ABSTRACT
TGF-beta-targeting structural and inflammatory cells has been implicated in the mechanisms leading to the inflammatory and restructuring processes in asthma, suggesting an impact of TGF-beta1 signaling on the development and persistency of this disease. We investigated the potential early involvement of TGF-beta1 activity in the immunological and molecular mechanisms underlying progression of inflammation in childhood asthma. We evaluated the levels of TGF-beta1 in induced sputum supernatants (ISSs) and the expression of small mother cell against decapentaplegic (Smad) 2 and Smad7 proteins in induced sputum cells (ISCs) from children with intermittent asthma (IA), moderate asthma (MA) and control subjects (C). Furthermore, we investigated the regulatory role of TGF-beta1 activity on eosinophil and neutrophil adhesion to epithelial cells using adhesion assay, and on the granulocyte expression of adhesion molecule CD11b/CD18 Macrophage-1 antigen (MAC-1), by flow cytometry. We found that the levels of TGF-beta1 are increased in ISSs of IA and MA in comparison to C, concomitantly to the activation of intracellular signaling TGFbeta/Smads pathway in ISCs. In MA, TGF-beta1 levels correlated with the number of sputum eosinophils and neutrophils. Furthermore, we showed the ability of sputum TGF-beta1 to promote eosinophil and neutrophil adhesion to epithelial cells, and to increase the expression of MAC-1 on the granulocyte surface. This study shows the activation of TGFbeta/Smad signaling pathway in the airways of children with IA and, despite the regular ICS treatment, in children with MA, and provides evidence for the contribution of TGF-beta1 in the regulation of granulocyte activation and trafficking.
Subject(s)
Asthma/metabolism , Lung/metabolism , Signal Transduction , Transforming Growth Factor beta1/metabolism , Administration, Inhalation , Adolescent , Adrenal Cortex Hormones/administration & dosage , Age Factors , Asthma/diagnosis , Asthma/drug therapy , Asthma/immunology , Asthma/physiopathology , Bronchodilator Agents/administration & dosage , Case-Control Studies , Cell Adhesion , Cell Line , Child , Eosinophils/immunology , Eosinophils/metabolism , Epithelial Cells/immunology , Epithelial Cells/metabolism , Female , Granulocytes/immunology , Granulocytes/metabolism , Humans , Lung/drug effects , Lung/immunology , Lung/physiopathology , Macrophage-1 Antigen/metabolism , Male , Neutrophils/immunology , Neutrophils/metabolism , Phosphorylation , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Severity of Illness Index , Signal Transduction/drug effects , Smad2 Protein/metabolism , Smad7 Protein/metabolism , Sputum/metabolismABSTRACT
A flow injection (FIA) method was designed for the determination of chlorophylls a and b in small in vitro Dieffenbachia maculata "Sublime" plants. In the first step, the pigments from spinach leaves were separated, purified by solvent extraction and freeze-dried, to obtain standards for the FIA optimization. The sample extraction procedure was optimized. Four solvents were tested: diethyl ether, methanol, acetone and ethanol. The ethanol 96% was the optimal solvent for FIA purposes. It allows to the efficient extraction of the pigments and water can be used as carrier. The best FIA conditions found for the quasi-simultaneous quantification of chlorophylls a and b were a flow rate of 10.84mLmin(-1), a sample injection volume of 1.45mL and a reactor length of 63cm. The detection was performed with the automatic wavelength scanning Cintra 10e spectrometer, at 649 and 665nm. The results obtained by the FIA method were compared to those obtained by the Arnon method. A deviation less than 5% was found between results for both methods. The concentration (mgg(-1)) of chlorophylls a and b during three periods of the plants (in vitro, acclimatization, and adult) was determined to evaluate the whole in vitro procedure. It was found an increment of both pigment concentrations since the in vitro step till the adult stage, while the chlorophylls a to b ratio decreases. The designed method is suitable especially for the determination of the pigments at low concentrations in small samples with appropriate analytical quality.
ABSTRACT
Cases of patients with markedly depressed CD4+ T-lymphocyte counts, with or without opportunistic infections, in the absence of any evidence of human immunodeficiency virus (HIV) have been described in recent years. In 1992, the definition of "idiopathic CD4+ T-lymphocytopenia" was formulated by the Centers for Disease Control and Prevention (CDC) of Atlanta (USA). The present case illustrates the occurrence of an unexplained Mycobacterium kansasii pneumonia in a white HIV-negative subject with a persistent depletion of CD4+ T-lymphocytes and suppression of cell-mediated immunity. To our knowledge, this is the first observation of idiopathic CD4+ T-lymphocytopenia with pulmonary mycobacteriosis due to Mycobacterium kansasii, and the sixth case of this kind of immunodeficiency described in Italy.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Lymphopenia/diagnosis , Mycobacterium Infections, Nontuberculous/diagnosis , Pneumonia, Bacterial/diagnosis , Adult , Antitubercular Agents/therapeutic use , Bronchoalveolar Lavage , Bronchoscopy , CD4 Lymphocyte Count , Diagnosis, Differential , HIV Seronegativity , Humans , Lymphopenia/drug therapy , Lymphopenia/immunology , Male , Mycobacterium Infections, Nontuberculous/drug therapy , Mycobacterium Infections, Nontuberculous/immunology , Pneumonia, Bacterial/drug therapy , Pneumonia, Bacterial/immunologyABSTRACT
Listeria monocytogenes is a facultative intracellular pathogen which enters cells by endocytosis and reaches phagolysosomes from where it escapes and multiplies in the cytosol of untreated cells. Exposure of macrophages to gamma interferon (IFN-gamma) restricts L. monocytogenes to phagosomes and prevents its intracellular multiplication. We have tested whether IFN-gamma also modulates the susceptibility of L. monocytogenes to antibiotics. We selected drugs from three different classes displaying marked properties concerning their cellular accumulation and subcellular distribution, namely, ampicillin (not accumulated by cells but present in cytosol), azithromycin (largely accumulated by cells but mostly restricted to lysosomes), and sparfloxacin (accumulated to a fair extent but detected only in cytosol). We used a continuous line of myelomonocytic cells (THP-1 macrophages), which display specific surface receptors for IFN-gamma, and examined the activity of these antibiotics against L. monocytogenes Hly+ (virulent variant) and L. monocytogenes Hly- (a nonvirulent variant defective in hemolysin production). Untreated THP-1 and phorbol myristate acetate-differentiated THP-1 were permissive for infection and multiplication of intracellular L. monocytogenes Hly+ (virulent variant). All three antibiotics tested were bactericidal against this Listeria strain when added to an extracellular concentration of 10x their MIC. After preexposure of THP-1 to IFN-gamma, L. monocytogenes Hly+ was still phagocytosed but no longer grew intracellularly. The activity of ampicillin became almost undetectable (antagonistic effect), and that of azithromycin was unchanged (additive effect with that of IFN-gamma), whereas that of sparfloxacin was markedly enhanced (synergy). A similar behavior (lack of bacterial growth, associated with a loss of activity of ampicillin, an enhanced activity of sparfloxacin, and unchanged activity of azithromycin) was observed in cells infected with L. monocytogenes Hly-. This modulation of antibiotic activity, which we ascribe to the change of subcellular localization of L. monocytogenes caused by IFN-gamma or by the lack of virulence factor, could result from a change in bacterial responsiveness to antibiotics, a modification of the drug activity, or differences in drug bioavailabilities between cytosol and phagosomes.
