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Rheumatic fever (RF) is an autoimmune disease which affects more than 20 million children in developing countries. It is triggered by Streptococcus pyogenes throat infection in untreated susceptible individuals. Carditis, the most serious manifestation of the disease, leads to severe and permanent valvular lesions, causing chronic rheumatic heart disease (RHD). We have been studying the mechanisms leading to pathological autoimmunity in RF/RHD for the last 15 years. Our studies allowed us a better understanding of the cellular and molecular pathogenesis of RHD, paving the way for the development of a safe vaccine for a post-infection autoimmune disease. We have focused on the search for protective T and B cell epitopes by testing 620 human blood samples against overlapping peptides spanning 99 residues of the C-terminal portion of the M protein, differing by one amino acid residue. We identified T and B cell epitopes with 22 and 25 amino acid residues, respectively. Although these epitopes were from different regions of the C-terminal portion of the M protein, they showed an identical core of 16 amino acid residues. Antibodies against the B cell epitope inhibited bacterial invasion/adhesion in vitro. Our results strongly indicated that the selected T and B cell epitopes could potentially be protective against S. pyogenes. [ABSTRACT FROM AUTHOR]

We have investigated the effect of methylprednisolone on the intracellular activity of erythromycin and clindamycin in vitro. An assay system was developed for the determination of intracellular activity of antibiotics against Legionella pneumophila using guinea pig resident alveolar macrophages. Erythromycin at a concentration of 0.625 mg/L (5 x MIC) and clindamycin at a concentration of 8 mg/L (MIC) inhibited the growth of a single strain of L. pneumophila in macrophages, whilst ceftizoxime at a concentration of 0.625 mg/L (5 x MIC) did not. Methylprednisolone at therapeutic concentrations did not affect the intracellular antibacterial activity of either erythromycin or clindamycin against L. pneumophila. We found no direct effect of methylprednisolone on the intracellular antibacterial activity of either erythromycin or clindamycin. [ABSTRACT FROM AUTHOR]

For prevention of nosocomial legionellosis, environmental investigation to identify possible infectious sources is essential. An environmental study in a ward of our hospital revealed that a steam towel warmer was contaminated with legionella whereas no legionella was detected in tap water supplies and shower heads. Water in the apparatus may be a reservoir of legionella. We abandoned the use of all steam towel warmers in our hospital. Based on this finding, we recommend that steam towel warmers in hospital settings be avoided. Otherwise, the apparatus should be drained, cleaned and dried every day.Copyright © 2012 by Elsevier Inc. [ABSTRACT FROM AUTHOR]

Abstract Article Retracted

Abstract Background Hepatocyte growth factor (HGF) is known to be involved in the resolution of pulmonary inflammation and repair of acute lung injury. Legionella pneumonia is sometimes complicated by acute lung injury. Our study aimed to determine the role of serum HGF levels in Legionella pneumonia. Methods Sera from patients with Legionella pneumonia (42 cases), other bacterial pneumonia (33 cases), pulmonary tuberculosis (19 cases), and normal controls (29 cases) were collected. The serum HGF levels for each serum sample were determined by sandwich ELISA. Clinical and laboratory data were collected by reviewing the medical charts. Results Serum HGF levels were higher in patients with Legionella pneumonia than in those with other bacterial pneumonia, pulmonary tuberculosis, and controls. The HGF levels were compared with white blood cell counts, C-reactive protein, Alanine amino- transferase, and lactate dehydrogenase (LDH). The HGF levels were correlated to serum LDH levels. Moreover, serum HGF levels were significantly higher in non-survivors than in survivors. Conclusions HGF levels increased in severer pneumonia caused by Legionella, suggesting that HGF might play a significant role in the Legionella pneumonia.

Abstract Background Legionella pneumophila is the causative agent of human Legionnaire's disease. During infection, the bacterium invades macrophages and lung epithelial cells, and replicates intracellularly. However, little is known about its interaction with T cells. We investigated the ability of L. pneumophila to infect and stimulate the production of interleukin-8 (IL-8) in T cells. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells. Results Wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T cells. On the other hand, wild-type L. pneumophila and Dot/Icm-deficient Legionella, but not flagellin-deficient Legionella or heat-killed Legionella induced IL-8 expression. L. pneumophila activated an IL-8 promoter through the NF-κB and AP-1 binding regions. Wild-type L. pneumophila but not flagellin-deficient Legionella activated NF-κB, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and transforming growth factor β-associated kinase 1 (TAK1). Transfection of dominant negative mutants of IκBα, IκB kinase, NF-κB-inducing kinase, TAK1, MyD88, and p38 MAPK inhibited L. pneumophila-induced IL-8 activation. Inhibitors of NF-κB, p38 MAPK, and JNK blocked L. pneumophila-induced IL-8 expression. In addition, c-Jun, JunD, cyclic AMP response element binding protein, and activating transcription factor 1, which are substrates of p38 MAPK and JNK, bound to the AP-1 site of the IL-8 promoter. Conclusions Taken together, L. pneumophila induced a flagellin-dependent activation of TAK1, p38 MAPK, and JNK, as well as NF-κB and AP-1, which resulted in IL-8 production in human T cells, presumably contributing to the immune response in Legionnaire's disease.

Abstract Background Legionella pneumophila pneumonia often exacerbates acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells. Methods Nuclear deoxyribonucleic acid (DNA) fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling method (TUNEL method) and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila. Results The virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining) and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1) protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 μM) did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells. Conclusion Infection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a major virulence factor of L. pneumophila, is involved in the effects we measured in alveolar epithelial cells. Methyl prednisolone may modulate the interaction of Legionella and these cells.

Abstract Background Legionella pneumophila is a facultative intracellular bacterium, capable of replicating within the phagosomes of macrophages and monocytes, but little is known about its interaction with human lung epithelial cells. We investigated the effect of L. pneumophila on the expression of interleukin-8 (IL-8) in human A549 alveolar and NCI-H292 tracheal epithelial cell lines. Results Infection of L. pneumophila strain, but not heat-killed strain, resulted in upregulation of IL-8. IL-8 mRNA expression was induced immediately after the infection and its signal became gradually stronger until 24 h after infection. On the other hand, IL-8 expression in A549 cells infected with L. pneumophila lacking a functional type IV secretion system was transient. The IL-8 expression was slightly induced at 16 h and increased at 24 h after infection with flagellin-deficient Legionella. Activation of the IL-8 promoter by L. pneumophila infection occurred through the action of nuclear factor-κB (NF-κB). Transfection of dominant negative mutants of NF-κB-inducing kinase, IκB kinase and IκB inhibited L. pneumophila-mediated activation of IL-8 promoter. Treatment with hsp90 inhibitor suppressed L. pneumophila-induced IL-8 mRNA due to deactivation of NF-κB. Conclusion Collectively, these results suggest that L. pneumophila induces activation of NF-κB through an intracellular signaling pathway that involves NF-κB-inducing kinase and IκB kinase, leading to IL-8 gene transcription, and that hsp90 acts as a crucial regulator in L. pneumophila-induced IL-8 expression, presumably contributing to immune response in L. pneumophila. The presence of flagellin and a type IV secretion system are critical for Legionella to induce IL-8 expression in lung epithelial cells.

Background: Legionella pneumophila pneumonia often exacerbates acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells.Methods: Nuclear deoxyribonucleic acid (DNA) fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling method (TUNEL method) and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila.Results: The virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining) and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1) protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 muM) did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells.Conclusion: Infection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a major virulence factor of L. pneumophila, is involved in the effects we measured in alveolar epithelial cells. Methyl prednisolone may modulate the interaction of Legionella and these cells. [ABSTRACT FROM AUTHOR]

Abstract: Streptococcus pyogenes causes severe invasive infections: the post-streptococcal sequelae of acute rheumatic fever (RF) and rheumatic heart disease (RHD), acute glomerulonephritis, and uncomplicated pharyngitis and pyoderma. Efforts to produce a vaccine against S. pyogenes began several decades ago, and different models have been proposed. Here, we describe the methodology used in the development of a new vaccine model, consisting of both T and B protective epitopes constructed as synthetic peptides and recombinant proteins. Two adjuvants were tested in an experimental inbred mouse model: a classical Freund’s adjuvant and a new adjuvant (AFCo1) that induces mucosal immune responses and is obtained by calcium precipitation of a proteoliposome derived from the outer membrane of Neisseria meningitides B. The StreptInCor vaccine epitope co-administrated with AFCo1 adjuvant induced mucosal (IgA) and systemic (IgG) antibodies as preferential Th1-mediated immune responses. No autoimmune reactions were observed, suggesting that the vaccine epitope is safe. [Copyright &y& Elsevier]