Kazuhiro Yatera

Particulate matter exposure induces persistent lung inflammation and endothelial dysfunction

Epidemiologic and animal studies have shown that exposure to particulate matter air pollution (PM) is a risk factor for the development of atherosclerosis. Whether PM-induced lung and systemic inflammation is involved in this process is not clear. We hypothesized that PM exposure causes lung and systemic inflammation, which in turn leads to vascular endothelial dysfunction, a key step in the initiation and progression of atherosclerosis. New Zealand White rabbits were exposed for 5 days (acute, total dose 8 mg) and 4 wk (chronic, total dose 16 mg) to either PM smaller than 10 mum (PM(10)) or saline intratracheally. Lung inflammation was quantified by morphometry; systemic inflammation was assessed by white blood cell and platelet counts and serum interleukin (IL)-6, nitric oxide, and endothelin levels. Endothelial dysfunction was assessed by vascular response to acetylcholine (ACh) and sodium nitroprusside (SNP). PM(10) exposure increased lung macrophages (P<0.02), macrophages containing particles (P<0.001), and activated macrophages (P<0.006). PM(10) increased serum IL-6 levels in the first 2 wk of exposure (P<0.05) but not in weeks 3 or 4. PM(10) exposure reduced ACh-related relaxation of the carotid artery with both acute and chronic exposure, with no effect on SNP-induced vasodilatation. Serum IL-6 levels correlated with macrophages containing particles (P=0.043) and ACh-induced vasodilatation (P=0.014 at week 1, P=0.021 at week 2). Exposure to PM(10) caused lung and systemic inflammation that were both associated with vascular endothelial dysfunction. This suggests that PM-induced lung and systemic inflammatory responses contribute to the adverse vascular events associated with exposure to air pollution.

Significance of Anaerobes and Oral Bacteria in Community-Acquired Pneumonia

Background Molecular biological modalities with better detection rates have been applied to identify the bacteria causing infectious diseases. Approximately 10–48% of bacterial pathogens causing community-acquired pneumonia are not identified using conventional cultivation methods. This study evaluated the bacteriological causes of community-acquired pneumonia using a cultivation-independent clone library analysis of the 16S ribosomal RNA gene of bronchoalveolar lavage specimens, and compared the results with those of conventional cultivation methods. Methods Patients with community-acquired pneumonia were enrolled based on their clinical and radiological findings. Bronchoalveolar lavage specimens were collected from pulmonary pathological lesions using bronchoscopy and evaluated by both a culture-independent molecular method and conventional cultivation methods. For the culture-independent molecular method, approximately 600 base pairs of 16S ribosomal RNA genes were amplified using polymerase chain reaction with universal primers, followed by the construction of clone libraries. The nucleotide sequences of 96 clones randomly chosen for each specimen were determined, and bacterial homology was searched. Conventional cultivation methods, including anaerobic cultures, were also performed using the same specimens. Results In addition to known common pathogens of community-acquired pneumonia [Streptococcus pneumoniae (18.8%), Haemophilus influenzae (18.8%), Mycoplasma pneumoniae (17.2%)], molecular analysis of specimens from 64 patients with community-acquired pneumonia showed relatively higher rates of anaerobes (15.6%) and oral bacteria (15.6%) than previous reports. Conclusion Our findings suggest that anaerobes and oral bacteria are more frequently detected in patients with community-acquired pneumonia than previously believed. It is possible that these bacteria may play more important roles in community-acquired pneumonia.

A Higher Significance of Anaerobes: The Clone Library Analysis of Bacterial Pleurisy

BACKGROUND The frequencies of etiologic bacterial agents of intrapleural infections reported until now have been widely varied, largely depending on the implemented detective methods. The aims of this study were to evaluate bacterial etiologies of bacterial pleurisy using a cultivation-independent method. METHODS Pleural fluids were collected from 42 febrile patients with hemipleural effusion. The bacterial flora was analyzed by a clone library method using amplified fragments of the 16S ribosomal RNA gene (rDNA) with universal primers in addition to conventional cultivation methods. RESULTS Forty-two specimens were obtained from 26 patients with bacterial pleurisy, seven with mycobacterial pleurisy, and nine with other pleural effusions. In the 26 bacterial cases, 16 (61.5%) showed positive results for 16S rDNA sequencing analysis, of which 11 (42.3%) were also positive for cultivation method. In seven (43.8%) of the 16 polymerase chain reaction-positive cases, anaerobic phylotypes were predominantly detected. Anaerobic phylotypes (six of these seven cases) were not detected by cultivation method. In nine (34.6%) of the 26 bacterial pleural cases, the results from the clone library methods were not accordant with those of the cultivation method. In seven of these nine cases, the discrepancies between the two detection methods were due to the existence of anaerobes. CONCLUSION The clone library analysis using the 16S rDNA of pleural fluid showed a higher incidence of anaerobic bacteria in infectious pleurisy than that previously expected and provided additional bacterial information for cultivation methods.

High-resolution CT scoring system-based grading scale predicts the clinical outcomes in patients with idiopathic pulmonary fibrosis.

BackgroundThe 2011 idiopathic pulmonary fibrosis (IPF) guidelines are based on the diagnosis of IPF using only high-resolution computed tomography (HRCT). However, few studies have thus far reviewed the usefulness of the HRCT scoring system based on the grading scale provided in the guidelines. We retrospectively studied 98 patients with respect to assess the prognostic value of changes in HRCT findings using a new HRCT scoring system based on the grading scale published in the guidelines.MethodsConsecutive patients with IPF who were diagnosed using HRCT alone between January 2008 and January 2012 were evaluated. HRCT examinations and pulmonary function tests were performed at six-month intervals for the first year after diagnosis. The HRCT findings were evaluated using the new HRCT scoring system (HRCT fibrosis score) over time. The findings and survival rates were analyzed using a Kaplan-Meier analysis.ResultsThe HRCT fibrosis scores at six and 12 months after diagnosis were significantly increased compared to those observed at the initial diagnosis (p < 0.001). The patients with an elevated HRCT fibrosis score at six months based on a receiver operating characteristic (ROC) curves analysis had a poor prognosis (log-rank, hazard ratio [HR] 2.435, 95% CI 1.196-4.962; p = 0.0142). Furthermore, among the patients without marked changes in %FVC, those with an elevated score above the cut-off value had a poor prognosis (HR 2.192, 95% CI 1.003-4.791; p = 0.0491).ConclusionsOur data demonstrate that the HRCT scoring system based on the grading scale is useful for predicting the clinical outcomes of IPF and identifying patients with an adverse prognosis when used in combination with spirometry.

Original ResearchDisorders of the PleuraA Higher Significance of Anaerobes: The Clone Library Analysis of Bacterial Pleurisy

BACKGROUND The frequencies of etiologic bacterial agents of intrapleural infections reported until now have been widely varied, largely depending on the implemented detective methods. The aims of this study were to evaluate bacterial etiologies of bacterial pleurisy using a cultivation-independent method. METHODS Pleural fluids were collected from 42 febrile patients with hemipleural effusion. The bacterial flora was analyzed by a clone library method using amplified fragments of the 16S ribosomal RNA gene (rDNA) with universal primers in addition to conventional cultivation methods. RESULTS Forty-two specimens were obtained from 26 patients with bacterial pleurisy, seven with mycobacterial pleurisy, and nine with other pleural effusions. In the 26 bacterial cases, 16 (61.5%) showed positive results for 16S rDNA sequencing analysis, of which 11 (42.3%) were also positive for cultivation method. In seven (43.8%) of the 16 polymerase chain reaction-positive cases, anaerobic phylotypes were predominantly detected. Anaerobic phylotypes (six of these seven cases) were not detected by cultivation method. In nine (34.6%) of the 26 bacterial pleural cases, the results from the clone library methods were not accordant with those of the cultivation method. In seven of these nine cases, the discrepancies between the two detection methods were due to the existence of anaerobes. CONCLUSION The clone library analysis using the 16S rDNA of pleural fluid showed a higher incidence of anaerobic bacteria in infectious pleurisy than that previously expected and provided additional bacterial information for cultivation methods.

Comparison of pulmonary inflammatory responses following intratracheal instillation and inhalation of nanoparticles

Abstract In order to examine whether intratracheal instillation studies can be useful for determining the harmful effect of nanoparticles, we performed inhalation and intratracheal instillation studies using samples of the same nanoparticles. Nickel oxide nanoparticles (NiO) and titanium dioxide nanoparticles (TiO2) were used as chemicals with high and low toxicities, respectively. In the intratracheal instillation study, rats were exposed to 0.2 or 1 mg of NiO or TiO2. Cell analysis and chemokines in bronchoalveolar lavage fluid (BALF) were analyzed from 3 days to 6 months following the single intratracheal instillation. In the inhalation study, rats were exposed to inhaled NiO or TiO2 (1.65, 1.84 mg/m3, respectively) for 4 weeks. The same endpoints were examined from 3 days to 3 months after the end of exposure. Inhalation of NiO induced an increase in the number of neutrophils in BALF and concentrations of cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2 and heme oxygenase (HO)-1. Intratracheal instillation of NiO induced persistent inflammation and upregulation of these cytokines was observed in the rats. However, inhalation of TiO2 did not induce pulmonary inflammation, and intratracheal instillation of TiO2 transiently induced an increase in the number of neutrophils in BALF and the concentrations of CINC-1, CINC-2 and HO-1. Taken together, a difference in pulmonary inflammation was observed between the high and low toxicity nanomaterials in the intratracheal instillation studies, as in the inhalation studies, suggesting that intratracheal instillation studies may be useful for ranking the harmful effects of nanoparticles.

Severe Pneumonia with Leptotrichia sp. Detected Predominantly in Bronchoalveolar Lavage Fluid by Use of 16S rRNA Gene Sequencing Analysis

ABSTRACT We present the first case of severe pneumonia possibly caused by Leptotrichia species with oral bacteria. This was found in a healthy but elderly subject whose bronchoalveolar lavage fluid was analyzed by 16S rRNA gene sequencing analysis. The combination of this method and microscopic observation provided useful information for diagnosis and treatment.

Detection of MALT1 Gene Rearrangements in BAL Fluid Cells for the Diagnosis of Pulmonary Mucosa-Associated Lymphoid Tissue Lymphoma

BACKGROUND Mucosa-associated lymphoid tissue (MALT) lymphoma constitutes approximately 90% of primary pulmonary lymphoma, and the diagnosis of pulmonary MALT lymphoma often requires invasive methods such as surgical lung biopsy. Chromosomal rearrangements involving MALT lymphoma translocation gene 1 (MALT1) have been reported to be specific for MALT lymphoma. The combination of BAL and cytologic approaches with molecular methods is useful for the diagnosis of lymphoproliferative disorders. Therefore, we examined the detection of MALT1 gene rearrangements in BAL fluid (BALF) cells for the diagnosis of MALT lymphoma. METHODS We determined the percentage of BALF cells with MALT1 gene rearrangements by using the fluorescence in situ hybridization (FISH) method in 10 patients suspected to have pulmonary MALT lymphoma. RESULTS MALT1 gene rearrangements in BALF cells were found in four of five cases with pulmonary MALT lymphoma (percentage of BALF cells with MALT1 gene rearrangements: 21.8% ± 6.8%). On the other hand, MALT1 gene rearrangements in BALF cells were negative in the five cases without pulmonary MALT lymphoma and one case with pulmonary MALT lymphoma. CONCLUSION These results suggest that the detection of MALT1 gene rearrangements in BALF cells is useful for the diagnosis of pulmonary MALT lymphoma, as it is a specific method that is less invasive than surgical biopsy. Because of the small number of patients in this study, further investigations are necessary to evaluate the detection rate of MALT1 gene rearrangements in BALF cells from patients with pulmonary MALT lymphoma.

Assessment of Pathologically Diagnosed Patients with Castleman’s Disease associated with Diffuse Parenchymal Lung Involvement Using the Diagnostic Criteria for IgG4-Related Disease

BackgroundIgG4-related disease (IgG4RD) is a recently recognized disease entity. Differentiating IgG4RD from plasma cell type Castleman’s disease (PCD) is important but also difficult using only pathological findings. In addition, little is known about the association between these two diseases with diffuse parenchymal lung involvement.MethodsWe analyzed the serum IgG4 levels and the ratio of IgG4/IgG-positive plasmacytes in the lung and lymph node specimens of eight patients previously pathologically diagnosed of PCD with diffuse parenchymal lung involvement (DL-PCD). We also compared the clinical and laboratory findings observed in these patients.ResultsSix of the eight patients exhibited abundant IgG4-positive plasmacytes in the lung and lymph node tissues and elevated serum IgG4 levels, thereby fulfilling the diagnostic criteria of IgG4RD with DL (DL-IgG4RD) in addition to having obstructive phlebitis and massive lymphoplasmacytic infiltration with fibrosis. However, three of these six patients exhibited higher levels of serum interleukin-6 and were still diagnosed with DL-PCD. Accordingly, three of these eight patients were considered as IgG4RD with DL (DL-IgG4RD), and the other five patients were ultimately given a diagnosis of DL-PCD. These two diseases have different characteristics in terms of age, symptoms, serum levels of C-reactive protein, and IgA, complicating allergic disorders, response to corticosteroids, and prognosis.ConclusionsThis is the first report to show a high prevalence of DL-IgG4RD in DL-PCD patients, although additional large investigations are necessary. Clinical and laboratory findings are important for distinguishing between these two diseases in other organs, as previously described.

Calcitonin gene-related peptide stimulates proliferation of alveolar epithelial cells.

BackgroundAlveolar epithelial cells are known as progenitor cells for the restoration from the damage in the lung. Calcitonin gene-related peptide (CGRP) has been reported to play an important role in the proliferation of various types of epithelial and endothelial cells. We investigated the effects of CGRP on the proliferation of alveolar epithelial cells in vitro and in vivo.MethodsA549 cells were cultured in Dulbecco Modified Eagle Medium with 5% fatal bovin serum for 24 hours, then CGRP was added in vitro. The proliferation of DNA synthesis was measured using 5-bromo-2-deoxyuridine, an analog of thymidine, by enzyme-linked immunosorbent assay.As one intracellular response to CGRP, we examined activation of p44/42- extracellular signal-regulated kinase (ERK) pathway by adding CGRP, using western blotting method.Recombinant adenovirus encoding nuclear-targeted-human β-CGRP (rhCGRP) was administered into Male Wister rat (n = 5, 10 weeks old) lungs by intratracheal instillation in vivo. 7 days after the administration of CGRP, rat lungs were harvested and histological findings and immunohistochemical staining of proliferating cell nuclear antigen (PCNA) were evaluated to examine cell proliferation.ResultsIn vitro study, CGRP increased the proliferation of A549 cells in a dose and time dependent manner. CGRP8-37 (inhibitor of CGRP receptor) decreased CGRP induced proliferation of DNA synthesis. Phosphorylation of ERK pathway was observed within 15 minutes and peaked in one hour. U0126 (inhibitor of ERK pathway) decreased CGRP induced proliferation of DNA synthesis.In vivo study, histological examination of the lung indicated proliferation of alveolar epithelial cells in the rhCGRP-treated group and the nuclei of alveolar epithelial cells were positive for PCNA immunostaining.ConclusionIn this study, we conclude that CGRP stimulates proliferation of human alveolar epithelial cells in vivo and in vitro.