-5 to 100-500 ng/g, depending on the analyte. Limits of detection and quantification ranged from 0.5 to 2.5 ng/g and 1 to 5 ng/g, respectively. Method imprecision was ≤15.3 %, except for MDMA at low quality control (18.1 %); accuracy, 87.1 to 114 %; extraction efficiency, 16.3 to 154.0 % (%CV = 1.8-39.4 %); matrix effect, -75.7 to 449.9 % (%CV = 3.5-50 %); and process efficiency, 8.7 to 316.0 %. The method was applied to authentic placenta and umbilical cord specimens from drug-user pregnant women.

Studies on the contribution of PPAR Gamma to tuberculosis physiopathology.

Introduction: Tuberculosis (TB) is a major health problem characterized by an immuno-endocrine imbalance: elevated plasma levels of cortisol and pro- and anti-inflammatory mediators, as well as reduced levels of dehydroepiandrosterone. The etiological agent, Mycobacterium tuberculosis (Mtb), is captured by pulmonary macrophages (Mf), whose activation is necessary to cope with the control of Mtb, however, excessive activation of the inflammatory response also leads to tissue damage. Glucocorticoids (GC) are critical elements to counteract the immunoinflammatory reaction, and peroxisome proliferator-activated receptors (PPARs) are also involved in this regard. The primary forms of these receptors are PPARϒ, PPARα, and PPARß/δ, the former being the most involved in anti-inflammatory responses. In this work, we seek to gain some insight into the contribution of PPARϒ in immuno-endocrine-metabolic interactions by focusing on clinical studies in pulmonary TB patients and in vitro experiments on a Mf cell line. Methods and results: We found that TB patients, at the time of diagnosis, showed increased expression of the PPARϒ transcript in their peripheral blood mononuclear cells, positively associated with circulating cortisol and related to disease severity. Given this background, we investigated the expression of PPARϒ (RT-qPCR) in radiation-killed Mtb-stimulated human Mf. The Mtb stimulation of Mf derived from the human line THP1 significantly increased the expression of PPARϒ, while the activation of this receptor by a specific agonist decreased the expression of pro- and anti-inflammatory cytokines (IL-1ß and IL-10). As expected, the addition of GC to stimulated cultures reduced IL-1ß production, while cortisol treatment together with the PPARϒ agonist lowered the levels of this proinflammatory cytokine in stimulated cultures. The addition of RU486, a glucocorticoid receptor antagonist, only reversed the inhibition produced by the addition of GC. Conclusion: The current results provide a stimulating background for further analysis of the interconnection between PPARs and steroid hormones in the context of Mtb infection.

The Immunoregulatory Actions of DHEA in Tuberculosis, A Tool for Therapeutic Intervention?

Dehydroepiandrosterone (DHEA) is an androgen synthesized by the adrenal cortex, which is an intermediary in the biosynthesis of sex hormones, such as testosterone and estradiol. DHEA mostly circulates as a conjugated ester, in the form of sulfate (DHEA-S). There exist several endogenous factors able to influence its synthesis, the most common ones being the corticotrophin-releasing hormone (CRH), adrenocorticotrophin (ACTH), growth factors, and proinflammatory cytokines, among others. Like other steroid hormones, DHEA, can alter the functioning of immune cells and therefore the course of diseases exhibiting an immune-inflammatory component, mostly from autoimmune or infectious nature. We herein review the role played by DHEA during a major infectious disease like tuberculosis (TB). Data recorded from TB patients, mouse models, or in vitro studies show that DHEA is likely to be implied in better disease control. This provides a stimulating background for carrying out clinical studies aimed at assessing the usefulness of DHEA as an adjuvant in TB patients.

Differential expression of genes regulated by the glucocorticoid receptor pathway in patients with pulmonary tuberculosis.

AIMS: Previous studies in TB patients showed an immuno-endocrine imbalance characterized by a disease-severity associated increase in plasma levels of proinflammatory cytokines and glucocorticoids (GCs). To analyze the potential immunomodulatory effect of circulating GCs over peripheral blood mononuclear cells (PBMC) from TB patients, we investigated the expression of positively (anti-inflammatory-related genes ANXA1; FKBP51; GILZ, NFKBIA, and NFKBIB) and negatively (inflammatory genes: IL-6, IL-1ß, and IFN-γ) Glucocorticoids Receptors (GR)-regulated genes. Plasma concentrations of cytokines and hormones, together with specific lymphoproliferation were also assessed. MATERIALS AND METHODS: Gene expression was quantified by RT-qPCR, specific lymphoproliferation by 3H-thymidine incorporation, whereas plasma cytokines and hormones levels by ELISA. KEY FINDINGS: Transcripts of ANXA1, GILZ, NFKBIB, and NFKBIA appeared significantly increased in patients, whereas FKBP51, IL-6, IL-1ß, and NF-κB remained unchanged. Upon analyzing according to disease severity, mRNA levels for ANXA1 and NFKBIB were even higher in moderate and severe patients. GILZ was increased in moderate cases, with NFKBIA and IL-1 ß being higher in severe ones, who also displayed increased GRß transcripts. TB patients had reduced plasma DHEA concentrations together with increased pro and anti-inflammatory cytokines (IFN-γ, IL-6, and IL-10) cortisol and cortisol/DHEA ratio, more evident in progressive cases, in whom their PBMC also showed a decreased mycobacterial-driven proliferation. The cortisol/DHEA ratio and GRα expression were positively correlated with GR-regulated genes mainly in moderate patients. SIGNIFICANCE: The increased expression of cortisol-regulated anti-inflammatory genes in TB patients-PBMC, predominantly in progressive disease, seems compatible with a relatively insufficient attempt to downregulate the accompanying inflammation.

SCAMP3 Regulates EGFR and Promotes Proliferation and Migration of Triple-Negative Breast Cancer Cells through the Modulation of AKT, ERK, and STAT3 Signaling Pathways.

Triple-negative breast cancer (TNBC) is the most aggressive, metastatic, and lethal breast cancer subtype. To improve the survival of TNBC patients, it is essential to explore new signaling pathways for the further development of effective drugs. This study aims to investigate the role of the secretory carrier membrane protein 3 (SCAMP3) in TNBC and its association with the epidermal growth factor receptor (EGFR). Through an internalization assay, we demonstrated that SCAMP3 colocalizes and redistributes EGFR from the cytoplasm to the perinucleus. Furthermore, SCAMP3 knockout decreased proliferation, colony and tumorsphere formation, cell migration, and invasion of TNBC cells. Immunoblots and degradation assays showed that SCAMP3 regulates EGFR through its degradation. In addition, SCAMP3 modulates AKT, ERK, and STAT3 signaling pathways. TNBC xenograft models showed that SCAMP3 depletion delayed tumor cell proliferation at the beginning of tumor development and modulated the expression of genes from the PDGF pathway. Additionally, analysis of TCGA data revealed elevated SCAMP3 expression in breast cancer tumors. Finally, patients with TNBC with high expression of SCAMP3 showed decreased RFS and DMFS. Our findings indicate that SCAMP3 could contribute to TNBC development through the regulation of multiple pathways and has the potential to be a target for breast cancer therapy.

Ganoderma lucidum enhances carboplatin chemotherapy effect by inhibiting the DNA damage response pathway and stemness.

Inflammatory Breast Cancer (IBC) is a rare and aggressive type of breast cancer with a poor prognosis. Its management is challenging because of a lack of targeted therapies, increased metastatic potential, and high recurrence rates. Interest in using platinum agents such as carboplatin emerged from data suggesting frequent DNA repair defects in breast cancer. Because studies show that medicinal mushroom Ganoderma lucidum (GLE) sensitizes cancer cells to radiation and other drugs; herein, we aimed to investigate the therapeutic potential of GLE, alone or in combination with carboplatin in breast cancer models. Our studies were focused on the regulation of the DNA Damage Response (DDR) and on cancer cell stemness. Carboplatin and GLE were tested in vitro using the IBC cell line, SUM-149, breast cancer non-IBC cells, MDA-MB-231, and in vivo using IBC xenograft models. Our results show that the GLE/carboplatin combination decreased cell viability, induced cell death by two different mechanisms, and delayed the response to DNA damage. Furthermore, the combination suppressed mammosphere formation and the expression of cancer stemness proteins. In xenograft models, the combination showed significant tumor growth inhibitory effects without systemic toxicity. This study emphasizes the potential of this dual therapy for IBC patients.

Increased levels of circulating LPS during Tuberculosis prevails in patients with advanced pulmonary involvement.

Our earlier studies in tuberculosis (TB) patients indicate that in those where the process evolves to a larger pulmonary involvement, the immune endocrine response may promote an unfavorable environment. Chronic infectious diseases, and their persistent proinflammatory response, may affect mucosal barriers integrity favoring the translocation of gastrointestinal bacteria, leading to an increase of circulating lipopolysaccharides (LPS). Consequently, we quantified LPS levels in TB patients, with different degrees of pulmonary involvement, and controls (Co) and analyzed the possible relationship between LPS and inflammatory mediators i.e., C reactive protein (CRP), interleukin 6 (IL-6) and Interferon-gamma (IFN-γ), Erythrocyte Sedimentation Rate (ESR), steroid hormones (Cortisol and Dehydroepiandrosterone, DHEA), and inflammatory transcripts from peripheral blood mononuclear cells (IL-1ß, IL-6, IFN-γ). LPS was assessed by the Limulus amoebocyte lysate assay and the ELISA technique was used to quantify hormones and cytokines in the plasma samples. Cytokine transcripts from PBMC were evaluated by qRT-PCR. Non-parametric tests were used. LPS levels were increased in TB patients, as did levels of CRP, IL-6, IFN-γ, cortisol and ESR. Severe patients had the highest amounts of circulating LPS; with moderate and severe cases showing much higher levels of CRP, ESR, IL-6, IFN-γ and cortisol/DHEA ratio, as an endocrine imbalance. Only in PBMC from severe cases was mRNA for IL-1ß increased. Correlation analysis showed that levels of LPS from severe patients were positively associated with IL-6 and IFN-γ plasma concentrations and with IL-1ß transcripts, while IL-6 had a positive correlation with the cortisol/DHEA ratio. The higher levels of circulating LPS during progressive TB may emerge as a contributing factor for the persistence of the greater immune endocrine imbalance distinctive of advanced disease, which might suggest a vicious cycle among LPS, inflammation and endocrine imbalance.

HIF2 Regulates Intestinal Wnt5a Expression.

Radiation therapy for abdominal tumors is challenging because the small intestine is exquisitely radiosensitive. Unfortunately, there are no FDA-approved therapies to prevent or mitigate GI radiotoxicity. The EGLN protein family are oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs). Our group has previously shown that stabilization of HIF2 through genetic deletion or pharmacologic inhibition of the EGLNs mitigates and protects against GI radiotoxicity in mice by improving intestinal crypt stem cell survival. Here we aimed to elucidate the molecular mechanisms by which HIF2 confers GI radioprotection. We developed duodenal organoids from mice, transiently overexpressed non-degradable HIF2, and performed bulk RNA sequencing. Interestingly, HIF2 upregulated known radiation modulators and genes involved in GI homeostasis, including Wnt5a. Non-canonical Wnt5a signaling has been shown by other groups to improve intestinal crypt regeneration in response to injury. Here we show that HIF2 drives Wnt5a expression in multiple duodenal organoid models. Luciferase reporter assays performed in human cells showed that HIF2 directly activates the WNT5A promoter via a hypoxia response element. We then evaluated crypt regeneration using spheroid formation assays. Duodenal organoids that were pre-treated with recombinant Wnt5a had a higher cryptogenic capacity after irradiation, compared to vehicle-treated organoids. Conversely, we found that Wnt5a knockout decreased the cryptogenic potential of intestinal stem cells following irradiation. Treatment with recombinant Wnt5a prior to irradiation rescued the cryptogenic capacity of Wnt5a knockout organoids, indicating that Wnt5a is necessary and sufficient for duodenal radioprotection. Taken together, our results suggest that HIF2 radioprotects the GI tract by inducing Wnt5a expression.

Comparative Untargeted Metabolomic Profiling of Induced Mitochondrial Fusion in Pancreatic Cancer.

Mitochondria are dynamic organelles that constantly alter their shape through the recruitment of specialized proteins, like mitofusin-2 (Mfn2) and dynamin-related protein 1 (Drp1). Mfn2 induces the fusion of nearby mitochondria, while Drp1 mediates mitochondrial fission. We previously found that the genetic or pharmacological activation of mitochondrial fusion was tumor suppressive against pancreatic ductal adenocarcinoma (PDAC) in several model systems. The mechanisms of how these different inducers of mitochondrial fusion reduce pancreatic cancer growth are still unknown. Here, we characterized and compared the metabolic reprogramming of these three independent methods of inducing mitochondrial fusion in KPC cells: overexpression of Mfn2, genetic editing of Drp1, or treatment with leflunomide. We identified significantly altered metabolites via robust, orthogonal statistical analyses and found that mitochondrial fusion consistently produces alterations in the metabolism of amino acids. Our unbiased methodology revealed that metabolic perturbations were similar across all these methods of inducing mitochondrial fusion, proposing a common pathway for metabolic targeting with other drugs.

Evidence for a More Disrupted Immune-Endocrine Relation and Cortisol Immunologic Influences in the Context of Tuberculosis and Type 2 Diabetes Comorbidity.

Pulmonary tuberculosis (PTB), caused by Mycobacterium tuberculosis (Mtb), is a major health problem worldwide, further aggravated by the convergence of type 2 diabetes mellitus (DM) which constitutes an important risk factor for TB development. The worse scenario of patients with PTB and DM may be partly related to a more unbalanced defensive response. As such, newly diagnosed PTB patients with DM (TB+DM, n = 11) or not (TB, n = 21), as well as DM (n = 18) patients and pair matched controls (Co, n = 22), were investigated for the circulating immuno-endocrine-metabolic profile (ELISA), along with studies in peripheral blood mononuclear cells (PBMC) analyzing transcript expression (RT-qPCR) of mediators involved in glucocorticoid functionality. Given the hyperglycemic/hypercortisolemic scenario of TB+DM patients, PBMC were also exposed to stress-related cortisol concentrations (0.1 and 1 µM) and supraphysiologic glucose doses (10, 20, and 40 mM) and assessed for the specific response against Mtb stimulation (lymphoproliferation, -thymidine incorporation-, and cytokine production -bead-cytometry). All TB patients displayed increased plasma amounts of cortisol, growth hormone -hGH-, and proinflammatory mediators. In turn, TB+DM showed even higher levels of interferon gamma -IFN-γ- and hGH (vs. TB), or IL-6, C reactive protein, cortisol and hGH (vs. DM). Both DM groups had equally augmented values of IL-10. All TB patients showed decreased dehydroepiandrosterone- sulfate concentrations, even more in TB+DM cases. Leptin was also decreased in both TB cases, particularly in the TB group, revealing a lower body mass index, as well. Unlike PBMC from TB cases showing a decreased relationship between the glucocorticoids receptor (GR) isoforms (GRα/GRß; functional isoform/negative isoform), cells from TB+DM patients had no changes in this regard, along with an increased expression of 11-beta hydroxysteroid dehydrogenase type-1, the enzyme facilitating intracellular cortisone to cortisol conversion. TB+DM patients also showed an increased Mtb antigen-driven lymphoproliferation. Compared to TB, DM and HCo counterparts, PBMC from TB+DM patients had a biased Th1 response to Mtb stimulation (increased IL-2 and IFN-γ production), even when exposed to inhibitory cortisol doses. TB+DM patients show a more unbalanced immuno-endocrine relationship, respect the non-diabetic counterparts, with a relative deficiency of cortisol immunomodulatory influences, despite their more favorable microenvironment for cortisol-mediated immune effects.

Ganoderma lucidum Extract Reduces the Motility of Breast Cancer Cells Mediated by the RAC⁻Lamellipodin Axis.

Breast cancer (BC) is the second leading cause of cancer death among women worldwide. The main cause of BC morbidity and mortality is the invasiveness capacity of cancer cells that may lead to metastasis. Here, we aimed to investigate the therapeutic efficacy of Ganoderma lucidum extract (GLE)-a medicinal mushroom with anticancer properties-on BC motility via the Rac/Lamellipodin pathway. GLE treatment effects were tested on MDA-MB-231 breast cancer cells. The effects were tested on cell viability, migration and invasion. Pulldowns, immunoblotting, and immunofluorescence were used to measure Rac activity and the expression of proteins involved in cell migration and in lamellipodia formation, respectively. As a result, GLE suppressed BC cell viability, migration, and invasion capacity. GLE impaired Rac activity, as well as downregulated Lamellipodin, ENA/VASP, p-FAK (Tyr925), Cdc42, and c-Myc expression. Lamellipodia formation was significantly reduced by GLE. In conclusion, we demonstrate that GLE reduces Rac activity and downregulates signaling molecules involved in lamellipodia formation. These novel findings serve as basis for further studies to elucidate the potential of GLE as a therapeutic agent regulating the Rac/Lamellipodin pathway in BC metastasis.

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.

miR-30c is specifically repressed in patients with active pulmonary tuberculosis.

Tuberculous pleurisy (PLTB) is a common form of extrapulmonary tuberculosis. It often resolves without chemotherapy being hence considered a rather benign manifestation of the disease. Patients with PLTB mount an effective anti-mycobacterial response, unlike those with active pulmonary TB (pTB) that were shown to present an imbalance in plasma immune and endocrine mediators. In this work, we explored whether expression of the active isoform of the glucocorticoid receptor (hGRα) in the context of the inflammatory-anti-inflammatory responses of TB patients may be associated to microRNA levels. As expected, the inflammatory response triggered in patients coexists with increased circulating cortisol and altered hGRα levels in the peripheral blood mononuclear cells. However, while hGRα expression is significantly downregulated in PLTB, its levels in pTB patients are higher within the control values. These results point out to the existence of an additional mechanism tending to preserve hGRα levels probably to deal with the chronic inflammation observed in pTB. In this regard, we found that miR-30c is strongly downregulated in mononuclear cells of pTB patients compared to PLTB cases, showing an expression profile opposite to that seen with hGRα. Interestingly, low levels of miR-30c are specific for this active form of TB, as its expression is not altered in mononuclear cells from either healthy controls or patients with tuberculous or non-tuberculous pleurisy. Moreover, miR-30c and hGRα also showed an inverse expression pattern in M. tuberculosis-stimulated THP-1 macrophage cultures. In sum, our studies identify miR-30c as a specific correlate of pulmonary manifestations of TB, potentially involved in the altered glucocorticoid sensitivity observed in these patients.

Increased frequency of CD4+ CD25+ FoxP3+ T regulatory cells in pulmonary tuberculosis patients undergoing specific treatment and its relationship with their immune-endocrine profile.

Tuberculosis (TB) is a major health problem requiring an appropriate cell immune response (IR) to be controlled. Since regulatory T cells (Tregs) are relevant in IR regulation, we analyzed Tregs variations throughout the course of TB treatment and its relationship with changes in immune-endocrine mediators dealing with disease immunopathology. The cohort was composed of 41 adult patients, 20 of them completing treatment and follow-up. Patients were bled at diagnosis (T0) and at 2 (T2), 4 (T4), 6 (T6), and 9 months following treatment initiation. Twenty-four age- and sex-matched healthy controls (HCo) were also included. Tregs (flow cytometry) from TB patients were increased at T0 (versus HCo P < 0.05), showing even higher values at T2 (versus T0 P < 0.01) and T4 (versus T0 P < 0.001). While IL-6, IFN-γ, TGF-ß (ELISA), and Cortisol (electrochemiluminescence, EQ) were augmented, DHEA-S (EQ) levels were diminished at T0 with respect to HCo, with cytokines and Cortisol returning to normal values at T9. Tregs correlated positively with IFN-γ (R = 0.868, P < 0.05) at T2 and negatively at T4 (R = -0.795, P < 0.05). Lowered levels of proinflammatory cytokines together with an increased frequency of Tregs of patients undergoing specific treatment might reflect a downmodulatory effect of these cells on the accompanying inflammation.

The implication of pro-inflammatory cytokines in the impaired production of gonadal androgens by patients with pulmonary tuberculosis.

BACKGROUND: The chronic nature of tuberculosis and the protracted immuno-inflammatory reactions are implied in a series of metabolic and immune-endocrine changes accompanying the disease. We explored components from the hypothalamous-pituitary-gonadal axis and their relationship with cytokines involved in disease immunopathology, in male TB patients. METHODS: Plasma samples from 36 active untreated pulmonary TB male patients were used to determine TNF-α, IFN-γ, TGF-ß, IL-6, cortisol, dehydroepiandrosterone, testosterone, progesterone, estradiol, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by ELISA. Healthy controls corresponded to 21 volunteers without contact with TB patients and similar age (40 ± 16,8 years). Testicular histological samples from necropsies of patients dying from TB were immune-stained for IL-1ß, TNF-α, IL-6 and IFN-γ. The TM3 mouse Leydig cell line was incubated with recombinants TNF-α, IFN-γ and TGF-ß, supernatants were collected and used to measure testosterone by ELISA. RESULTS: Patients showed decreased levels of testosterone in presence of high amounts of LH, together with augmented IFN-γ, IL-6 and TGF-ß levels. Testicular histological sections showed abundant presence of IL-1ß, TNF-α, IL-6 and IFN-γ in interstitial macrophages, Sertoli cells and some spermatogonia. In vitro treatment of Leydig cells with these cytokines led to a remarkable reduction of testosterone production.

Levels of inflammatory cytokines, adrenal steroids, and mRNA for GRα, GRß and 11ßHSD1 in TB pleurisy.

Our previous work on the immune-endocrine features of patients with pulmonary tuberculosis (TB) showed markedly decreased plasma levels of dehydroepiandrosterone (DHEA) together with augmented concentrations of Cortisol and pro- and anti-inflammatory cytokines. Studies in peripheral blood mononuclear cells (PBMC) indicated a lower mRNA α/ß ratio of glucocorticoid receptors -GR- together with a higher 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) mRNA expression in cases with severe pulmonary TB. Since Pleural TB (PLTB) is a rather benign manifestation of TB, we now analyzed the systemic and local immune-endocrine profile as well as the GRα, GRß, 11ßHSD1 and 11ßHSD2 transcripts in PBMC and pleural effusion mononuclear cells (PEMC) of patients with PLTB. PLTB patients had increased levels of IL-1ß, IL-6 and IFNγ together with reduced Cortisol and DHEA concentrations in pleural fluids. Also, a significantly increased expression of 11ßHSD1 and GRα was found in PEMC compared to PBMC. Findings point out to an appropriate immune response and a substantial inflammatory reaction, wherein the low Cortisol concentrations may be equally effective, because of the increased expression of GRα and 11ßHSD1 transcripts which may optimize the immunomodulatory properties of Cortisol.

Altered microRNA expression levels in mononuclear cells of patients with pulmonary and pleural tuberculosis and their relation with components of the immune response.

Different lines of evidence demonstrate that microRNAs (miRNAs) play an important role in host-pathogen interactions. In this study we investigated the expression patterns of several miRNAs, most of them involved in regulating inflammatory responses, in patients with tuberculosis (TB). In order to understand the events occurring at the site of infection, we employed mononuclear cells obtained from both peripheral blood (PBMC) and pleural fluids (PFMC) of patients. Interestingly, we found that the miRNA signature of each compartment is different, with a strong down-regulation in PFMCs of miR-223, miR-144* and miR-421. In addition, we observed that miR-146a expression is also down-regulated in tuberculosis patients, both in PBMCs and PFMCs while miR-424 levels are elevated only in the peripheral compartments. We also showed that systemic expression of these miRNAs changes upon specific treatment and is associated with IL-6 levels, a cytokine playing a substantial role in TB immunopathology. Present results contribute to a better knowledge of the host responses in TB pathogenesis, pointing out the role of miRNAs in this disease.