The neuro-endocrine-immune relationship in pulmonary and pleural tuberculosis: a better local profile in pleural fluid.

BACKGROUND: Tuberculosis (TB) is a major health problem worldwide. In TB, the immune and central nervous systems modulate each other. The two main components of this network are the hypothalamic-pituitary-adrenal axis (HPA) and autonomic nervous system (ANS). OBJECTIVE: To elucidate neuro-endocrine-immune (NEI) interactions in pulmonary (PTB) or pleural (PLTB) TB, we analysed the relationship among compounds from these systems. METHODS: We quantified levels of catecholamines, hormones and cytokines in plasma from patients with PTB (n = 46) or PLTB (n = 12) and controls (n = 32), and in the pleural fluid from PLTB patients. Transcript expression for genes involved in glucocorticoid-related function (quantitative real-time polymerase chain reaction) was also analysed in mononuclear cells (MCs) from peripheral blood (PBMC) or pleural effusion (PEMC) compartments. RESULTS: Both patient groups had increased plasma levels of pro- and anti-inflammatory cytokines, cortisol, growth hormone (GH) and dopamine, whereas insulin-like growth factor 1 (IGF-1) and dehydroepiandrosterone levels were decreased. The pleural fluid contained increased levels of pro-inflammatory cytokines, GH and IGF-1 and reduced levels of steroid hormones compared with their plasma counterparts. PBMCs from PTB patients had increased expression of transcripts for 11ß-hydroxysteroid dehydrogenase (11ßHSD1) and a decreased glucocorticoid receptor (GR) ratio (GRα/GRß). In PLTB cases, expression of 11ßHSD1 and GRα transcripts was higher in PEMCs. CONCLUSION: PTB patients seem to display adverse NEI dysregulation. Changes in pleural fluid are compatible with a more effective NEI reaction.

Melatonin decreases the oxidative stress produced by 2,4-dichlorophenoxyacetic acid in rat cerebellar granule cells.

2,4-Dichlorophenoxyacetic acid (2,4-D) is one of the most widely used herbicides due to its relatively moderate toxicity and to its biodegradability in the soil. In toxic concentrations, 2,4-D displays strong neurotoxicity, partly due to generation of free radicals. Since melatonin has remarkable antioxidant properties, the objective of this study was to assess to what extent it was effective in preventing the 2,4-D effect on redox balance of rat cerebellar granule cells (CGC) in vitro. Cellular viability, generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), reduced glutathione (GSH) levels, and the activities of the antioxidant enzymes Cu/Zn-superoxide dismutase (Cu/Zn-SOD), Mn-SOD, selenium-glutathione peroxidase (Se-GPx) and catalase (CAT) were measured in CGC exposed to 2,4-D and/or melatonin for 48 h. In CGC cultures exposed to 2,4-D, cell viability, GSH levels and CAT activity decreased significantly whereas ROS generation and Se-GPx activities were augmented. Except for Se-GPx activity, all these changes were counteracted by the concomitant addition of 0.1 or 0.5 mM melatonin. In addition, incubation of CGC with melatonin alone resulted in augmentation of cell viability, GSH levels and Se-GPx activity. RNS generation and SOD activity remained unaffected by either treatment. Since melatonin was able to counteract most of redox changes produced by 2,4-D in CGC in culture, the experimental evidence reported further support the efficacy of melatonin to act as a neuroprotector.

Detection of 2,4-dichlorophenoxyacetic acid in rat milk of dams exposed during lactation and milk analysis of their major components.

2,4-Dichlorophenoxyacetic acid (2,4-D) and its derivatives are herbicides widely used to control the growth of broadleaf and woody plant. Human and animal exposure to 2,4-D through agriculture use, food products, or use in lawn and garden care has been well documented, but little information is available on the transfer from serum to milk in exposed dams. In this study, we measured the content of 2,4-D in rat milk from mother exposed to 15, 25, 50 or 7 0mg 2,4-D/kg bw through the diet (4 treated groups, 8 dam each; 1 control group with 8 dams) over a period of 16 days starting on the post-natal day 1 (PND 1). The effect of 2,4-D on milk components was also evaluated. All doses tested caused a decrease in the body weight gain of the pups (4 groups, 64 pups each). It also produce a 30% in the content of total lipids and a changed the content of minor proteins in milk of the treated groups. 2,4-D produces an important decrease in some fatty acids content, being the polyunsaturated fatty acids the most affected. Further analysis showed that 2,4-D concentrations chromatographically detected both serum of dams and pups and milk were dose-dependent.

An adverse immune-endocrine profile in patients with tuberculosis and type 2 diabetes.

Diabetes is a risk factor for the development of pulmonary tuberculosis (TB) and both diseases present endocrine alterations likely to play a role in certain immuno-endocrine-metabolic associated disorders. Patients with TB, or with TB and type 2 diabetes (TB + T2DM) and healthy controls (HCo) were assessed for plasma levels of cortisol, dehydroepiandrosterone (DHEA), estradiol, testosterone, growth hormone (GH), prolactin, insulin-like growth factor-1 (IGF-1), cytokines (IL-6, IL-10, IFN-γ) and the specific lymphoproliferative capacity of peripheral blood mononuclear cells. All patients had higher levels of cortisol with a reduction in DHEA, thus resulting in an increased cortisol/DHEA ratio (Cort/DHEA). Increased prolactin and particularly GH levels were found in both groups of TB patients. This was not paralleled by increased concentrations of IGF, which remained within the levels of HCo. Estradiol levels were significantly augmented in patients TB, and significantly more in TB + T2DM, whereas testosterone levels were decreased in both groups of patients. IFN- γ and IL-6 concentrations were significantly increased in all TB, even further in TB + T2DM; while IL-10 was equally increased in both groups of TB patients. The in vitro specific proliferative capacity was decreased in both groups of patients as compared to that of HCo. The adverse immune-endocrine profile of TB seems to be slightly more pronounced in patients who also have T2DM.

Quinoxalinylethylpyridylthioureas (QXPTs) as potent non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors. Further SAR studies and identification of a novel orally bioavailable hydrazine-based antiviral agent.

Quinoxalinylethylpyridylthioureas (QXPTs) represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 6-FQXPT (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis of novel heteroarylethylpyridylthioureas which were tested as anti-HIV agents. Several compounds proved to be potent broad-spectrum enzyme inhibitors and significantly inhibited HIV-1 replication in vitro. Their potency depends on the substituents and the nature of the heterocyclic skeleton linked to the ethyl spacer, and structure-activity relationships are discussed in terms of the possible interaction with the RT binding site. Although the new QXPTs analogues show potent antiviral activity, none of the compounds tested overcome the pharmacokinetic disadvantages inherent to ethylpyridylthioureidic antiviral agents, which in general have very low oral bioavailability. Through an integrated effort involving synthesis, docking studies, and biological and pharmacokinetic evaluation, we investigated the structural dependence of the poor bioavailability and rapid clearance within the thioureidic series of antivirals. Replacing the ethylthioureidic moiety with a hydrazine linker led to a new antiviral lead, offering promising pharmacological and pharmacokinetic properties in terms of antiviral activity and oral bioavailability.

Leptin does not enhance cell-mediated immune responses following mycobacterial antigen stimulation.

BACKGROUND: Tuberculosis (TB) is a infectious disease characterised by a profound immune-endocrine metabolic imbalance, including a diminution in leptin plasma levels. Leptin appears to be the link between nutritional status and the development of a protective immune response. OBJECTIVE: To examine the effects of leptin on the proliferation and production of interferon-gamma (IFN-γ) by peripheral blood mononuclear cells (PBMC) in TB patients and healthy controls stimulated with mycobacterial antigens with or without leptin. As macrophages are key cells in mycobacterial containment, the effect of leptin on the production of interleukin (IL) 1ß and IL-1Ra by the monocytic cell line THP-1 was also studied. RESULTS: Leptin diminished the proliferative capacity of PBMC on mycobacterial stimulation, and had no effect on IFN-γ production in terms of measurements in culture supernatants or intracytoplasmic analysis using flow cytometry. Real-time polymerase chain reaction studies of PBMC from TB patients revealed a preserved expression of leptin receptor. Furthermore, IL-1ß and IL-1Ra secretion by THP-1 cells was not modified by leptin treatment. CONCLUSION: The study results do not support the utility of treatment with leptin to correct immune imbalances due to TB.

Adverse effects of 2,4-dichlorophenoxyacetic acid on rat cerebellar granule cell cultures were attenuated by amphetamine.

2,4-Dichlorophenoxyacetic acid (2,4-D), a worldwide-used herbicide, has been shown to produce a wide range of adverse effects in the health--from embryotoxicity and teratogenicity to neurotoxicity--of animals and humans. In this study, neuronal morphology and biochemical events in rat cerebellar granule cell (CGC) cultures have been analyzed to define some of the possible mechanisms involved in 2,4-D-induced cell death. For that purpose, amphetamine (AMPH) that has been shown to accelerate the recovery of several functions in animals with brain injury has been used as a pharmacologycal tool and was also investigated as a possible protecting agent. Addition of 2,4-D to CGC cultures produced a drastic decrease in cell viability, in association with an increased incidence of necrosis and apoptosis, and an increased level of reactive oxygen species, a decrease in glutathione content, and an abnormal activity of some enzymes with respect to the control group. The adverse effects of 2,4-D were partly attenuated in presence of AMPH. Some deleterious effects on several ultrastructural features of the cells, as well as the enhanced incidence of apoptosis, were partially preserved in AMPH-protected cultures as compared with those which were exposed to 2,4-D alone. The collected evidences (1) confirms the previously observed, deleterious effects of 2.4D on the same or a similar model; (2) suggests that the 2,4-D-induced apoptosis could have been mediated by or associated to an oxidative imbalance in the affected cells, and (3) shows some evidence of a protective effect of AMPH on 2,4-D-induced cell death, which could have been exerted through a reduction in the oxidative stress.

Suppressor cell function in atopic dermatitis associated with elevated immunoglobulin E.

Suppressor cell function was evaluated in 11 patients with atopic dermatitis (AD) and elevated IgE levels (mean, 4,554 IU/ml +/- 1,825 SEM) and compared to 11 matched nonatopic controls (135 IU/ml +/- 52 SEM). Two assays were employed to evaluate suppressor cell function. In the first assay, concanavalin A--activated suppressor cell activity of AD and control subjects were compared. In the second assay, peripheral blood mononuclear cells (PBM) from the same AD and control subjects were stimulated with varying doses of mitogen at day 0 and after 24 hr of preculture. In this system, increased proliferative response of precultured cells as compared to 0-hr cells has previously been shown in normals to represent loss of suppressor cell function in vitro. The lack of such an increase implies aberrant suppressor cell function. The data from both assays showed no significant difference in the degree of suppressor cell function of the patient population vs the control population. Thus, suppressor cell function as tested in these proliferative assays appears normal in AD patients with increased IgE.

Granulocyte-macrophage colony-stimulating factor regulates cytokine production in cultured macrophages through CD14-dependent and -independent mechanisms.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has multiple effects on the antigen phenotype and function of macrophages. In this study we investigated the effect of GM-CSF on cytokine production by macrophages. We found that GM-CSF may modify the tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) response to lipopolysaccharide (LPS) through two different mechanisms. Relatively early in culture, GM-CSF increases the amount of cytokines synthesized by responding cells; this effect appears to be unrelated to modulation of CD14 expression and LPS-binding capacity. After prolonged incubation, GM-CSF up-regulates both CD14 expression and LPS-binding capacity, and the frequency of cytokine-producing cells. Release of CD14 in the culture supernatant was decreased in the presence of GM-CSF, suggesting that a reduced shedding was responsible for the effect of GM-CSF on CD14 expression. Enhancement of cytokine production was also observed in GM-CSF-treated macrophages after stimulation by phorbol 12-myristate 13-acetate (PMA), thus indicating that GM-CSF affects both CD14-dependent and -independent cytokine production. Finally, GM-CSF did not modulate the LPS- and PMA-induced production of IL-10 and IL-12. We conclude that GM-CSF may play a role in manipulating the activation-induced expression of pro-inflammatory cytokines by macrophages. Enhanced production of these cytokines could play an important role in the pathogenesis of Gram-negative septic shock syndrome and in defence against infectious agents.

Selective deficiency of a T cell subpopulation in active atopic dermatitis.

The T cell subpopulation bearing a receptor for the Fc fragment of IgM (T mu) or the Fc fragment of IgG (T gamma) were studied in 12 patients with active atopic dermatitis (AD) and elevated IgE (5678 I.U./ml +/- 1382) and compared with a control group with normal IgE (33 +/- 17). Values for the total lymphocytes and for absolute and relative numbers of total T and T mu cells were comparable in both groups. However, AD patients had markedly reduced relative T gamma levels, 1.8% +/- 0.4%, compared with normals, 10.5% +/- 0.7% (p less than 0.001). The patients also had reduced absolute T gamma levels, 29 +/- 7, compared with 181 +/- 36 (p less than 0.0005). This abnormality was not indicative of allergic disease in general, nor did it appear to be caused directly by serum factors. The T gamma cell deficit did reflect disease activity and correlated inversely with the levels of serum IgE in the AD patients. Thus, patients with active AD and elevated IgE have a selective reduction in circulating T gamma cells.

Human immunodeficiency virus type 1 infection modulates the interleukin (IL)-1beta and IL-6 responses of human macrophages to CD40 ligand stimulation.

Better understanding of the mechanisms of proinflammatory cytokine production during human immunodeficiency virus (HIV) type 1 infection is of pivotal importance. The effect of HIV-1 infection on recombinant CD40 ligand (CD40L)-induced interleukin (IL)-1beta and IL-6 production by human macrophages was analyzed. ELISA and cytofluorometric analysis demonstrated that CD40L stimulation of HIV-1-infected macrophages resulted in substantial production of IL-1beta and IL-6. In contrast, no cytokine response was observed in uninfected cells. No modulation of the receptor for CD40 was found to account for the enhanced response to CD40L. The CD40L effect was not due to lipopolysaccharide contamination and was completely abrogated by preincubation with a monoclonal anti-CD40L antibody. mRNA studies indicated that the priming effect of HIV-1 on the macrophage response to CD40L was regulated at the transcriptional level. Finally, the effect of HIV-1 on the cytokine response could not be abolished by the HIV-1 protease inhibitor U75875 at concentrations that completely suppressed HIV-1 replication.

Treatment with ribavirin and interferon-alpha reduces interferon-gamma expression in patients with chronic hepatitis C.

Recent studies in vitro and in animals have suggested that ribavirin may potentiate the antihepatitis C virus (HCV) activity of interferon-alpha (IFN-alpha) by up-modulating the production of T cell-derived cytokines, such as interleukin (IL)-2 and IFN-gamma, which play a key role in the cellular immune response against HCV. To study the immune-modulatory mechanisms of ribavirin further, cytokine production by activated T cells and circulating cytokine levels were studied by FACS analysis and ELISA testing in 25 patients with chronic hepatitis C unresponsive to IFN-alpha, before and after treatment with either ribavirin plus IFN-alpha or IFN-alpha alone. After 16 weeks of treatment, both the expression of IFN-gamma by activated T cells and the blood levels of IFN-gamma, were significantly reduced with respect to pretreatment values in patients treated with ribavirin and IFN-alpha but not in those undergoing treatment with IFN-alpha alone. The expression of IFN-gamma was significantly lower in patients that gained normal ALT levels with respect to those that did not. No modification of the expression of IL-2, IL-4 and IL-10 was found before and after treatment in either group of patients. In conclusion, the results of this study do not support up-modulation of IFN-gamma and IL-2 production as the mechanism by which ribavirin potentiates IFN-alpha anti HCV activity. In addition, our findings suggest that ribavirin may exert an anti-inflammatory effect and may help reducing IFN-gamma-driven T cell activation and liver damage.

Lack of evidence for the Th2 predominance in patients with chronic hepatitis C.

A T helper (Th)1 to Th2 shift has been proposed to be a critical pathogenic determinant in chronic hepatitis C. Here, we evaluated mitogen-induced and hepatitis C virus (HCV) core antigen-induced cytokine production in 28 patients with biopsy-proven chronic hepatitis C. Flow cytometry demonstrated that after mitogenic stimulation the percentage of Th2 cells (IL-4 + or IL-13 +) and Th0 cells (IFN-gamma/IL-4 + or IL-2/IL-13 +) did not differ between patients and controls. In contrast, the percentage of Th1 cells (IFN-gamma + or IL-2 +) was significantly increased in CD4 +, CD8 +, 'naive'-CD45RA + and 'memory'-CD45RO + T-cell subsets from patients versus controls. Similar results were obtained by ELISA testing supernatants from mitogen-stimulated, unfractionated peripheral blood mononuclear cell (PBMC) cultures. Interferon-alpha treatment was associated with a reduction in the mitogen-induced Th1 cytokine response in those patients who cleared their plasma HCV-RNA. Analysis of cytokine expression by CD4 + T cells after HCV core antigen stimulation in a subgroup of 13 chronic hepatitis C patients demonstrated no cytokine response in 74% of these patients and an IFN-gamma-restricted response in 26%. Finally, no Th2 shift was found in lipopolysaccharide-stimulated monocytes. These data indicate that a Th1 to Th2 shift does not occur in chronic hepatitis C.