Intrinsic clarithromycin heteroresistance in Mycobacterium avium.

BACKGROUND: Mycobacterium avium is associated with pulmonary disease in otherwise healthy adults. Several clarithromycin-refractory cases have been reported, including some cases caused by clarithromycin-susceptible strains. OBJECTIVES: To characterize the reason for the discrepancy between clinical response and antibiotic susceptibility results. METHODS: We conducted population analysis of clarithromycin-tolerant and heteroresistant subpopulations of M. avium cultured in vitro and in homogenates of infected lungs of mice. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for 28 M. avium and two M. kansasii strains. Mice were intranasally infected with M. avium and treated with or without clarithromycin (100 mg/kg) thrice weekly. They were sacrificed on day 35 and the bacteria in lung homogenates were tested for clarithromycin resistance. Population analysis assays were performed based on colony growth on plates containing two-fold dilutions of clarithromycin. RESULTS: The MBC/MIC ratios were ≥8 in all 28 strains of M. avium tested. In the population analysis assay, several colonies were observed on the plates containing clarithromycin concentrations above the MIC (2-64 mg/L). No growth of M. kansasii colonies was observed on the plates containing clarithromycin concentrations ≥2 mg/L. M. avium in the homogenates of infected lungs showed clearer clarithromycin-resistant subpopulations than in vitro, regardless of clarithromycin exposure. CONCLUSION: M. avium shows intrinsic heterogeneous resistance (heteroresistance) to clarithromycin. This may explain the observed discrepancies between clarithromycin susceptibility testing results and clinical response to clarithromycin treatment. Further studies are needed to confirm a link between heteroresistance and clinical outcomes.

Metformin Mediates Protection against Legionella Pneumonia through Activation of AMPK and Mitochondrial Reactive Oxygen Species.

In Legionella pneumophila infection, macrophages play a critical role in the host defense response. Metformin, an oral drug for type 2 diabetes, is attracting attention as a new supportive therapy against a variety of diseases, such as cancer and infectious diseases. The novel mechanisms for metformin actions include modulation of the effector functions of macrophages and other host immune cells. In this study, we have examined the effects of metformin on L. pneumophila infection in vitro and in vivo. Metformin treatment suppressed growth of L. pneumophila in a time- and concentration-dependent fashion in bone marrow-derived macrophages, RAW cells (mouse), and U937 cells (human). Metformin induced phosphorylation of AMP-activated protein kinase (AMPK) in L. pneumophila-infected bone marrow-derived macrophages, and the AMPK inhibitor Compound C negated metformin-mediated growth suppression. Also, metformin induced mitochondrial reactive oxygen species but not phagosomal NADPH oxidase-derived reactive oxygen species. Metformin-mediated growth suppression was mitigated in the presence of the reactive oxygen species scavenger glutathione. In a murine L. pneumophila pneumonia model, metformin treatment improved survival of mice, which was associated with a significant reduction in bacterial number in the lung. Similar to in vitro observations, induction of AMPK phosphorylation and mitochondrial ROS was demonstrated in the infected lungs of mice treated with metformin. Finally, glutathione treatment abolished metformin effects on lung bacterial clearance. Collectively, these data suggest that metformin promotes mitochondrial ROS production and AMPK signaling and enhances the bactericidal activity of macrophages, which may contribute to improved survival in L. pneumophila pneumonia.

Overlapping Roles for Interleukin-36 Cytokines in Protective Host Defense against Murine Legionella pneumophila Pneumonia.

Legionella pneumophila causes life-threatening pneumonia culminating in acute lung injury. Innate and adaptive cytokines play an important role in host defense against L. pneumophila infection. Interleukin-36 (IL-36) cytokines are recently described members of the larger IL-1 cytokine family known to exert potent inflammatory effects. In this study, we elucidated the role for IL-36 cytokines in experimental pneumonia caused by L. pneumophila Intratracheal (i.t.) administration of L. pneumophila induced the upregulation of both IL-36α and IL-36γ mRNA and protein production in the lung. Compared to the findings for L. pneumophila-infected wild-type (WT) mice, the i.t. administration of L. pneumophila to IL-36 receptor-deficient (IL-36R-/-) mice resulted in increased mortality, a delay in lung bacterial clearance, increased L. pneumophila dissemination to extrapulmonary organs, and impaired glucose homeostasis. Impaired lung bacterial clearance in IL-36R-/- mice was associated with a significantly reduced accumulation of inflammatory cells and the decreased production of proinflammatory cytokines and chemokines. Ex vivo, reduced expression of costimulatory molecules and impaired M1 polarization were observed in alveolar macrophages isolated from infected IL-36R-/- mice compared to macrophages from WT mice. While L. pneumophila-induced mortality in IL-36α- or IL-36γ-deficient mice was not different from that in WT animals, antibody-mediated neutralization of IL-36γ in IL-36α-/- mice resulted in mortality similar to that observed in IL-36R-/- mice, indicating redundant and overlapping roles for these cytokines in experimental murine L. pneumophila pneumonia.

Interleukin-36γ and IL-36 receptor signaling mediate impaired host immunity and lung injury in cytotoxic Pseudomonas aeruginosa pulmonary infection: Role of prostaglandin E2.

Pseudomonas aeruginosa is a Gram-negative pathogen that can lead to severe infection associated with lung injury and high mortality. The interleukin (IL)-36 cytokines (IL-36α, IL-36ß and IL-36γ) are newly described IL-1 like family cytokines that promote inflammatory response via binding to the IL-36 receptor (IL-36R). Here we investigated the functional role of IL-36 cytokines in the modulating of innate immune response against P. aeruginosa pulmonary infection. The intratracheal administration of flagellated cytotoxic P. aeruginosa (ATCC 19660) upregulated IL-36α and IL-36γ, but not IL-36ß, in the lungs. IL-36α and IL-36γ were expressed in pulmonary macrophages (PMs) and alveolar epithelial cells in response to P. aeruginosa in vitro. Mortality after bacterial challenge in IL-36 receptor deficient (IL-36R-/-) mice and IL-36γ deficient (IL-36γ-/-) mice, but not IL-36α deficient mice, was significantly lower than that of wild type mice. Decreased mortality in IL-36R-/- mice and IL-36γ-/- mice was associated with reduction in bacterial burden in the alveolar space, bacterial dissemination, production of inflammatory cytokines and lung injury, without changes in lung leukocyte influx. Interestingly, IL-36γ enhanced the production of prostaglandin E2 (PGE2) during P. aeruginosa infection in vivo and in vitro. Treatment of PMs with recombinant IL-36γ resulted in impaired bacterial killing via PGE2 and its receptor; EP2. P. aeruginosa infected EP2 deficient mice or WT mice treated with a COX-2-specific inhibitor showed decreased bacterial burden and dissemination, but no change in lung injury. Finally, we observed an increase in IL-36γ, but not IL-36α, in the airspace and plasma of patients with P. aeruginosa-induced acute respiratory distress syndrome. Thus, IL-36γ and its receptor signal not only impaired bacterial clearance in a possible PGE2 dependent fashion but also mediated lung injury during P. aeruginosa infection.

Cerebral infarction in advanced non-small cell lung cancer: a case control study.

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) patients often develop thromboembolic events, including cerebral infarction (CI). However, the relationship between advanced NSCLC and CI has not been thoroughly investigated. We examined the association between advanced NSCLC and CI and risk factors for CI in advanced or post-operative recurrent NSCLC patients. METHODS: We retrospectively investigated 515 patients diagnosed with advanced or post-operative recurrent NSCLC at Juntendo University Hospital between April 2009 and March 2014. RESULTS: Among the 515 patients evaluated, 15 patients (2.9%) developed CI after diagnosis of advanced or post-operative recurrent NSCLC. Univariate and multivariate analyses were conducted, and brain metastasis was the only significant independent risk factor for CI (odds ratio 5.24, 95% confidence interval 1.72-16.10, p = 0.004). The incidence was 6.3% in these patients. The median survival time was 36 days, and 1-year survival rate was 6.7% after development of CI. Overall survival from diagnosis of advanced NSCLC or post-operative recurrence was significantly shorter in patients with CI than in patients without CI (223 days versus 895 days; HR, 3.46; 95% confidence interval, 2.04-6.02; p = 0.001). CONCLUSIONS: The incidence of CI is high in advanced or post-operative recurrent NSCLC, and is especially higher in patients with brain metastasis than in those without brain metastasis. Moreover, CI may affect patient's prognosis. Careful monitoring for the development of CI in patients with advanced or post-operative recurrent NSCLC is needed, especially for patients with brain metastasis.

Effects of slow-releasing colistin microspheres on endotoxin-induced sepsis.

Lipopolysaccharide (LPS) is a major contributing factor to endotoxic shock. Colistin specifically binds to LPS. However, it has the disadvantages that adverse reactions are common and it has a short half-life. To overcome these disadvantages, we prepared slow-releasing colistin microspheres and examined the efficacy of these colistin microspheres in a mouse model of endotoxin-induced sepsis. We prepared the colistin microspheres using poly-lactic-co-glycolic acid. For acute toxicity investigations, mice were overdosed with colistin sulfate or colistin microspheres. The group administered with colistin microspheres was associated with less acute toxicity and fewer nephrotoxic changes on histopathological examination compared to the group administered with colistin sulfate alone. For pharmacokinetic analysis, mice were subcutaneously administered with colistin microspheres or colistin sulfate alone. The plasma concentration of colistin was higher in the colistin microspheres group than in the colistin sulfate group at 12 and 24 h after administration. Moreover, mice were intraperitoneally injected with LPS and then immediately subcutaneously administered with blank microspheres, colistin microspheres or colistin sulfate alone. The levels of endotoxin in the sera and cytokine in the spleens were then measured. A significant reduction in the serum endotoxin level in the colistin microspheres group was observed at 24 h. The reduced endotoxin levels in the sera were correlated with the lower cytokine levels in the spleens of mice treated with colistin microspheres. Our results suggest that the use of colistin microspheres may help to maintain a higher colistin concentration in blood, reduce the levels of endotoxin and cytokines in endotoxin-induced sepsis, and lead to decreased toxicity.

ß2-adrenergic receptor haplotype may be associated with susceptibility to desensitization to long-acting ß2-agonists in COPD patients.

PURPOSE: Τhat ß2-adrenergic receptor (ß2AR) haplotypes may play a key role in clinical response to ß2-agonists and haplotype Cys-19Gly16Gln27 (CysGlyGln) is reported to be associated with desensitization of ß2AR to ß-agonists in lymphocytes isolated from patients with asthma and septic shock. We sought to determine whether haplotypic variation of the ß2AR affects the functional outcomes of long-acting ß2-agonist (LABA) treatment for chronic obstructive pulmonary disease (COPD) when used as monotherapy. METHODS: Treatment-naïve patients with COPD (n = 36) were prospectively treated with two kinds of LABA--inhaled salmeterol and transdermal tulobuterol patch--for 12 weeks in crossover study, and changes in pulmonary function data and 6-minute walk distance (6 MWD) were compared between groups stratified by the CysGlyGln. RESULTS: Frequencies of haplotype and diplotype for the CysGlyGln were 0.51 and 0.36, respectively. The individuals homozygous for CysGlyGln showed less improvement in FEV(1), %FEF(25-75 %), and IC/TLC than those with 0 or 1 copy of CysGlyGln after treatment with both LABAs despite initial bronchodilator responses to albuterol being similar in these groups. The response in these parameters was not significantly different between two types of LABA. Overall changes in 6 MWD in individuals with 2 copies of CysGlyGln versus 0 or 1 copy for salmeterol were 2.8 and 11 m, and for tulobuterol were -1.3 and 16 m, respectively. CONCLUSIONS: Homozygous haplotype for the CysGlyGln of ß2AR may be associated with susceptibility to desensitization to LABA in patients with COPD.

Multiple Types of Taste Disorders among Patients with COVID-19.

Objective Based on the increasing incidence of smell and taste dysfunction among coronavirus disease 2019 (COVID-19) patients, such issues have been considered an early symptom of infection. However, few studies have investigated the type of taste components that are most frequently affected in COVID-19 patients. This study investigated the difference in frequencies of the types of taste component disorders among hospitalized COVID-19 patients. Methods In this retrospective, single-center, observational study, patients' background characteristics, clinical course, laboratory and radiological findings, and details on taste and/or smell disorders were collected and analyzed from medical records. Patients A total of 227 COVID-19 patients were enrolled, among whom 92 (40.5%) complained of taste disorders. Results Multiple types of taste disorders (hypogeusia/ageusia and hypersensitivity, or hypersensitivity and changing tastes) were reported in 10 patients. In particular, 23 patients reported hypersensitivity to at least 1 type of taste, and 2 patients complained of a bitter taste on consuming sweet foods. Impairment of all taste components was found in 48 patients (52.2%). The most frequent taste disorder was salty taste disorder (81 patients, 89.0%). Hypersensitivity to salty taste was most frequently observed (19 patients, 20.9%). Conclusion Patients with COVID-19 develop multiple types of taste disorders, among which salty taste disorder was the most frequent, with many patients developing hypersensitivity to salty taste. As smell and taste are subjective senses, further studies with the combined use of objective examinations will be required to confirm the findings.

Patients with mild-to-moderate asthma may develop clinically significant chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: Little is known about the aetiology and clinical characteristics of COPD among people who have never smoked. METHODS: A case-control study was conducted to describe the potential risk factors for clinically significant COPD among smokers and people who had never smoked. Medical record reviews and scoring of high-resolution computed tomography (HRCT) findings were performed in patients with clinically significant COPD (defined as having post-bronchodilator FEV(1)/FVC < 0.7 and FEV(1) < 60% of the predicted values). Pathological analyses were performed in some patients following autopsy. RESULTS: Among the 9493 subjects screened, 424 (4.5%) were diagnosed with clinically significant COPD. Forty-nine (11.6% of clinically significant COPD) were never smokers (NSCOPD), and a comparative group of 98 smoking patients with COPD was randomly selected (SMCOPD). NSCOPD was characterized by predominantly female sex (87.8%) and a high prevalence of physician-diagnosed asthma (61.2%). Similar levels of reduction in %FEV(1) and %FEF(25-75%) were found in NSCOPD and SMCOPD, but there were higher %DL(CO) values and fewer low attenuation areas on HRCT in NSCOPD. More than half of the NSCOPD patients without a history of asthma had features of asthma. More than one-third of NSCOPD patients with an asthma history had never had a severe attack. At autopsy, both NSCOPD and SMCOPD showed wall thickening and inflammatory cell infiltration in small airways, and the number of CD4(+)-T cells was increased in NSCOPD. CONCLUSION: In elderly Japanese, COPD among never smokers is largely attributable to asthma. Airflow limitation in NSCOPD results from small airway disease (airway predominant phenotype) rather than parenchymal destruction (emphysematous phenotype).

Rapid, simple, and clinically applicable high-performance liquid chromatography method for clinical determination of plasma colistin concentrations.

BACKGROUND: Since both the antibacterial effects and common adverse effects of colistin are concentration-dependent, determination of the most appropriate dosage regimen and administration method for colistin therapy is essential to ensure its efficacy and safety. We aimed to establish a rapid and simple high-performance liquid chromatography (HPLC)-based system for the clinical determination of colistin serum concentrations. METHODS: Extraction using a solid-phase C18 cartridge, derivatisation with 9-fluorenylmethyl chloroformate, and elution with a short reversed-phase Cl8 column effectively separated colistin from an internal standard. The HPLC apparatus and conditions were as follows: analytical column, Hydrosphere C18; sample injection volume, 50 µL; column temperature, 40 °C; detector, Shimadzu RF-5300 fluorescence spectrophotometer (excitation wavelength, 260 nm; emission wavelength, 315 nm); mobile phase, acetonitrile/tetrahydrofuran/distilled water (50,14,20, v/v/v); flow-rate, 1.6 mL/min. RESULTS: The calibration curves obtained for colistin were linear in the concentration range of 0.10-8.0 µg/mL. The regression equation was y = 0.6496× - 0.0141 (r2 = 0.9999). The limit of detection was ~ 0.025 µg/mL, and the assay intra- and inter-day precisions were 0.87-3.74% and 1.97-6.17%, respectively. The analytical peaks of colistin A, colistin B, and the internal standard were resolved with adequate peak symmetries, and their retention times were approximately 8.2, 6.8, and 5.4 min, respectively. Furthermore, the assay was successfully applied to quantify the plasma colistin levels of a haemodialysis patient. CONCLUSION: The assay is a simple, rapid, accurate, selective, clinically applicable HPLC-based method for the quantification of colistin in human plasma.

Better adherence to a transdermal tulobuterol patch than inhaled salmeterol in elderly chronic obstructive pulmonary disease patients.

BACKGROUND: Inhaled long-acting ß2-agonists (LABA) are often poorly adhered to by elderly patients with chronic obstructive pulmonary disease (COPD). We hypothesized that older age and compromised cognitive function might contribute to poor adherence to inhaled medications among COPD patients, and that transdermally delivered medications could improve adherence, exercise tolerance and quality of life (QOL). OBJECTIVE: To compare adherence and effects on health outcomes between transdermal and inhaled LABA. METHODS: A total of 44 treatment-naïve, elderly Japanese patients with moderate-to-severe COPD were treated with a transdermal tulobuterol patch (TP; 2 mg, once a day) or inhaled salmeterol (50 µg, twice a day) in a randomized crossover manner. The primary outcomes were adherence to the LABA medications and changes in QOL measured by the St George's Respiratory Questionnaire. Secondary outcomes were changes in 6-min walk distance (6MWD) and spirometric values. RESULTS: The overall adherence rate was 90.3 ± 1.6% for TP and 75.5 ± 2.9% for salmeterol (P < 0.001). Adherence to salmeterol was correlated with age and Mini-Mental State Examination (MMSE) score (P < 0.05 and P < 0.01, respectively), although that to TP was relatively constant regardless of age and MMSE score. 6MWD and QOL were significantly improved from baseline after TP, but not after salmeterol treatment (P < 0.05). Similar degrees of increase in spirometric values occurred after treatment with TP and salmeterol. CONCLUSIONS: Adherence levels were higher overall with TP than with inhaled salmeterol, and more stable across age groups and MMSE levels. TP might be a favorable treatment option for COPD patients with poor adherence to an inhaled LABA.