Molecular epidemiological and clinical infection characteristics analysis of Ralstonia.

PURPOSE: This study was to clarify the molecular epidemiology and clinical infection characteristics of Ralstonia pickettii and establish sequence typing system. METHODS: 48 nonrepetitive Ralstonia pickettii strains were collected from January 2008 to December 2013 at the Chinese People's Liberation Army General Hospital (PLAGH) and were identified through a specific PCR experiment, 16 S rDNA experiment and VITEK 2 system to compare the identification accuracy. The sequence types of the strains were analyzed by multilocus sequence typing (MLST) method. The antibiotic sensitivity of these strains was determined with disc diffusion tests and broth microdilution method. The clinical data of Ralstonia pickettii infected patients were collected. RESULTS: All of the 48 strains were identified as Ralstonia pickettii by VITEK 2 system. 30 and 34 strains were identified as Ralstonia pickettii by PCR and 16 S rDNA experiment respectively. ST9 was the most sequence types (STs) in these 18 STs of 42 strains. 42 strains were divided into 2 groups (A and B) and 18 genotypes. Ralstonia pickettii was sensitive to some cephalosporins, ß-lactam/ß-lactamase inhibitor, levofloxacin and trimethoprim/sulfamethoxazole. Cough, sputum, shortness of breath and pulmonary rales were the common clinical symptoms of most Ralstonia pickettii infected patients. CONCLUSION: We established a sequence typing system with a relatively fine resolution and the PCR assay is a faster and more sensitive method for clinical identification of Ralstonia pickettii. ST9 is the most common sequence types of Ralstonia pickettii. The most common clinical characteristics of Ralstonia pickettii infected patients were cough, sputum, shortness of breath and pulmonary rales.

Epitranscriptome analysis of NAD-capped RNA by spike-in-based normalization and prediction of chronological age.

Nicotinamide adenine dinucleotide (NAD) can be used as an initiating nucleotide in RNA transcription to produce NAD-capped RNA (NAD-RNA). RNA modification by NAD that links metabolite with expressed transcript is a poorly studied epitranscriptomic modification. Current NAD-RNA profiling methods involve multi-steps of chemo-enzymatic labeling and affinity-based enrichment, thus presenting a critical analytical challenge to remove unwanted variations, particularly batch effects. Here, we propose a computational framework, enONE, to remove unwanted variations. We demonstrate that designed spike-in RNA, together with modular normalization procedures and evaluation metrics, can mitigate technical noise, empowering quantitative and comparative assessment of NAD-RNA across different datasets. Using enONE and a human aging cohort, we reveal age-associated features of NAD-capping and further develop an accurate RNA-based aging clock that combines signatures from both transcriptome and NAD-modified epitranscriptome. enONE facilitates the discovery of NAD-RNA responsive to physiological changes, laying an important foundation for functional investigations into this modification.

Lactobacillus fermentum CECT5716 Alleviates the Inflammatory Response in Asthma by Regulating TLR2/TLR4 Expression.

Background: Asthma is a chronic disease, which is harmful to the health of the body and the quality of life. Supplementation of Lactobacillus can affect the immune environment of the lungs through the gut-lung axis. This study aimed to explore the potential regulatory targets of Lactobacillus to relieve inflammation in asthma and determine a new approach for improving asthma. Methods: A mouse ovalbumin (OVA)-induced model was constructed. OVA mice were supplemented with Lactobacillus fermentum CECT5716 by gavage. The gut microbiota composition of normal and OVA mice was analyzed using 16S ribosomal DNA identification. BALF, serum, lung tissues, and duodenal tissues were collected. Wright's staining was performed to determine the cell content of the alveolar lavage fluid. Hematoxylin-eosin staining, Masson staining, and periodic acid-Schiff staining were performed to observe the improvement in the lungs of OVA mice supplemented with Lactobacillus. Immunofluorescence was performed to measure the severity of the intestinal barrier leakage. Enzyme-linked immunosorbent assay was carried out to determine the expression levels of inflammatory cell factors, while quantitative reverse transcription-polymerase chain reaction and western blotting were performed to detect the levels of toll-like receptor 2 (TLR2)/TLR4 expression and cell adhesion factors. Results: Compared with Control mice, OVA mice exhibited malignant conditions, such as intestinal leakage and lung edema. After supplementation with Lactobacillus, the inflammatory cell content in the bronchoalveolar lavage fluid decreased, and the inflammatory response was alleviated. The level of TLR2/TLR4 expression was reduced. The inflammatory cell infiltration in the airway mucosa of OVA mice was improved, alveolar swelling was reduced and the basement membrane appeared thinner. Conclusion: The Lactobacillus inhibited the TLR2/TLR4 expression in OVA mice. Supplementation with Lactobacillus can alleviate the inflammatory response in OVA mice, inhibit pulmonary fibrosis, and treat asthma.

The Pathogenesis of Eosinophilic Asthma: A Positive Feedback Mechanism That Promotes Th2 Immune Response via Filaggrin Deficiency.

Eosinophilic asthma (EA) is a common subtype of asthma and often progresses to severe disease. In order to understand its pathogenesis, targeted next-generation gene sequencing was performed on 77 Chinese EA patients and 431 Chinese healthy controls to obtain differential genomic variations. Among the 41 Single Nucleotide Polymorphisms (SNPs) screened for mutation sites in more than 3 patients, filaggrin gene FLG rs192116923 T>G and FLG rs75235053 C>G were newly found to be associated with EA patients with atopic dermatitis (AD) (P <0.001) and severe EA (P=0.032), respectively. Filaggrin has been shown to be mainly expressed in epithelial cells and plays an important role in formation of an effective skin barrier. Bioinformatic analysis indicated FLG rs192116923 T>G may increase the binding of Smad3 to transmit TGF-ß1 signaling, and thereby inhibit filaggrin expression, and FLG rs75235053 C>G may add new splicing sites to reduce filaggrin monomers. It has been known that the level of Th2 cytokine IL-4 is increased in EA patients, and IL-4 increases airway epithelial permeability and enhances inflammatory response through some unclear mechanisms. To figure out whether filaggrin is involved in immune responses in asthma, we have treated human respiratory epithelial cell line BEAS-2B cells with IL-4 and found that the expression levels of filaggrin and E-cadherin decreased significantly in a time and dose-dependent manner, suggesting that IL-4 increased airway epithelial permeability by reducing filaggrin and adhesion molecule. In addition, in our study, IL-4 increased the expression of epithel-derived inflammatory cytokines IL-33 and TSLP which further enhanced the Th2 inflammatory response. To investigate the role of filaggrin in development of EA, knockdown filaggrin with siRNA revealed a decrease in E-cadherin levels, which were further down-regulated by IL-4 stimulation. Knockdown of filaggrin alone did not affect the levels of IL-33 and TSLP, but further exacerbated the decrease of IL-33/TSLP caused by IL-4, suggesting that filaggrin may involve in IL-4R signaling pathway to regulate the level of IL-33/TSLP. In conclusion, in the Th2 cytokine milieu of asthma, FLG deficient mutation in airway epithelial cells may increase the epithelial permeability and the expression of IL-33/TSLP which positively feedback the Th2 inflammation response.

Clinical characteristics and biomarkers analysis of asthma inflammatory phenotypes.

Aim: Asthma inflammatory phenotypes facilitate treatment, their clinical characteristics and biomarkers were analyzed. Materials & methods: A total of 176 asthmatics were divided into eosinophilic asthma (EA), neutrophilic asthma (NA), paucigranulocytic asthma and mixed-granulocytic asthma by induced sputum. Results: EA, NA and paucigranulocytic asthma patients were 65 (36.9%), 31 (17.6%) and 75 (42.6%). Sputum IL-5 and IL-13, blood IL-13, fractional exhaled nitric oxide (FeNO) were related to EA, the areas under the receiver operating characteristic curve were 0.790, 0.846, 0.828, 0.830, combined FeNO with blood IL-13 was 0.872. Sputum and blood IL-8, blood IL-17 were related to NA, their area under the receiver operating characteristic curve was 0.939, 0.844, 0.821, combined blood IL-8 with IL-17 was 0.882. Conclusion: Blood IL-13 and FeNO, blood IL-8 and IL-17 were alternative biomarkers of EA and NA, respectively.

Analysis of lncRNA Expression in Patients With Eosinophilic and Neutrophilic Asthma Focusing on LNC_000127.

Long non-coding RNA (lncRNA) is important in many diseases. Some studies have shown that lncRNA affects the pathogenesis of systemic inflammation of asthma. lncRNA regulates gene transcription, protein expression, and epigenetic regulation. However, lncRNAs associated with different airway phenotypes, such as eosinophilic (Eos) and neutrophilic (Neu) asthma have not been identified. The goal of this study was to determine the differences in circulating lncRNA signatures in Eos and Neu samples. Using RNA-sequencing (RNA-seq), lncRNA expression was evaluated in peripheral whole blood samples among Eos patients, Neu patients, and healthy individuals (Control). Bioinformatic analysis was used to predict relevant biological pathways. Quantitative PCR (qPCR) was used to measure gene expression in whole blood samples, Jurkat cells, and human CD4+ T cells. Finally, a novel lncRNA, LNC_000127, was inhibited by transfection of Jurkat cells with a lentiviral vector, and the effect was examined by Human Asthma RT2 Profiler™ PCR Array and western blotting. Compared to control samples, Eos samples contained 190 unique lncRNAs and Neu samples had 166 unique lncRNAs (difference ≥2-fold). KEGG pathway annotation data and GO terms revealed that different lncRNAs are involved in different mechanisms. LNC_000127, was highly expressed in Eos samples before treatment; its expression was increased in Jurkat cells and human CD4+ T cells following stimulation with PMA/CD28. Subsequent analyses revealed that LNC_000127 functions in the Th2 inflammation pathway. The results suggest that lncRNAs are involved in different phenotypes of asthma. Whether the different phenotypes of asthma can be recognized based on these lncRNAs (as biomarkers) requires further analysis. Targeting LNC_000127 may be effective for reducing Th2 inflammation in Eos asthma.

[Survey of macrolide resistance in Mycoplasma pneumoniae in adult patients with community-acquired pneumonia in Beijing, China].

OBJECTIVE: To explore the tendency of macrolide resistance in Mycoplasma pneumoniae infection in community-acquired pneumonia (CAP) patients in Beijing. METHODS: Adult CAP patients of ≥ 18 yrs were enrolled in 3 medical centers in Beijing , China. Throat swab samples were taken from all the patients to perform the culture of M. pneumoniae . All the isolated M. pneumoniae strains were subjected to susceptibility evaluation for 6 agents, including macrolides such as erythromycin and azithromycin. In strains showing macrolide resistance, the 23S rRNA gene was analyzed. RESULTS: A total 53 strains of M. pneumoniae were isolated from 321 enrolled patients. Thirty-eight of the isolated strains (71.7%) were resistant to erythromycin and 32 of them (60.4%) were resistant to azithromycin. Six strains with moderate or low level of erythromycin-resistance were still susceptible to azithromycin. No fluoroquinolone-resistant or tetracycline-resistant strains were observed in our study. Point transition of A2063G in the 23S ribosomal RNA gene was the main reason for the high prevalence of macrolide resistance. CONCLUSIONS: The prevalence of macrolide resistance in M. pneumoniae is very high in adult CAP patients in Beijing. Studies are needed to clarify the clinical meaning of prevalence of macrolide-resistant M. pneumoniae in adults CAP patients.

[A clinical analysis of 7 cases of allergic bronchopulmonary aspergillosis].

OBJECTIVE: To study the clinical features, diagnosis and treatment of allergic bronchopulmonary aspergillosis (ABPA). METHODS: The clinical presentations, serologic results, chest radiology, pathological results and treatment of 7 patients with ABPA in Chinese PLA General Hospital were retrospectively analyzed. RESULTS: There were 4 males and 3 females, with a mean age of (33 ± 16) years. Before the diagnosis of ABPA, 6 cases had been misdiagnosed as bronchial asthma, 3 as pulmonary infection, 2 as tuberculosis and 1 as bronchiectasis. The main clinical manifestations included cough (n = 6), sputum production (n = 5), hemoptysis (n = 4), wheeze (n = 3), dyspnea(n = 3) and fever(n = 2). All cases had increased total serum IgE levels (median 3040 U/ml) and peripheral blood eosinophil count (median 0.19). Six of them showed increased peripheral eosinophil count median 1.84 × 10(9)/L, and skin test positive for Aspergillus antigen. Five of them had increased serum IgE antibodies specific to A. fumigatus (22 ± 15) kU/L, and 4 had increased serum IgG antibodies specific to A. fumigatus (108 ± 96) mg/L. The chest CT scan findings included transient or fixed pulmonary opacities, central bronchiectasis and finger-in-glove opacities. Five patients were treated with corticosteroids combined with antifungal therapy. CONCLUSIONS: Clinical features of ABPA include a history of asthma, elevation of the total serum IgE levels, presence of aspergillus IgE antibodies, peripheral eosinophilia, and transient or fixed pulmonary opacities and central bronchiectasis. Patients with asthma complicated with bronchiectasis should be routinely screened for Aspergillus skin test, and measurement of total serum IgE levels and chest CT scan are useful for confirmation of the diagnosis of ABPA. Oral glucocorticoids and anti-fungal drugs are effective in treatment of ABPA. Regular follow-up is needed for prevention of recurrence.