Leukotrienes Produced in Allergic Lung Inflammation Activate Alveolar Macrophages

It has been well-documented that leukotrienes (LTs) are released in allergic lung inflammation and that they participate in the physiopathology of asthma. A role for LTs in innate immunity has recently emerged: Cys-LTs were shown to enhance Fc gamma R-mediated phagocytosis by alveolar macrophages (AMs). Thus, using a rat model of asthma, we evaluated Fc gamma R-mediated phagocytosis and killing of Klebsiella pneumoniae by AMs. The effect of treatment with a cys-LT antagonist (montelukast) on macrophage function was also investigated. Male Wistar rats were immunized twice with OVA/alumen intraperitoneally and challenged with OVA aerosol. After 24 h, the animals were killed, and the AMs were obtained by bronchoalveolar lavage. Macrophages were cultured with IgG-opsonized red blood cells (50: 1) or IgG-opsonized K. pneumoniae (30: 1), and phagocytosis or killing was evaluated. Leukotriene C(4) and nitric oxide were quantified by the EIA and Griess methods, respectively. The results showed that AMs from sensitized and challenged rats presented a markedly increased phagocytic capacity via Fc gamma R (10X compared to controls) and enhanced killing of K. pneumoniae (4X higher than controls). The increased phagocytosis was inhibited 15X and killing 3X by treatment of the rats with montelukast, as compared to the non-treated group. cys-LT addition increased phagocytosis in control AMs but had no effect on macrophages from allergic lungs. Montelukast reduced nitric oxide (39%) and LTC(4) (73%). These results suggest that LTs produced during allergic lung inflammation potentiate the capacity of AMs to phagocytose and kill K. pneumonia via Fc gamma R. Copyright (C) 2010 S. Karger AG, Basel

Apoptosis of macrophages during pulmonary Mycobacterium bovis infection: correlation with intracellular bacillary load and cytokine levels

P>Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette-Guerin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) and IL-12 and of the anti-apoptotic protein Bcl-2 were measured at day 3 and day 7 post-infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high-dose group, while on the 7th day post-infection macrophage apoptosis was reduced in the low-dose group. Inhibition of apoptosis was correlated with increased production of IL-10, reduced production of TNF-alpha and increased production of Bcl-2. In addition, the production of IL-12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.

Reactive nitrogen intermediate susceptibility of Mycobacterium tuberculosis genotypes in an urban setting

BACKGROUND: Mycobacterium tuberculosis genotypes resistant to reactive nitrogen intermediates (RNI) predominate in certain urban communities, suggesting that this phenotype influences disease transmission. OBJECTIVE: To compare different M. tuberculosis genotypes for resistance to RNI generated in vitro. DESIGN: We genotyped 420 M. tuberculosis isolates from a neighborhood in Sao Paulo, Brazil, and analyzed them for susceptibility to RNI generated in acidified sodium nitrite (ASN) solution. RESULTS: Seventy-one (43%) of 167 recent-infection strains and 68 (43%) of 158 endogenous infection strains showed moderate- to high-level ASN resistance. CONCLUSION: ASN resistance of M. tuberculosis is not necessarily a determining factor for enhanced transmission.

Pulmonary Surfactant in Respiratory Syncytial Virus Bronchiolitis: The Role in Pathogenesis and Clinical Implications

Respiratory syncytial virus (RSV) bronchiolitis is the leading cause of lower respiratory tract infection, and the most frequent reason for hospitalization among infants throughout the world. In addition to the acute consequences of the disease, RSV bronchiolitis in early childhood is related to further development of recurrent wheezing and asthma. Despite the medical and economic burden of the disease, therapeutic options are limited to supportive measures, and mechanical ventilation in severe cases. Growing evidence suggests an important role of changes in pulmonary surfactant content and composition in the pathogenesis of severe RSV bronchiolitis. Besides the well-known importance of pulmonary surfactant in maintenance of pulmonary homeostasis and lung mechanics, the surfactant proteins SP-A and SP-D are essential components of the pulmonary innate immune system. Deficiencies of such proteins, which develop in severe RSV bronchiolitis, may be related to impairment in viral clearance, and exacerbated inflammatory response. A comprehensive understanding of the role of the pulmonary surfactant in the pathogenesis of the disease may help the development of new treatment strategies. We conducted a review of the literature to analyze the evidences of pulmonary surfactant changes in the pathogenesis of severe RSV bronchiolitis, its relation to the inflammatory and immune response, and the possible role of pulmonary surfactant replacement in the treatment of the disease. Pediatr Pulmonol. 2011; 46:415-420. (c) 2010 Wiley-Liss, Inc.

Thiol and Sulfenic Acid Oxidation of AhpE, the One-Cysteine Peroxiredoxin from Mycobacterium tuberculosis: Kinetics, Acidity Constants, and Conformational Dynamics

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen`s antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

Research on Candida dubliniensis in a Brazilian yeast collection obtained from cardiac transplant, tuberculosis, and HIV-positive patients, and evaluation of phenotypic tests using agar screening methods

The aim of this study was to research Candida dubliniensis among isolates present in a Brazilian yeast collection and to evaluate the main phenotypic methods for discrimination between C. albicans and C. dubliniensis from oral cavity. A total of 200 isolates, presumptively identified as C. albicans or C. dubliniensis obtained from heart transplant patients under immunosuppressive therapy, tuberculosis patients under antibiotic therapy, HIV-positive patients under antiretroviral therapy, and healthy subjects, were analyzed using the following phenotypic tests: formation and structural arrangement of chlamydospores on corn meal agar, casein agar, tobacco agar, and sunflower seed agar; growth at 45 degrees C; and germ tube formation. All strains were analyzed by polymerase chain reaction (PCR). In a preliminary screen for C. dubliniensis, 48 of the 200 isolates on corn meal agar, 30 of the 200 on casein agar, 16 of the 200 on tobacco agar, and 15 of the 200 on sunflower seed agar produced chlamydoconidia; 27 of the 200 isolates showed no or poor growth at 45 degrees C. All isolates were positive for germ tube formation. These isolates were considered suggestive of C. dubliniensis. All of them were subjected to PCR analysis using C. dubliniensis-specific primers. C. dubliniensis isolates were not found. C. dubliniensis isolates were not recovered in this study done with immunocompromised patients. Sunflower seed agar was the medium with the smallest number of isolates of C. albicans suggestive of C. dubliniensis. None of the phenotypic methods was 100% effective for discrimination between C. albicans and C. dubliniensis. (C) 2011 Elsevier Inc. All rights reserved.

CD28 Exerts Protective and Detrimental Effects in a Pulmonary Model of Paracoccidioidomycosis

T-cell immunity has been claimed as the main immunoprotective mechanism against Paracoccidioides brasiliensis infection, the most important fungal infection in Latin America. As the initial events that control T-cell activation in paracoccidioidomycosis (PCM) are not well established, we decided to investigate the role of CD28, an important costimulatory molecule for the activation of effector and regulatory T cells, in the immunity against this pulmonary pathogen. Using CD28-deficient (CD28(-/-)) and normal wild-type (WT) C57BL/6 mice, we were able to demonstrate that CD28 costimulation determines in pulmonary paracoccidioidomycosis an early immunoprotection but a late deleterious effect associated with impaired immunity and uncontrolled fungal growth. Up to week 10 postinfection, CD28(-/-) mice presented increased pulmonary and hepatic fungal loads allied with diminished production of antibodies and pro-and anti-inflammatory cytokines besides impaired activation and migration of effector and regulatory T (Treg) cells to the lungs. Unexpectedly, CD28-sufficient mice progressively lost the control of fungal growth, resulting in an increased mortality associated with persistent presence of Treg cells, deactivation of inflammatory macrophages and T cells, prevalent presence of anti-inflammatory cytokines, elevated fungal burdens, and extensive hepatic lesions. As a whole, our findings suggest that CD28 is required for the early protective T-cell responses to P. brasiliensis infection, but it also induces the expansion of regulatory circuits that lately impair adaptive immunity, allowing uncontrolled fungal growth and overwhelming infection, which leads to precocious mortality of mice.

TLR2 Is a Negative Regulator of Th17 Cells and Tissue Pathology in a Pulmonary Model of Fungal Infection

To study the role of TLR2 in a experimental model of chronic pulmonary infection, TLR2-deficient and wild-type mice were intratracheally infected with Paracoccidioides brasiliensis, a primary fungal pathogen. Compared with control, TLR2(-/-) mice developed a less severe pulmonary infection and decreased NO synthesis. Equivalent results were detected with in vitro-infected macrophages. Unexpectedly, despite the differences in fungal loads both mouse strains showed equivalent survival times and severe pulmonary inflammatory reactions. Studies on lung-infiltrating leukocytes of TLR2(-/-) mice demonstrated an increased presence of polymorphonuclear neutrophils that control fungal loads but were associated with diminished numbers of activated CD4(+) and CD8(+) T lymphocytes. TLR2 deficiency leads to minor differences in the levels of pulmonary type 1 and type 2 cytokines, but results in increased production of KC, a CXC chemokine involved in neutrophils chemotaxis, as well as TGF-beta, IL-6, IL-23, and IL-17 skewing T cell immunity to a Th17 pattern. In addition, the preferential Th17 immunity of TLR2(-/-) mice was associated with impaired expansion of regulatory CD4(+)CD25(+)FoxP3(+) T cells. This is the first study to show that TLR2 activation controls innate and adaptive immunity to P. brasiliensis infection. TLR2 deficiency results in increased Th17 immunity associated with diminished expansion of regulatory T cells and increased lung pathology due to unrestrained inflammatory reactions. The Journal of Immunology, 2009, 183: 1279-1290.

Different mechanisms underlie the effects of acute and long-term inhibition of nitric oxide synthases in antigen-induced pulmonary eosinophil recruitment in BALB/C mice

Nitric oxide synthase (NOS) inhibitors are largely used to evaluate the NO contribution to pulmonary allergy, but contrasting data have been reported. In this study, pharmacological, biochemical and pharmacokinetic assays were performed to compare the effects of acute and long-term treatment of BALB/C mice with the non-selective NOS inhibitor L-NAME in ovalbumin (OVA)-challenged mice. Acute L-NAME treatment (50 mg/kg, gavage) significantly reduced the eosinophil number in bronchoalveolar lavage fluid (BALF). The inducible NOS (iNOS) inhibitor aminoguanidine (20 mg/kg/day in the drinking water) also significantly reduced the eosinophil number in BALF In contrast, 3-week L-NAME treatment (50 and 150 mg/kg/day in the drinking water) significantly increased the pulmonary eosinophil influx. The constitutive NOS (cNOS) activity in brain and lungs was reduced by both acute and 3-week L-NAME treatments. The pulmonary iNOS activity was reduced by acute L-NAME (or aminoguanidine), but unaffected by 3-week L-NAME treatment. Acute L-NAME (or aminoguanidine) treatment was more efficient to reduce the NO(x) levels compared with 3-week L-NAME treatment. The pharmacokinetic study revealed that L-NAME is not bioavailable when given orally. After acute L-NAME intake, serum concentrations of the metabolite N(omega)-nitro-L-arginine decreased from 30 min to 24 h. In the 3-week L-NAME treatment, the N(omega)-nitro-L-arginine concentration was close to the detection limit. In conclusion, 3-week treatment with L-NAME yields low serum N(omega)-nitro-L-arginine concentrations, causing preferential inhibition of cNOS activity. Therefore, eosinophil influx potentiation by 3-week L-NAME treatment may reflect removal of protective cNOS-derived NO, with no interference on the ongoing inflammation due to iNOS-derived NO. (c) 2008 Elsevier Ltd. All rights reserved.

Low level laser therapy (LLLT) decreases pulmonary microvascular leakage, neutrophil influx and IL-1 beta levels in airway and lung from rat subjected to LPS-induced inflammation

Background and Objective. Low level laser therapy (LLLT) is a known anti-inflammatory therapy. Herein we studied the effect of LLLT on lung permeability and the IL-1 beta level in LPS-induced pulmonary inflammation. Study Design/Methodology. Rats were divided into 12 groups (n = 7 for each group). Lung permeability was measured by quantifying extravasated albumin concentration in lung homogenate, inflammatory cells influx was determined by myeloperoxidase activity, IL-1P in BAL was determined by ELISA and IL-1P mRNA expression in trachea was evaluated by RT-PCR. The rats were irradiated on the skin over the upper bronchus at the site of tracheotomy after LPS. Results. LLLT attenuated lung permeability. In addition, there was reduced neutrophil influx, myeloperoxidase activity and both IL-1 beta in BAL and IL-1 beta mRNA expression in trachea obtained from animals subjected to LPS-induced inflammation. Conclusion. LLLT reduced the lung permeability by a mechanism in which the IL-1 beta seems to have an important role.

Vanadium complexes with thiosemicarbazones: Synthesis, characterization, crystal structures and anti-Mycobacterium tuberculosis activity

The development of more efficient anti-tuberculosis drugs is of interest. Three oxovanadium(IV) and three cis-dioxovanadium(V) complexes with thiosemicarbazone derivatives bearing moieties with different lipophilicity have been prepared and had their inhibitory activity against Mycobacterium tuberculosis H(37)Rv ATCC 27294 evaluated. The analytical methods used by the complexes` characterization included IR, EPR, (1)H, (13)C and (51)V NMR spectroscopies, elemental analysis, cyclic voltammetry, magnetic susceptibility measurement and single crystal X-ray diffractometry. [VO(acac)(aptsc)], [VO(acac)(apmtsc)] and [VO(acac)(apptsc)] (acac = acetylacetonate; Haptsc = 2-acetylpyridinethiosemicarbazone; Hapmtsc = 2-acetylpyridine-N(4)-methyl-thiosemicarbazone and Happtsc = 2-acetylpyridine-N(4)-phenyl-thiosemicarbazone) are paramagnetic and their EPR spectra are consistent with the monoanionic N,N,S-tridentate coordination of the thiosemicarbazone ligands, resulting in octahedral structures of rhombic symmetry and with the oxidation state +IV for the vanadium atom. As result of oxidation of the vanadium(IV) complexes above, the diamagnetic cis-dioxovanadium(V) complexes [VO(2)(aptsc)[, [VO(2)(apmtsc)[ and [VO(2)(apptsc)] are formed. Their (1)H, (13)C and (51)V NMR spectra were acquired and support a distorted square pyramidal geometry for them, in accord with the solid state X-ray structures determined for [VO(2)(aptsc)] and [VO(2)(apmtsc)]. In general, the vanadium compounds show comparable or larger anti-M. tuberculosis activities than the free thiosemicarbazone ligands, with MIC values within 62.5-1.56 (mu g/mL). (C) 2008 Elsevier Ltd. All rights reserved.

Inhibition of Mycobacterium tuberculosis tyrosine phosphatase PtpA by synthetic chalcones: Kinetics, molecular modeling, toxicity and effect on growth

Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world, and it is estimated that one-third of the world`s population is infected with Mycobacterium tuberculosis. Among a series of tested compounds, we have recently identified five synthetic chalcones which inhibit the activity of M. tuberculosis protein tyrosine phosphatase A (PtpA), an enzyme associated with M. tuberculosis infectivity. Kinetic studies demonstrated that these compounds are reversible competitive inhibitors. In this work we also carried out the analysis of the molecular recognition of these inhibitors on their macromolecular target, PtpA, through molecular modeling. We observed that the predominant determinants responsible for the inhibitory activity of the chalcones are the positions of the two methoxyl groups at the A-ring, that establish hydrogen bonds with the amino acid residues Arg17, His49, and Thr12 in the active site of PtpA, and the substitution of the phenyl ring for a 2-naphthyl group as B-ring, that undergoes p stacking hydrophobic interaction with the Trp48 residue from PtpA. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel therapeutics. The biological activity, synthetic versatility, and low cost are clear advantages of this new class of potential tuberculostatic agents. (C) 2010 Elsevier Ltd. All rights reserved.

Structural studies of prephenate dehydratase from Mycobacterium tuberculosis H37Rv by SAXS, ultracentrifugation, and computational analysis

Tuberculosis (TB) is one of the most common infectious diseases known to man and responsible for millions of human deaths in the world. The increasing incidence of TB in developing countries, the proliferation of multidrug resistant strains, and the absence of resources for treatment have highlighted the need of developing new drugs against TB. The shikimate pathway leads to the biosynthesis of chorismate, a precursor of aromatic amino acids. This pathway is absent from mammals and shown to be essential for the survival of Mycobacterium tuberculosis, the causative agent of TB. Accordingly, enzymes of aromatic amino acid biosynthesis pathway represent promising targets for structure-based drug design. The first reaction in phenylalanine biosynthesis involves the conversion of chorismate to prephenate, catalyzed by chorismate mutase. The second reaction is catalyzed by prephenate dehydratase (PDT) and involves decarboxylation and dehydratation of prephenate to form phenylpyruvate, the precursor of phenylalanine. Here, we describe utilization of different techniques to infer the structure of M. tuberculosis PDT (MtbPDT) in solution. Small angle X-ray scattering and ultracentrifugation analysis showed that the protein oligomeric state is a tetramer and MtbPDT is a flat disk protein. Bioinformatics tools were used to infer the structure of MtbPDT A molecular model for MtbPDT is presented and molecular dynamics simulations indicate that MtbPDT i.s stable. Experimental and molecular modeling results were in agreement and provide evidence for a tetrameric state of MtbPDT in solution.

Thiosemicarbazones, semicarbazones, dithiocarbazates and hydrazide/hydrazones: Anti-Mycobacterium tuberculosis activity and cytotoxicity

The aim of this study was to identify a candidate drug for the development of anti-tuberculosis therapy from previously synthesized compounds based on the thiosemicarbazones, semicarbazones, dithio-carbazates and hydrazide/hydrazones compounds. The minimal inhibitory concentration (MIC) of these compounds against Mycobacterium tuberculosis was determined. Their in vitro cytotoxicity to J774 cells (IC(50)) was determined to establish a selectivity index (SI) (SI = IC(50)/MIC). The best compounds were the thiosemicarbazones (2, 3 and 4) and the hydrazide/hydrazones (14, 15, 16 and 18). The results are comparable to or better than those of ""first line"" or ""second line"" drugs commonly used to treat TB, suggesting these compounds as anti-TB drug candidates. (C) 2010 Elsevier Masson SAS. All rights reserved.