Inactivation of M111 Protein Gene Modifies Streptococcus Pyogenes Interactions with Mouse Macrophages In Vitro.

Immunomodulatory properties of S. pyogenes protein M111 were studied on the model of Gurov strain and its isogenic mutant not expressing M protein. Mouse resident peritoneal macrophages were incubated with bacteria and generation of nitroxide and superoxide anions and production of IL-6, IL-10, and IL-17 were evaluated. Protein M111 modified macrophage response: it exhibited antiphagocytic activity, prevented ROS formation, and stimulated the production of anti-inflammatory cytokine IL-10. The results suggested that this protein could serve in the bacteria as a factor suppressing the host defense forces and promoting the realization of the strategy beneficial for pathogens - escape from the host immune defense.

Adaptation of Mycobacterium tuberculosis to Impaired Host Immunity in HIV-Infected Patients.

BACKGROUND: It is unknown whether immunosuppression influences the physiologic state of Mycobacterium tuberculosis in vivo. We evaluated the impact of host immunity by comparing M. tuberculosis and human gene transcription in sputum between human immunodeficiency virus (HIV)-infected and uninfected patients with tuberculosis. METHODS: We collected sputum specimens before treatment from Gambians and Ugandans with pulmonary tuberculosis, revealed by positive results of acid-fast bacillus smears. We quantified expression of 2179 M. tuberculosis genes and 234 human immune genes via quantitative reverse transcription-polymerase chain reaction. We summarized genes from key functional categories with significantly increased or decreased expression. RESULTS: A total of 24 of 65 patients with tuberculosis were HIV infected. M. tuberculosis DosR regulon genes were less highly expressed among HIV-infected patients with tuberculosis than among HIV-uninfected patients with tuberculosis (Gambia, P < .0001; Uganda, P = .037). In profiling of human genes from the same sputa, HIV-infected patients had 3.4-fold lower expression of IFNG (P = .005), 4.9-fold higher expression of ARG1 (P = .0006), and 3.4-fold higher expression of IL10 (P = .0002) than in HIV-uninfected patients with tuberculosis. CONCLUSIONS: M. tuberculosis in HIV-infected patients had lower expression of the DosR regulon, a critical metabolic and immunomodulatory switch induced by NO, carbon monoxide, and hypoxia. Our human data suggest that decreased DosR expression may result from alternative pathway activation of macrophages, with consequent decreased NO expression and/or by poor granuloma formation with consequent decreased hypoxic stress.

Water balance of lung and nitrogen oxide in blood at experimental autoimmune encephalomyelitis after capsaicin blockade of vagus nerve.

The purpose of the research: To study the water balance of lung and NO level in blood in experimental autoimmune encephalomyelitis combined with capsaicin blockade of vagus nerve. Methods: Experiments were conducted on 47 adult (16-week-old) male rats weighing 220-280 g. To simulate the experimental autoimmune encephalomyelitis (EAE) rats were subcutaneously injected with encephalitogenic mixture in complete Freund's adjuvant (0.2 ml; the content of inactivated Mycobacterium tuberculosis was 5 mg/ml) at the rate of 100 mg of homologous spinal cord homogenate per animal. Сapsaicin blockade was performed by bilateral application of 50 uM capsaicin («Sigma¼) on the neck portions of vagus nerves. The animals were divided into 4 groups: intact rats - control group1; rats with EAE; rats with capsaicin application on vagus nerve + EAE; sham operated rats subjected to vagus nerves allocation without the subsequent capsaicin application + EAE - control group 2. The next parameters were detected: the content of nitric oxide in blood plasma; protein content in broncho-alveolar lavage fluid; lung water balance indices including the amount of total, extra- and intravascular fluid and blood supply of lungs, which were calculated based on wet and dry lung mass and the hemoglobin content in blood and lung tissue determined by hemiglobincyanide method. Results: It was found that EAE is accompanied by an increase of total fluid, extravascular fluid (EVF) and blood supply of lungs on the background of increasing content of nitric oxide in arterial (art) and venous (ven) blood. In EAE and its combination with bilateral capsaicin blockade of vagus nerve a strong negative correlation between the NOart / NOven coefficient and EVF amount was found out. The blockade of capsaicin-sensitive vagal afferents normalized lung water balance impaired in EAE and restored the levels of nitric oxide in blood plasma. Conclusion: The obtained results suggest that capsaicin-sensitive vagal afferents with NO-ergic mechanisms involvment take part in the development of pulmonary hyperhydration during experimental autoimmune encephalomyelitis.

Immuno-spin trapping from biochemistry to medicine: advances, challenges, and pitfalls. Focus on protein-centered radicals.

BACKGROUND: Immuno-spin trapping (IST) is based on the reaction of a spin trap with a free radical to form a stable nitrone adduct, followed by the use of antibodies, rather than traditional electron paramagnetic resonance spectroscopy, to detect the nitrone adduct. IST has been successfully applied to mechanistic in vitro studies, and recently, macromolecule-centered radicals have been detected in models of drug-induced agranulocytosis, hepatotoxicity, cardiotoxicity, and ischemia/reperfusion, as well as in models of neurological, metabolic and immunological diseases. SCOPE OF THE REVIEW: To critically evaluate advances, challenges, and pitfalls as well as the scientific opportunities of IST as applied to the study of protein-centered free radicals generated in stressed organelles, cells, tissues and animal models of disease and exposure. MAJOR CONCLUSIONS: Because the spin trap has to be present at high enough concentrations in the microenvironment where the radical is formed, the possible effects of the spin trap on gene expression, metabolism and cell physiology have to be considered in the use of IST and in the interpretation of results. These factors have not yet been thoroughly dealt with in the literature. GENERAL SIGNIFICANCE: The identification of radicalized proteins during cell/tissue response to stressors will help define their role in the complex cellular response to stressors and pathogenesis; however, the fidelity of spin trapping/immuno-detection and the effects of the spin trap on the biological system should be considered. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Heteroarylimino-4-thiazolidinones as inhibitors of cartilage degradation.

2-Benzo[d]thiazolyl- and 2-benzo[d]isothiazolyl-imino-5-benzylidene-4-thiazolidinone derivatives were investigated as potential metalloproteinases (MMPs) inhibitors and evaluated for their antidegenerative activity on human chondrocyte cultures stimulated by IL-1ß, using an experimental model that reproduces the mechanisms involved in osteoarthritic (OA) diseases. Cell viability, the amount of glycosaminoglycans (GAGs) and the production of nitric oxide (NO) were measured. The most potent compound, 5-(4-methoxy-benzylidene)-2-(benzo[d]isothiazol-3-ylimino)-thiazolidin-4-one (4b), a MMP-13 inhibitor at nanomolar concentration (IC(50)=0.036 µM), could be considered as a lead compound for the development of novel clinical agents, inhibitors of cartilage degradation, for the treatment of OA.

Oxidized low-density lipoprotein-induced proinflammatory cytokine response in macrophages are suppressed by CD4CD25(+)Foxp3(+) regulatory T cells through downregulating toll like receptor 2-mediated activation of NF-kappaB.

CD4(+)CD25(+) regulatory T cells (Tregs) exert a suppressive activity on atherosclerosis but the underlying mechanism remains unclear. Here, we investigated whether and how Tregs affect oxLDL-induced proinflammatory response in macrophages. Tregs were isolated by magnetic cell sorting-column and analyzed by flow cytometry. Macrophages were cultured with or without Tregs in the presence of oxLDL for 48 hours to induce proinflammatory response. Our data showed that with oxLDL challenge, the Treg-modulated macrophages have decreased NO production and iNOS expression, decreased HLA-DR and CD86 expression, and down-regulated proinflammatory cytokine/chemokine production. Tregs can inhibit the pro-inflammatory properties of macrophages and steer macrophage differentiation toward an anti-inflammatory cytokine producing phenotype. Mechanistic studies reveal that Treg-mediated suppression of the monocyte response to oxLDL was reflected by a reduction in the up-regulation of NF-kappaB activity accompanied by a decreased expression of TLR2 but not TLR4 at the transcriptional level. These results suggest that CD4(+)CD25(+)Foxp3(+) regulatory T cells may exert its suppressive functions on pro-inflammatory properties of OxLDL induced-macrophages partly through TLR2-NF-kappaB signaling pathway.

Immuno-spin trapping of macromolecules free radicals in vitro and in vivo - One stop shopping for free radical detection.

The only general technique that allows the unambiguous detection of free radicals is electron spin resonance (ESR). However, ESR spin trapping has severe limitations especially in biological systems. The greatest limitation of ESR is poor sensitivity relative to the low steady-state concentration of free radical adducts, which in cells and in vivo is much lower than the best sensitivity of ESR. Limitations of ESR have led to an almost desperate search for alternatives to investigate free radicals in biological systems. Here we explore the use of the immuno-spin trapping technique, which combine the specificity of the spin trapping to the high sensitivity and universal use of immunological techniques. All of the immunological techniques based on antibody binding have become available for free radical detection in a wide variety of biological systems.

Effect of dehydroepiandrosterone on the immune function of mice in vivo and in vitro.

Dehydroepiandrosterone (DHEA) has anti-inflammatory, anti-oxidant and immune-regulating properties, while the mechanism of DHEA actions remains unclear. The present study aims to investigate the effect and possible mechanism of DHEA on immune function of mice in vivo and in vitro. In vivo, a lipopolysaccharide (LPS)-induced experimental inflammation model was constructed to analyze the regulation of DHEA on anti-oxidative and immune function in ICR mice; In vitro, the effects of DHEA on the biological functions of lymphocytes and macrophages were studied. The results showed that DHEA increased the activity of total antioxidant capacity and superoxide dismutase, while it decreased the level of reactive oxygen species in LPS-induced mice. Meanwhile, DHEA increased the proportion of T lymphocytes and decreased that of B lymphocytes in primary cultured spleen lymphocytes, and markedly enhanced the Th1/Th2 ratio in spleen T lymphocytes. Furthermore, DHEA significantly increased the Th1 type cytokine (IL-2 and IFN-α) and decreased the Th2 type cytokine (IL-4 and IL-10) levels in LPS-induced mice or in primary cultured spleen T lymphocytes. In addition, DHEA improved the phagocytic ability, enhanced the NO production and increased the iNOS activity in peritoneal macrophages. Our data indicates that DHEA increases the macrophages function via improving NO content and up-regulating TNF-α expression levels; and it evoked a Th1 immuno-response and repressed a Th2 immuno-response through promoting a shift in Th1/Th2 balance toward Th1-dominant immunity in vivo and in vitro. These results provide substantial evidence on the mechanism of DHEA-mediated immune function and the efficient protection against infectious and inflammatory response in animals and humans.

The role of type I interferons (IFNs) in the regulation of chicken macrophage inflammatory response to bacterial challenge.

Mammalian type I interferons (IFNα/ß) are known to modulate inflammatory processes in addition to their antiviral properties. Indeed, virus-induced type I interferons regulate the mammalian phagocyte immune response to bacteria during superinfections. However, it remains unresolved whether type I IFNs similarly impact the chicken macrophage immune response. We first evidenced that IFNα and IFNß act differently in terms of gene expression stimulation and activation of intracellular signaling pathways in chicken macrophages. Next, we showed that priming of chicken macrophages with IFNα increased bacteria uptake, boosted bacterial-induced ROS/NO production and led to an increased transcriptional expression or production of NOS2/NO, IL1B/IL-1ß and notably IFNB/IFNß. Neutralization of IFNß during bacterial challenge limited IFNα-induced augmentation of the pro-inflammatory response. In conclusion, we demonstrated that type I IFNs differently regulate chicken macrophage functions and drive a pro-inflammatory response to bacterial challenge. These findings shed light on the diverse functions of type I IFNs in chicken macrophages.

Monoclonal antibodies to a nitroxide lipid hapten.

The isolation and characterization of a hybridoma cell line producing a monoclonal IgG1 antibody against a spin-label nitroxide group is described. The antibody recognizes a synthetic hapten containing linked dinitrophenyl and 2,2,6,6-tetramethylpiperidinyl-1-oxy groups, having an affinity of 3.6 +/- 1.0 . 10(6) M-1 for the soluble hapten at 25 degrees. The antibody binds to phospholipid vesicles containing 2 mol% of spin label-derivitized lipid (lipid hapten) with an affinity of 1.5 +/- 0.2 . 10(8) M-1. This monoclonal IgG1 mediates the binding of hapten-bearing lipid vesicles to mouse macrophage RAW264 cells bearing Fc receptors. The cellular responses to this binding are similar to those observed previously using polyclonal rabbit anti-hapten IgG. As with the heterogeneous antibodies, the monoclonal IgG1 is more efficient in mediating cellular uptake when the vesicles are in the "fluid' physical state (dimyristoylphosphatidylcholine at 37 degrees C) compared to "solid' (dipalmitoylphosphatidylcholine at 37 degrees C). Despite the enhanced binding of "fluid' phospholipid vesicles to cells, only the "solid' vesicles triggered a significant respiratory burst in Raw264 macrophages.

Identification of the myoglobin tyrosyl radical by immuno-spin trapping and its dimerization.

5,5-Dimethyl-1-pyrroline N-oxide (DMPO) spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation. The present study demonstrates that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosyl radical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct. The reaction of sperm whale myoglobin with equimolar hydrogen peroxide resulted in the formation of a myoglobin dimer detectable by electrophoresis/protein staining. Addition of DMPO resulted in the trapping of the globin radical, which was detected by Western blot. The location of this adduct was demonstrated to be at tyrosine-103 by MS/MS and site-specific mutagenicity. Interestingly, formation of the myoglobin dimer, which is known to be formed primarily by cross-linkage of tyrosine-151, was inhibited by the addition of DMPO.

Immunostimulating activity of the polysaccharides isolated from Cordyceps militaris.

In this study, the signaling mechanism of the polysaccharides isolated from fruiting body of Cordyceps militaris (CM) was investigated in macrophages to evaluate its immuno-stimulating properties. We found that CM was capable of upregulation of NO, ROS, TNF-α and phagocytic uptake in mouse peritoneal macrophages and RAW264.7 macrophages. Macrophages activation by CM seemed to occur via activation of NF-κB and all three MAPKs pathways through dectin-1 and TLR2 macrophage receptors. Additionally, we showed that CM suppressed in vivo growth of melanoma in an experimental mouse model. Based on these data, we suggested that CM may potentially regulate the immune response.

Tocotrienols suppress proinflammatory markers and cyclooxygenase-2 expression in RAW264.7 macrophages.

Tocotrienols are powerful chain breaking antioxidant. Moreover, they are now known to exhibit various non-antioxidant properties such as anti-cancer, neuroprotective and hypocholesterolemic functions. This study was undertaken to investigate the anti-inflammatory effects of tocotrienol-rich fraction (TRF) and individual tocotrienol isoforms namely delta-, gamma-, and alpha-tocotrienol on lipopolysaccharide-stimulated RAW264.7 macrophages. The widely studied vitamin E form, alpha-tocopherol, was used as comparison. Stimulation of RAW264.7 with lipopolysaccharide induced the release of various inflammatory markers. 10 mcirog/ml of TRF and all tocotrienol isoforms significantly inhibited the production of interleukin-6 and nitric oxide. However, only alpha-tocotrienol demonstrated a significant effect in lowering tumor necrosis factor-alpha production. Besides, TRF and all tocotrienol isoforms except gamma-tocotrienol reduced prostaglandin E(2) release. It was accompanied by the down-regulation of cyclooxygenase-2 gene expression by all vitamin E forms except alpha-tocopherol. Collectively, the data suggested that tocotrienols are better anti-inflammatory agents than alpha-tocopherol and the most effective form is delta-tocotrienol.

Airway acidification: interactions with nitrogen oxides and airway inflammation.

Airway acidification is increasingly appreciated to occur in inflammatory obstructive airway diseases, resulting from acid reflux and aspiration and from direct acid formation in the airways. Acidity activates oxidants and nitrogen oxides to create a potent antimicrobial environment. Neurogenic inflammation is triggered by airway or esophageal acidification, innate immune cells are affected by acidity, and there are pathways by which the acquired immune system also can be activated by the chemistry of an acidic airway. Measuring airway acidity is now readily achievable with noninvasive breath assays, a procedure that has opened a window on the need to understand airway pH homeostasis in health and pH dysregulation in disease.

Immunological biomarkers in salt miners exposed to salt dust, diesel exhaust and nitrogen oxides.

OBJECTIVES: Air pollutants can affect lung function and also the immune system. In a study about lung function of salt miners in relation to the complex exposure in a salt mine, we also analysed selected immunological parameters and inflammation markers in the blood of miners. Effect of salt dust, diesel exhaust, nitrogen oxides (NOx) and smoking on the biomarkers was analysed. METHODS: Blood was drawn from 286 salt miners, and the soluble intercellular adhesion molecule-1 (s-ICAM), monocyte chemotactic protein (MCP-1) and clara cell protein (CC16) were analysed by an immunoassay, blood profile was done and lymphocyte subpopulations (CD3, CD3/CD4, CD3/CD8, CD19, NK-cells, CD3/HLA-DR) were determined by flow cytometry. Salt dust was measured by two-step gravimetry (personal sampling). Diesel exhaust was measured as elemental carbon concentration by coulometry. NOx were determined by an electrochemical cell method. Differences between non-smokers, former smokers and active smokers were analysed by analysis of variance. Linear regression analysis to describe exposure-response relationships was done with regard to confounding factors [smoking, inflammatory diseases, time of blood drawing, respiratory infection and body-mass index (BMI)]. RESULTS: Significant differences between non-smokers and active smokers were found for most of the leukocyte types (e.g. granulocytes P = 0.000, lymphocytes P = 0.002, T-cells P = 0.033) and for some soluble parameters (ICAM P = 0.000, IgM P = 0.007, IgE P = 0.035). Increasing numbers of total lymphocytes, T-cells and HLA-DR positive T-cells in relation to exposure were found by linear regression analysis (e.g. for inhalable dust:total lymphocytes P = 0.011, T-cells P = 0.061, HLA-DR positive T-cells P = 0.007). CONCLUSION. Comparison of immunological markers in non-smokers and active smokers confirms leukocytosis and inflammation following tobacco consumption. The combined exposure of salt dust, diesel exhaust and NOx seems to influence the immune system. Together, the results suggest that the analysis of leukocytes and their subsets can complete other investigations (lung function, questionnaire) to monitor exposure-response relationships in occupational studies investigating the effect of inhaled substances. Longitudinal studies will be necessary to determine the predictive value of the immunological changes.