12-OH-17,18-Epoxyeicosatetraenoic acid alleviates eosinophilic airway inflammation in murine lungs.

BACKGROUND: Asthma is characterized by airway inflammation and obstruction with eosinophil infiltration into the airway. Arachidonic acid, an omega-6 fatty acid, is metabolized into cysteinyl leukotriene with pro-inflammatory properties for allergic inflammation, whereas the omega-3 fatty acid eicosapentaenoic acid (EPA) and its downstream metabolites are known to have anti-inflammatory effects. In this study, we investigated the mechanism underlying the counter-regulatory roles of EPA in inflamed lungs. METHODS: Male C57BL6 mice were sensitized and challenged by ovalbumin (OVA). After EPA treatment, we evaluated the cell count of Bronchoalveolar lavage fluid (BALF), mRNA expressions in the lungs by q-PCR, and the amounts of lipid mediators by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics. We investigated the effect of the metabolite of EPA by in vivo and in vitro studies. RESULTS: Eicosapentaenoic acid treatment reduced the accumulation of eosinophils in the airway and decreased mRNA expression of selected inflammatory mediators in the lung. Lipidomics clarified the metabolomic profile in the lungs. Among EPA-derived metabolites, 12-hydroxy-17,18-epoxyeicosatetraenoic acid (12-OH-17,18-EpETE) was identified as one of the major biosynthesized molecules; the production of this molecule was amplified by EPA administration and allergic inflammation. Intravenous administration of 12-OH-17,18-EpETE attenuated airway eosinophilic inflammation through downregulation of C-C chemokine motif 11 (CCL11) mRNA expression in the lungs. In vitro, this molecule also inhibited the release of CCL11 from human airway epithelial cells stimulated with interleukin-4. CONCLUSION: These results demonstrated that EPA alleviated airway eosinophilic inflammation through its conversion into bioactive metabolites. Additionally, our results suggest that 12-OH-17,18-EpETE is a potential therapeutic target for the management of asthma.

Annexin V decreases PS-mediated macrophage efferocytosis and deteriorates elastase-induced pulmonary emphysema in mice.

Efferocytosis is believed to be a key regulator for lung inflammation in chronic obstructive pulmonary disease. In this study we pharmacologically inhibited efferocytosis with annexin V and attempted to determine its impact on the progression of pulmonary emphysema in mouse. We first demonstrated in vitro and in vivo efferocytosis experiments using annexin V, an inhibitor for phosphatidylserine-mediated efferocytosis. We then inhibited efferocytosis in porcine pancreatic elastase (PPE)-treated mice. PPE-treated mice were instilled annexin V intranasally starting from day 8 until day 20. Mean linear intercept (Lm) was measured, and cell apoptosis was assessed in lung specimen obtained on day 21. Cell profile, apoptosis, and mRNA expression of matrix metalloproteinases (MMPs) and growth factors were evaluated in bronchoalveolar lavage (BAL) cells on day 15. Annexin V attenuated macrophage efferocytosis both in vitro and in vivo. PPE-treated mice had a significant higher Lm, and annexin V further increased that by 32%. More number of macrophages was found in BAL fluid in this group. Interestingly, cell apoptosis was not increased by annexin V treatment both in lung specimens and BAL fluid, but macrophages from mice treated with both PPE and annexin V expressed higher MMP-2 mRNA levels and had a trend for higher MMP-12 mRNA expression. mRNA expression of keratinocyte growth factor tended to be downregulated. We showed that inhibited efferocytosis with annexin V worsened elastase-induced pulmonary emphysema in mice, which was, at least partly, attributed to a lack of phenotypic change in macrophages toward anti-inflammatory one.

Relationship between improved airflow limitation and changes in airway calibre induced by inhaled anticholinergic agents in COPD.

BACKGROUND: Although airflow limitation improved by inhaled anticholinergic drugs varies among individuals with chronic obstructive pulmonary disease (COPD), the relationship between actual bronchodilation and improved pulmonary function and where in the lung such bronchodilation occurs remains unknown. A study was undertaken to determine the relationship between improved pulmonary function and changes in airway calibre at various sites in the airways in response to inhaled anticholinergic agents in patients with COPD using three-dimensional computed tomography (CT). METHODS: CT scans were performed at deep inspiration and detailed pulmonary function tests before and 1 week after daily inhalations of tiotropium bromide in 15 patients with clinically stable COPD. The airway luminal area was examined at the third (segmental) to the sixth generations of eight bronchi in the right lung. RESULTS: Bronchodilation was demonstrated by an overall average increase of 39% in the inner luminal area, and the mean (SE) forced expiratory volume in 1 s (FEV(1)) increased from 1.23 (0.11) l to 1.47 (0.13) l. The magnitude of bronchodilation was closely correlated with improved pulmonary function, particularly with that of FEV(1) (r = 0.843, p<0.001). Such correlations were significant at the fourth to the sixth generation but not at the third generation of bronchi, and the slope of the regression lines became steeper from the third to the sixth generation. CONCLUSIONS: Inhaled anticholinergic agents induce overall bronchodilation which is in proportion to improvements in FEV(1) in patients with COPD. Bronchodilation at the distal rather than the proximal airways is the determinant of functional improvement.

First report of hepatobiliary Mycobacterium avium infection developing obstructive jaundice in a patient with neutralizing anti-interferon-gamma autoantibodies.

This study describes a patient who experienced hepatobiliary Mycobacterium avium infection associated with neutralizing anti-interferon gamma (IFN-γ) autoantibodies during treatment for disseminated M. avium disease. Hepatobiliary M. avium infection should be considered in jaundiced patients with neutralizing anti-IFN-γ autoantibodies, including those receiving antimycobacterial therapy for disseminated M. avium disease.

Functional single nucleotide polymorphisms of the CCL5 gene and nonemphysematous phenotype in COPD patients.

It was previously reported that the gain-of-function -28 guanine allele of the promoter single nucleotide polymorphism (SNP; cytosine to guanine substitution of nucleotide -28 (-28C>G)) in the CC chemokine ligand 5 gene (CCL5) was associated with susceptibility to late-onset asthma in patients who developed asthma at age > or =40 yrs. The clinical diagnosis of chronic obstructive pulmonary disease (COPD) includes emphysema and small airway disease, and upregulation of CCL5 has been described in the airways of patients with COPD. It was hypothesised that CCL5 has a genetic impact upon the variable expression of emphysema in patients with COPD. Patients with COPD were studied (n = 267). All of the patients underwent pulmonary high-resolution computed tomography (CT), and visual scoring (CT score) was performed to determine emphysema severity. Three SNPs of CCL5 were genotyped, including -403G>A, -28C>G and 375T>C. A significant difference was found in CT score according to CCL5 genotype; the -28G allele was inversely associated with CT score. When the analysis was confined to 180 patients with bronchial reversibility of <15%, even stronger evidence for this association was noted. Functional single nucleotide polymorphisms in the CC chemokine ligand 5 gene were associated with milder emphysema. Together with previous findings, the present study may identify the CC chemokine ligand 5 gene as part of a common pathway in the pathogenesis of late-onset asthma and chronic obstructive pulmonary disease with milder emphysema.

Health-related QOL of elderly patients with pulmonary M. avium complex disease in a university hospital.

BACKGROUND: Little is known about the clinical characteristics and health-related quality of life (HQOL) of elderly patients with pulmonary Mycobacterium avium complex (pMAC) disease. OBJECTIVES: To evaluate HQOL using the 36-Item Short-Form Health Survey and St George's Respiratory Questionnaire (SGRQ) and to investigate the predictors of HQOL changes among elderly patients with pMAC disease. METHODS: This prospective cohort registry was conducted at Keio University Hospital, Tokyo, Japan, between May 2012 and July 2015 and included 84 patients with pMAC disease aged 75 years who had completed the HQOL questionnaire and 48 patients with pMAC disease who had been followed up and completed the HQOL questionnaire in cross-sectional and longitudinal analyses, respectively. RESULTS: In cross-sectional analyses, elderly patients with pMAC disease had significantly lower role-physical, general health, vitality, social functioning, role-emotional and role/social component scores than the general Japanese elderly population. Analysis of covariance revealed that patients with cavitary lesions had significantly worse physical functioning and SGRQ scores (P < 0.05). Longitudinal analysis showed that under-treatment, short duration of disease and positive sputum smear at baseline were predictors of worse HQOL at 12 months. CONCLUSIONS: Elderly patients with pMAC disease have reduced HQOL. Further large studies on HQOL are required to refine the use of this parameter in the treatment of these patients.

A functional granulocyte colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is widely used to treat neutropenia. In addition to stimulating polymorphonuclear neutrophil (PMN) production, G-CSF may have significant effects on PMN function. Because G-CSF receptor (G-CSFR)-deficient mice do not have the expected neutrophilia after administration of human interleukin-8 (IL-8), we examined the effect of the loss of G-CSFR on IL-8-stimulated PMN function. Compared with wild-type PMNs, PMNs isolated from G-CSFR-deficient mice demonstrated markedly decreased chemotaxis to IL-8. PMN emigration into the skin of G-CSFR-deficient mice in response to IL-8 was also impaired. Significant chemotaxis defects were also seen in response to N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, or macrophage inflammatory protein-2. The defective chemotactic response to IL-8 does not appear to be due to impaired chemoattractant receptor function, as the number of IL-8 receptors and chemoattractant-induced calcium influx, actin polymerization, and release of gelatinase B were comparable to those of wild-type PMNs. Chemoattractant-induced adhesion of G-CSFR-deficient PMNs was significantly impaired, suggesting a defect in beta2-integrin activation. Collectively, these data demonstrate that selective defects in PMN activation are present in G-CSFR-deficient mice and indicate that G-CSF plays an important role in regulating PMN chemokine responsiveness.

Tolerance for ATP-insensitive K(ATP) channels in transgenic mice.

To examine the role of sarcolemmal K(ATP) channels in cardiac function, we generated transgenic mice expressing GFP-tagged Kir6.2 subunits with reduced ATP sensitivity under control of the cardiac alpha-myosin heavy chain promoter. Four founder mice were isolated, and both founders and progeny were all apparently normal and fertile. Electrocardiograms from conscious animals also appeared normal, although mean 24-hour heart rate was approximately 10% lower in transgenic animals compared with littermate controls. In excised membrane patches, K(ATP) channels were very insensitive to inhibitory ATP: mean K(1/2) ([ATP] causing half-maximal inhibition) was 2.7 mmol/L in high-expressing line 4 myocytes, compared with 51 micromol/L in littermate control myocytes. Counterintuitively, K(ATP) channel density was approximately 4-fold lower in transgenic membrane patches than in control. This reduction of total K(ATP) conductance was confirmed in whole-cell voltage-clamp conditions, in which K(ATP) was activated by metabolic inhibition. K(ATP) conductance was not obvious after break-in of either control or transgenic myocytes, and there was no action potential shortening in transgenic myocytes. In marked contrast to the effects of expression of similar transgenes in pancreatic beta-cells, these experiments demonstrate a profound tolerance for reduced ATP sensitivity of cardiac K(ATP) channels and highlight differential effects of channel activity in the electrical activity of the 2 tissues.

Topoisomerase II alpha content and topoisomerase II catalytic activity cannot explain drug sensitivities to topoisomerase II inhibitors in lung cancer cell lines.

PURPOSE: Topoisomerase II alpha content, topoisomerase II catalytic activity and drug sensitivities to the topoisomerase II inhibitors, doxorubicin and etoposide, were examined in a panel of 14 unselected human lung cancer cell lines in order to determine the relationship between topoisomerase II and drug sensitivities to the topoisomerase II inhibitors. METHODS: Drug sensitivities were determined using a microculture tetrazolium assay. The topoisomerase II alpha levels were determined by Western blot analysis and the topoisomerase II catalytic activity was determined using a decatenation assay of kinetoplast DNA, using nuclear protein from cells of each cell line. RESULTS: Drug sensitivity tests revealed that small-cell lung cancer (SCLC) cell lines were more sensitive to drugs than non-small-cell lung cancer (NSCLC) cell lines. The relative topoisomerase II alpha levels and relative topoisomerase II catalytic activity from SCLC cell lines (mean +/- SD 0.89 +/- 0.54 and 5.3 +/- 3.4, respectively) were slightly higher than those from NSCLC cell lines (0.78 +/- 0.56 and 4.0 +/- 2.8, respectively), but the differences were not statistically significant, and not sufficient to account for the variation in drug sensitivities. Moreover, no clear association was observed between the topoisomerase II alpha levels or the topoisomerase II catalytic activity and drug sensitivities in the cell lines studied. CONCLUSIONS: These findings suggest that the difference in drug sensitivities to doxorubicin and etoposide in human lung cancer cell lines might not be explainable by the topoisomerase II alpha levels and topoisomerase II catalytic activity. Moreover, our results suggest that the topoisomerase II alpha levels and topoisomerase II catalytic activity may play a minor role in the determination of clinical drug resistance of human lung cancers.

A secondary amyloidosis associated with systemic lupus erythematosus.

Secondary amyloidosis associated with systemic lupus erythematosus has rarely been reported. A 57-year-old female had been diagnosed as having possible systemic lupus erythematosus, although her clinical course was not typical. About one year after the diagnosis, treatment was begun with prednisolone because of progressive renal dysfunction, thrombocytopenia and low serum levels of complements. Recurrent diarrhea and gastrointestinal bleeding soon developed, then amyloidosis was revealed in the stomach and duodenum. Postmortem examination confirmed systemic amyloidosis. We discuss the significance of this rare association of systemic lupus erythematosus and secondary amyloidosis.

[A comparative study of computed tomographic techniques for the detection of emphysema in middle-aged and older patient populations].

Helical-scan computed tomography (CT) is now widely utilized as a mass screening procedure for lung cancer. By adding 3 slices of high-resolution CT (HRCT) to the standard screening procedure, we were able to compare the efficacy of helical-scan CT and HRCT in detecting pulmonary emphysema. Additionally, the prevalence of emphysema detected by HRCT was examined as a function of patient age and smoking history. The subjects (106 men and 28 women) were all community-based middle-aged and older volunteers who participated in a mass lung cancer screening program. Based on visual assessments of the CT films, emphysema was detected in 29 subjects (22%) by HRCT, but in only 4 (3%) by helical-scan CT. Although the prevalence of emphysema was higher among subjects with a higher smoking index, no correlations with age were observed. We concluded that the efficacy of helical scan CT in detecting pulmonary emphysema can be significantly improved with the inclusion of 3 slices of HRCT, and confirmed that cigarette smoking is linked to the development of pulmonary emphysema.

[A study of microtubules in hypertrophied cardiomyocytes].

Microtubules, components of the cytoskeleton, play an important role in the maintenance of cellular shape, in intracellular transport of organelles and membrane vesicles, and in signal transduction in the cell. Studies on the role of microtubules in hypertrophied myocardial cells have been carried out using an acute pressure overloading model in which stenosis was produced in the aorta. Clinically, however cardiac hypertrophy is most frequently caused by a gradual increase in blood pressure due to essential hypertension. We evaluated the role of microtubulas by observing their serial changes in cells from spontaneously hypertensive rats (SHR), which are used as a model of essential hypertension. Blood pressure, body weight, left ventricular weight, and the cell cross-sectional area were measured in SHR and Wistar-Kyoto (WKY) rats aged 4, 6, 10, and 16 weeks (5 rats each). Microtubules in cardiocytes were observed using confocal laser scanning microscopy by the immunofluorescence method against beta-tubulin. Microtubules in cardiocytes run primarily in a longitudinal direction, thinly through the intermyofibrillar spaces and densely around the nuclei. The numbers of microtubules were measured separately in the perinuclear region and the nonperinuclear region, and their total was calculated. The cross-sectional area of a whole cell and nucleus was measured at the level of the nucleus, and microtubule density was calculated by the number of nonperinuclear microtubules in the cytosolic area. Five myocardial cells were randomly selected in each rat, and the mean density (/micron2) was observed. For comparison, similar analysis was done in an acute pressure loading model. This study showed that: 1) In the acute pressure overload model, hypertrophy was immediately observed, and microtubules were increased in 16% of cardiocytes. In SHR, an increase in blood pressure and hypertrophy of hearts were observed at the age of 6 weeks and after. The density of microtubules at the age of 4, 6, 10, and 16 weeks was 0.69 +/- 0.03 (/micron2), 0.73 +/- 0.05 (/micron2), 0.64 +/- 0.02 (/micron2), respectively, in WKY rats, and 0.54 +/- 0.03 (/micron2), 0.54 +/- 0.02 (/micron2), and 0.51 +/- 0.02 (/micron2), respectively, in the SHR, showing a relatively stability in the latter. This suggested that a gradual increase of blood pressure could not be a stimulus that changes the density and distribution of microtubules. 2) The immunofluorescence method against beta-tubulin using confocal laser scanning microscopy seems to be good method for quantitative analysis of microtubules in cardiocytes.

[Studies on recurrent and metastatic cases of the early stomach cancer--from diagnostic aspect].

There were 256 cases of the early stomach cancer resected, from 1967 to '77 in National Hospital-Sapporo. First, 4 recurrent cases of these 256 cases (1.6%) are analyzed and concluded. 1) Common items of these recurrent cases were male and submucosal invasion in depth of cancer. 2) Modes of recurrence of the gastric remnant were shown in 3 of 4 recurrent cases. These 3 cases died within 6 months, on the average, from the diagnosis of recurrence. Therefore, earlier detection and operation of recurrent cancer is important. 3) There are some cases in which the possibility of multiple or double cancers exist, so careful examination before initial operation is most necessary. Second, the early stomach cancers with metastasis to the regional lymph nodes (n+ group) are analyzed. 1) There were 20 cases of n+ group (7.8%). 2) There were 12 cases of depressed type early cancers with metastasis to the regional nodes (depressed type n+ group), and 137 cases of the single depressed type early cancers without metastasis to the nodes (depressed type n- group). Statistical comparison between the depressed type n+ group and n- group disclosed that the maximum diameter of the lesion of the n+ group was longer than that of the n- group, and histological type of cancer of the n+ group was more undifferentiated than that of the n- group. Items of the depth of invasion and site of the lesion are not statistically significant at this time, however, close attention should be paid to these items.

[A case of vasovagal syncope associated with venipuncture].

A 33 year-old-man presented recurrent syncopal episodes after venipuncture. This was sometimes associated with seizure. It was also noted that the syncope was aroused by mental stress. The patient was admitted to the department of neurological medicine because of epilepsy. Cardiac arrest of more than 15 seconds was detected during the venipuncture-prooshed syncope test. He had normal routine testing, holter monitoring, head CT scan, carotid sinus massage, valsalva maneuver etc. The syncope was similar to a malignant vasovagal one which has no typical warning signs. He had recurrent syncopal episodes without typical prodrome. Therefore a DDD pacemaker was implanted. It has not been completely established as effective in the treatment of vasovagal syncope, but for the treatment of syncope involving cardioinhibitory action, dual chamber pacing in considered as the main treatment available.

Relationship between neutrophil influx and oxidative stress in alveolar space in lipopolysaccharide-induced lung injury.

We intratracheally administered lipopolysaccharide (LPS) to ICR mice and then collected BAL fluid and lung tissue to determine whether levels of neutrophils and/or myeloperoxidase (MPO) in bronchoalveolar lavage (BAL) fluid reflect lung tissue damage. Robust neutrophil accumulation into the alveolar space and lung tissue were almost completely abolished at seven days along with oxidative stress markers in the lung. However, lung injury scores and lung wet/dry ratios, as well as MPO and oxidative stress markers in BAL fluid were significantly increased at five and seven days after LPS administration. At later time points, BAL neutrophils generated more MPO activity and ROS than those harvested sooner after LPS administration. Although elevated neutrophil levels in BAL fluid reflected oxidative stress in the lungs, MPO might serve as a useful marker to evaluate damage sustained by epithelial cells over the long term.

Long term smoking with age builds up excessive oxidative stress in bronchoalveolar lavage fluid.

BACKGROUND: Epithelial lining fluid plays a critical role in protecting the lung from oxidative stress, in which the oxidised status may change by ageing, smoking history, and pulmonary emphysema. METHODS: Bronchoalveolar lavage (BAL) was performed on 109 young and older subjects with various smoking histories. The protein carbonyls, total and oxidised glutathione were examined in BAL fluid. RESULTS: By Western blot analysis, the major carbonylated protein in the BAL fluid was sized at 68 kDa, corresponding to albumin. The amount of carbonylated albumin per mg total albumin in BAL fluid was four times higher in older current smokers and three times higher in older former smokers than in age matched non-smokers (p<0.0001, p=0.0003, respectively), but not in young smokers. Total glutathione in BAL fluid was significantly increased both in young (p=0.006) and older current smokers (p=0.0003) compared with age matched non-smokers. In contrast, the ratio of oxidised to total glutathione was significantly raised (72%) only in older current smokers compared with the other groups. There was no significant difference in these parameters between older smokers with and without mild emphysema. CONCLUSIONS: Oxidised glutathione associated with excessive protein carbonylation accumulates in the lung of older smokers with long term smoking histories even in the absence of lung diseases, but they are not significantly enhanced in smokers with mild emphysema.