Futoshi Higa

Molecular characterization of Legionella pneumophila-induced interleukin-8 expression in T cells

BackgroundLegionella pneumophila is the causative agent of human Legionnaires 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.ResultsWild-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.ConclusionsTaken 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 Legionnaires disease.

Differential Roles of Toll-Like Receptors 2 and 4 in In Vitro Responses of Macrophages to Legionella pneumophila

ABSTRACT The role of Toll-like receptors (TLRs) in innate immunity to Legionella pneumophila, a gram-negative facultative intracellular bacterium, was studied by using bone marrow-derived macrophages and dendritic cells from TLR2-deficient (TLR2−/−), TLR4−/−, and wild-type (WT) littermate (C57BL/6 × 129Sv) mice. Intracellular growth of L. pneumophila was enhanced within TLR2−/− macrophages compared to WT and TLR4−/− macrophages. There was no difference in the bacterial growth within dendritic cells from WT and TLR-deficient mice. Production of interleukin-12p40 (IL-12p40) and IL-10 after infection with L. pneumophila was attenuated in TLR2−/− macrophages compared to WT and TLR4−/− macrophages. Induction of IL-12p40, IL-10, and tumor necrosis factor alpha secretion from macrophages by the L. pneumophila dotO mutant, which cannot multiply within macrophages, and heat-killed bacteria, was similar to that caused by a viable virulent strain. There was no difference between the WT and its mutants in susceptibility to the cytopathic effect of bacteria. An L. pneumophila sonicated lysate induced IL-12p40 production by macrophages, but that of TLR2−/− macrophages was significantly lower than those of WT and TLR4−/− macrophages. Treatment of L. pneumophila sonicated lysate with proteinase K and heating did not abolish TLR2-dependent IL-12p40 production. Our results show that TLR2, but not TLR4, is involved in murine innate immunity against L. pneumophila, although other TLRs may also contribute to innate immunity against this organism.

Mechanisms of Legionella pneumophila-induced interleukin-8 expression in human lung epithelial cells

BackgroundLegionella 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.ResultsInfection 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.ConclusionCollectively, 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.

Alcoholic liver cirrhosis complicated with torsade de pointes during plasma exchange and hemodiafiltration

A 36-year-old man with severe alcoholic hepatitis was treated with plasma exchange combined with hemodiafiltration to remove endotoxins and inflammatory cytokines. During the treatment, he had critical arrhythmia (torsade de pointes [TdP]). His laboratory data showed hypomagnesemia, which was suspected to be responsible for the development of TdP. Patients with alcoholic liver disease tend to have hypomagnesemia and Q-T interval prolongation. Furthermore, hemodiafiltration may cause hypomagnesemia. Careful observation for electrolytic imbalance is necessary when clinicians treat patients with alcoholic liver failure with a liver support system.

Adjunctive Systemic Corticosteroids for Hospitalized Community-Acquired Pneumonia: Systematic Review and Meta-Analysis 2015 Update

Previous randomized controlled trials (RCTs) and meta-analyses evaluated the efficacy and safety of adjunctive corticosteroids for community-acquired pneumonia (CAP). However, the results from them had large discrepancies. The eligibility criteria for the current meta-analysis were original RCTs written in English as a full article that evaluated adjunctive systemic corticosteroids adding on antibiotic therapy targeting typical and/or atypical pathogen for treating hospitalized human CAP cases. Four investigators independently searched for eligible articles through PubMed, Embase, and Cochrane databases. Random model was used. The heterogeneity among original studies and subgroups was evaluated with the I2 statistics. Of 54 articles that met the preliminary criteria, we found 10 eligible RCTs comprising 1780 cases. Our analyses revealed following pooled values by corticosteroids. OR for all-cause death: 0.80 (95% confidence interval (95% CI) 0.53–1.21) from all studies; 0.41 (95% CI 0.19–0.90) from severe-case subgroup; 0.21 (95% CI 0.0–0.74) from intensive care unit (ICU) subgroup. Length of ICU stay: −1.30 days (95% CI (−3.04)−0.44). Length of hospital stay: −0.98 days (95% CI (−1.26)–(−0.71)). Length to clinical stability: −1.16 days (95% CI (−1.73)–(−0.58)). Serious complications do not seem to largely increase by steroids. In conclusion, adjunctive systemic corticosteroids for hospitalized patients with CAP seems preferred strategies.

Endoscopic and radiographic features of gastrointestinal involvement in vasculitis

Vasculitis is an inflammation of vessel walls, followed by alteration of the blood flow and damage to the dependent organ. Vasculitis can cause local or diffuse pathologic changes in the gastrointestinal (GI) tract. The variety of GI lesions includes ulcer, submucosal edema, hemorrhage, paralytic ileus, mesenteric ischemia, bowel obstruction, and life-threatening perforation.The endoscopic and radiographic features of GI involvement in vasculitisare reviewed with the emphasis on small-vessel vasculitis by presenting our typical cases, including Churg-Strauss syndrome, Henoch-Schönlein purpura, systemic lupus erythematosus, and Behçets disease. Important endoscopic features are ischemic enterocolitis and ulcer. Characteristic computed tomographic findings include bowel wall thickening with the target sign and engorgement of mesenteric vessels with comb sign. Knowledge of endoscopic and radiographic GI manifestations can help make an early diagnosis and establish treatment strategy.

Computed tomographic features of 23 sporadic cases with Legionella pneumophila pneumonia

OBJECTIVE To describe the chest computed tomographic (CT) findings of Legionella pneumophila pneumonia. METHODS CT scans obtained from 23 sporadic cases of L. pneumophila pneumonia were retrospectively reviewed. Chest CT findings were analyzed with regard to the patterns and distributions of pulmonary abnormalities. We also analyzed the histopathology of lungs from guinea pigs with experimentally induced L. pneumophila pneumonia. RESULTS Consolidation and ground-glass opacity (GGO) were the main findings of CT scans in L. pneumophila pneumonia. The distribution of opacities was categorized as non-segmental (n=20) and segmental (n=4). Non-segmental distribution may follow an onset of segmental distribution. Pleural effusion was observed in 14 (58.3%) patients, of which 13 were accompanied with non-segmental distribution. Abscess formation was observed in only one immunocompromised patient. In the animal pneumonia model, the lesions comprised of terminal bronchioles, alveolar spaces, and interstitia. Small bacilli were observed to be contained by many macrophages within the alveoli. CONCLUSION Non-segmental distribution was significantly more frequent than segmental distribution in L. pneumophila pneumonia. It is possible that L. pneumophila infection initially results in segmental pneumonia, which progresses to typical non-segmental distribution.

Legionella pneumophila infection induces programmed cell death, caspase activation, and release of high-mobility group box 1 protein in A549 alveolar epithelial cells: inhibition by methyl prednisolone

BackgroundLegionella 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.MethodsNuclear 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.ResultsThe 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.ConclusionInfection 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.

Detection of Legionella Species in Clinical Samples: Comparison of Polymerase Chain Reaction and Urinary Antigen Detection Kits

Background:Recently, two excellent methods have been used for the diagnosis of Legionnaires’ disease: urinary antigen detection and PCR. The purpose of the present study is to analyze and evaluate the sensitivity and specificity of three different urinary antigen detection kits as well as PCR.Materials and Methods:A total of 148 samples were collected from 33 patients between 1993 and 2004. These consisted of 73 urine samples obtained from 33 patients, 57 serum samples provided by 29 patients, and 18 respiratory tract specimens from 13 patients. Three commercially available kits were used to detect urinary antigen. For the 5S PCR reaction, primers L5SL2 and L5SR84 were used.Results:Positive results were shown in all patients’ urine (representing 79.5% of total samples) using the Binax EIA kit, in 93.9% patients (representing 75.3% samples) using the Binax NOW immunochromatographic kit, and in 90.9% (representing 72.6% samples) using the Biotest EIA kit. Urine samples from 12.1% patients (representing 6.8% of total samples), serum samples from 41.4% patients (representing 35.1% of total samples), and respiratory samples from 84.6% patients (representing 88.9% of total samples) showed positive results with PCR.Conclusion:In testing urine of legionellosis patients, it was suggested that three kits were all valuable tools for diagnosis of legionellosis. Since over one-third of patients’ serum samples and most respiratory specimens showed positive results with PCR, the addition of PCR for testing of these samples might be useful, particularly in cases of culture negative and serum antibody negative patients.

Comparison of Polymerase Chain Reaction and Two Urinary Antigen Detection Kits for Detecting Legionella in Clinical Samples

In the study presented here, two urinary antigen tests (Legionella Urinary Antigen EIA; Binax, Portland, ME, USA and Biotest Legionella Urin Antigen EIA; Biotest, Dreieich, Germany) and one PCR with a 5S-primer were used to detect Legionella in the clinical samples of six patients diagnosed with legionellosis between 1997 and 1999. Initially, clinical specimens that could be collected in a series were obtained from each patient, resulting in a total of 68 samples (34 urine, 24 serum, 7 sputum, and 3 respiratory). These samples were tested with culture, serum antibody and urinary antigen tests, then stored at – 80�C until further testing in the present study. Upon retesting with the two urinary antigen tests and the 5Sprimer PCR system, Legionella infections were identified using culture and urinary antigen tests in three cases, with culture alone in one case, and with urinary antigen tests alone (both Binax and Biotest) in two cases. The 5Sprimer PCR system detected 26 of 34 Legionella spp. tested. Patient 1 had undergone a cone resection for earlystage uterine cervical carcinoma 1 year prior to presentation. A diagnosis of Pneumocystis carinii pneumonia and pulmonary infiltration of adult T-cell leukemia cells was made. Legionella pneumophila serogroup 1 was later isolated from bronchial lavage fluid and, over time, the serum indirect immunofluorescent antibody (IFA) titer against Legionella pneumophila serogroup 1 increased from 1:128 to 1:512. The IFA test was performed as described previously [1]. The patient was treated with antibiotics (erythromycin, sparfloxacin, and trimethoprim-sulfamethoxazole) and antineoplastic agents (vincristine, cyclophosphamide and adriamycin) and was discharged 5 5 months after admission, with ATL in