Jiro Fujita

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.

Non-specific interstitial pneumonia as pulmonary involvement of systemic sclerosis

The pathological features of lung disease in nine patients with systemic sclerosis (SSc) were evaluated. The patients comprised one man and eight women, with a median age of 58 years. SSc was diagnosed according to the criteria of the American Rheumatism Association. In all patients, high resolution computed radiographic scanning of the lungs (HRCT) was performed, and apparent honeycomb formation was seen in four patients. Pathologically, four patients were diagnosed with usual interstitial pneumonia (UIP), three with non-specific interstitial pneumonia (NSIP) group II, one NSIP group II–III, and one NSIP group II with diffuse alveolar damage. HRCT showed no apparent honeycomb formations in patients diagnosed with NSIP. This is the first report describing NSIP as a pulmonary complication of SSc.

Dysbiosis of Salivary Microbiota in Inflammatory Bowel Disease and Its Association With Oral Immunological Biomarkers

Analysis of microbiota in various biological and environmental samples under a variety of conditions has recently become more practical due to remarkable advances in next-generation sequencing. Changes leading to specific biological states including some of the more complex diseases can now be characterized with relative ease. It is known that gut microbiota is involved in the pathogenesis of inflammatory bowel disease (IBD), mainly Crohns disease and ulcerative colitis, exhibiting symptoms in the gastrointestinal tract. Recent studies also showed increased frequency of oral manifestations among IBD patients, indicating aberrations in the oral microbiota. Based on these observations, we analyzed the composition of salivary microbiota of 35 IBD patients by 454 pyrosequencing of the bacterial 16S rRNA gene and compared it with that of 24 healthy controls (HCs). The results showed that Bacteroidetes was significantly increased with a concurrent decrease in Proteobacteria in the salivary microbiota of IBD patients. The dominant genera, Streptococcus, Prevotella, Neisseria, Haemophilus, Veillonella, and Gemella, were found to largely contribute to dysbiosis (dysbacteriosis) observed in the salivary microbiota of IBD patients. Analysis of immunological biomarkers in the saliva of IBD patients showed elevated levels of many inflammatory cytokines and immunoglobulin A, and a lower lysozyme level. A strong correlation was shown between lysozyme and IL-1β levels and the relative abundance of Streptococcus, Prevotella, Haemophilus and Veillonella. Our data demonstrate that dysbiosis of salivary microbiota is associated with inflammatory responses in IBD patients, suggesting that it is possibly linked to dysbiosis of their gut microbiota.

pp60c-src activation in lung adenocarcinoma

Nine src family members are known including c-Src, c-Yes, c-Lck, c-Fyn, c-Hck, c-Lyn, c-Blk, c-Fgr and c-Yrk. They encode proteins with molecular weights of 55-62 kilodaltons (kDa), which are either cytoplasmic or membrane-associated protein tyrosine kinases. A close correlation exists between an elevated pp60c-src tyrosine kinase activity and cell transformation. However, the level of activation of pp60c-src in non-small cell lung cancers (NSCLC) remains obscure. The aim of this study was to examine the level of activity of pp60c-src in NSCLC. pp60c-src expression and in vitro protein tyrosine kinase activity in lung cancer tissue samples were measured by western blotting and in vitro kinase assays and compared with those in the surrounding non-tumour lung tissue from the same patient. pp60c-src phosphorylation was assessed by two-dimensional tryptic phosphopeptide mapping. The kinase activity of pp60c-src was significantly activated in NSCLC, especially in adenocarcinomas. In addition, the pp60c-src kinase activity increased with the size of the adenocarcinoma. Two-dimensional tryptic phosphopeptide mapping showed dephosphorylation of pp60c-src at Tyr 530 in adenocarcinomas. The proto-oncogene product, pp60c-src, was activated in NSCLC, especially in adenocarcinomas, in part through the dephosphorylation of Tyr 530. Our results suggest that activation of pp60c-src might play an important role in the progression of lung adenocarcinomas.

Deoxynucleic Acids from Cryptococcus neoformans Activate Myeloid Dendritic Cells via a TLR9-Dependent Pathway

The mechanism of host cell recognition of Cryptococcus neoformans, an opportunistic fungal pathogen in immunocompromised patients, remains poorly understood. In the present study, we asked whether the DNA of this yeast activates mouse bone marrow-derived myeloid dendritic cells (BM-DCs). BM-DCs released IL-12p40 and expressed CD40 upon stimulation with cryptococcal DNA, and the response was abolished by treatment with DNase, but not with RNase. IL-12p40 production and CD40 expression were attenuated by chloroquine, bafilomycin A, and inhibitory oligodeoxynucleotides (ODN) that suppressed the responses caused by CpG-ODN. Activation of BM-DCs by cryptococcal DNA was almost completely abrogated in TLR9 gene-disrupted (TLR9−/−) mice and MyD88−/− mice, similar to that by CpG-ODN. In addition, upon stimulation with whole yeast cells of acapsular C. neoformans, TLR9−/− BM-DCs produced a lower amount of IL-12p40 than those from wild-type mice, and TLR9−/− mice were more susceptible to pulmonary infection with this fungal pathogen than wild-type mice, as shown by increased number of live colonies in lungs. Treatment of cryptococcal DNA with methylase resulted in reduced IL-12p40 synthesis by BM-DCs. Furthermore, using a luciferase reporter assay, cryptococcal DNA activated NF-κB in HEK293 cells transfected with the TLR9 gene. Finally, confocal microscopy showed colocalization of fluorescence-labeled cryptococcal DNA with CpG-ODN and the findings merged in part with the distribution of TLR9 in BM-DCs. Our results demonstrate that cryptococcal DNA causes activation of BM-DCs in a TLR9-dependent manner and suggest that the CpG motif-containing DNA may contribute to the development of inflammatory responses after infection with C. neoformans.

Sequential changes of KL-6 in sera of patients with interstitial pneumonia associated with polymyositis/dermatomyositis

OBJECTIVE KL-6 is a mucin-like high molecular weight glycoprotein, which is strongly expressed on type II alveolar pneumocytes and bronchiolar epithelial cells. It has been demonstrated that the KL-6 antigen is a useful marker for estimating the activity of interstitial pneumonia. In this study, it is hypothesised that serum KL-6 is a useful marker to evaluate the activity of interstitial pneumonia associated with polymyositis/dermatomyositis (PM/DM). METHODS KL-6 was measured in sera in 16 patients diagnosed with PM/DM. Five had non-specific interstitial pneumonia (NSIP), three had diffuse alveolar damage (DAD), and eight had no pulmonary involvement, and 10 were normal non-smokers as a control group. The correlation was also evaluated between the KL-6 level and each clinical course in patients with pulmonary involvement associated with PM/DM. Immunohistochemical analysis using monoclonal anti-KL-6 antibody was also performed. RESULTS KL-6 concentrations in sera of patients with interstitial pneumonia associated with PM/DM were significantly high compared with those of PM/DM without interstitial pneumonia, and normal non-smokers. KL-6 concentrations in sera in patients with DAD significantly increased compared with those of other groups. KL-6 values in sera changed according to the progression or improvement of interstitial pneumonia. Immunohistochemical study using pulmonary tissues obtained from patients with DAD demonstrated that the hyaline membrane, proliferating type II pneumocytes, bronchial epithelial cells and some endothelial cells in pulmonary veins were stained by antihuman KL-6 antibody. CONCLUSION These data demonstrate that measurement of serum KL-6 was a useful marker to evaluate the activity of acute interstitial pneumonia associated with PM/DM.

Dectin‐1 Is Not Required for the Host Defense to Cryptococcus neoformans

Dectin‐1 is known as a sole receptor for β‐glucan, a major cell wall component of fungal microorganisms. In the current study, we examined the role of this molecule in the host defense to Cryptococcus neoformans, an opportunistic fungal pathogen in AIDS patients. There was no significant difference in the clinical course and cytokine production between dectin‐1 gene‐deficient and control mice. These results indicate that dectin‐1 is not likely essential for the development of host protective responses to C. neoformans.

Nationwide surveillance of bacterial respiratory pathogens conducted by the Japanese Society of Chemotherapy in 2007: general view of the pathogens' antibacterial susceptibility.

For the purpose of nationwide surveillance of the antimicrobial susceptibility of bacterial respiratory pathogens collected from patients in Japan, the Japanese Society of Chemotherapy conducted a third year of nationwide surveillance during the period from January to April 2008. A total of 1,097 strains were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections. Susceptibility testing was evaluable with 987 strains (189 Staphylococcus aureus, 211 Streptococcus pneumoniae, 6 Streptococcus pyogenes, 187 Haemophilus influenzae, 106 Moraxella catarrhalis, 126 Klebsiella pneumoniae, and 162 Pseudomonas aeruginosa). A total of 44 antibacterial agents, including 26 β-lactams (four penicillins, three penicillins in combination with β-lactamase inhibitors, four oral cephems, eight parenteral cephems, one monobactam, five carbapenems, and one penem), three aminoglycosides, four macrolides (including a ketolide), one lincosamide, one tetracycline, two glycopeptides, six fluoroquinolones, and one oxazolidinone were used for the study. Analysis was conducted at the central reference laboratory according to the method recommended by the Clinical and Laboratory Standard Institute (CLSI). The incidence of methicillin-resistant S. aureus (MRSA) was as high as 59.8%, and those of penicillin-intermediate and penicillin-resistant S. pneumoniae (PISP and PRSP) were 35.5 and 11.8%, respectively. Among H. influenzae, 13.9% of them were found to be β-lactamase-non-producing ampicillin (ABPC)-intermediately resistant (BLNAI), 26.7% to be β-lactamase-non-producing ABPC-resistant (BLNAR), and 5.3% to be β-lactamase-producing ABPC-resistant (BLPAR) strains. A high frequency (76.5%) of β-lactamase-producing strains was suspected in Moraxella catarrhalis isolates. Four (3.2%) extended-spectrum β-lactamase-producing K. pneumoniae were found among 126 strains. Four isolates (2.5%) of P.aeruginosa were found to be metallo β-lactamase-producing strains, including three (1.9%) suspected multidrug-resistant strains showing resistance to imipenem, amikacin, and ciprofloxacin. Continual national surveillance of the antimicrobial susceptibility of respiratory pathogens is crucial in order to monitor changing patterns of susceptibility and to be able to update treatment recommendations on a regular basis.

The role of caspase 3 in producing cytokeratin 19 fragment (CYFRA21-1) in human lung cancer cell lines

The CYFRA 21‐1 assay, which detects cytokeratin 19 (CK19) fragment, is widely used as a tumor marker for lung cancer, particularly non‐small cell lung cancer. However, the reason that some lung cancer cell lines release CYFRA 21‐1 in culture supernatants and others do not remains unclear. We hypothesized that the release of CYFRA 21‐1 might be related to the expression of CK19 and caspase 3. In order to prove this, the quantities of mRNA for CK19 were evaluated by the competitive reverse transcriptase‐polymerase chain reaction (RT‐PCR). CK19 protein synthesis was also evaluated by Western blotting and immunohistochemistry, and the levels of CYFRA 21‐1 in the culture supernatant were measured by an immunoradiometric assay. The expression of mRNA for caspase 3 was evaluated by the RT‐PCR, and caspase 3 protein synthesis was also evaluated by immunohistochemistry. In 13 lung cancer cell lines, the amounts of mRNA for CK19 correlated with the levels of CYFRA 21‐1 in culture supernatants, results of Western blotting for CK19, and positivities of immunohistochemistry for CK19. In 5 cell lines that produced a significant amount of CYFRA 21‐1, the level of CYFRA 21‐1 correlated with the positivity of RT‐PCR for caspase 3 and immunohistochmistry for caspase 3. This suggests that caspase 3 played a role in the formation of CYFRA 21‐1. In addition, the specific inhibitor of caspase 3 significantly inhibited the release of CYFRA 21‐1 in culture supernatants. In conclusion, we demonstrate that caspase 3, which cleaves several intermediate filaments and carries out cell apoptosis, played an important role in producing CYFRA 21‐1 in human lung cancer cell lines.

Histologic and immunohistochemical analyses of α-fetoprotein--producing cancer of the stomach.

Background and Aim:As the histogenesis and development of &agr;-fetoprotein–producing gastric cancer (AFPGC) have not yet been elucidated, we analyzed the histologic and immunologic relationship between the histologic type of the mucosal lesion considered to be the primary lesion, and that of its invasive lesion, in 36 cases of AFPGC. Patients and Methods:We reviewed 23 AFPGCs with mucosal lesions (1 mucosal and 22 submucosal or deeper invasive tumors) among 36 AFPGCs that had been resected endoscopically or surgically between 1970 and 2005. AFPGC was defined as a tumor showing immunohistochemical positivity for either &agr;-fetoprotein (AFP) or glypican-3. Histologic types were divided into hepatoid (HPT), enteroblastic (ENT), yolk sac tumor, and common (COM) adenocarcinoma type. The tumor phenotypes were classified into gastric, gastrointestinal, and intestinal types on the basis of immunohistochemical analysis. Results:Among the histologic types of mucosal lesions, the COM and ENT mixed type was observed in 65.2% of cases (15/23 patients), COM alone in 26.1% (6/23), and ENT alone in 8.7% (2/23) of cases. Among the invasive lesions, 16 cases (72.7%) were HPT. Both AFP and glypican-3 were positive in 60.9% (14/23) of mucosal lesions and in 90.9% (20/22) of invasive lesions. With regard to phenotypic expression, 82.6% (19/23) of mucosal lesions were the intestinal type, compared with 95.5% (21/22) of invasive lesions. Conclusions:These findings suggest that many cases of AFPGC develop as COM or ENT in the mucosa, which differentiate into ENT and HPT during the process of tumor invasion and proliferation, acquiring AFP production ability.