[Progranulin attenuated asthma inflammation by inhibiting the expression of IL-6].

Objective: To investigate the role and mechanism of progranulin (PGRN) in asthma. Methods: Control group and model group were set up in wild and IL-6 knockout (IL-6 ko) mice, respectively. For asthma model, mice were intraperitoneally sensitized with 100 µg OVA on days 0 and 7, followed by aerosol challenges with 5% OVA for 30 min per day from day 14 to 21, and mice were sacrificed 24 h after the last challenge. The mice in control group were treated in the same way with PBS. Bronchoalveolar lavage fluid (BALF) was collected for leukocytes count and differential count. The pathological changes of lung tissues were observed by H&E staining. The cytokines in lung homogenate, serum and BALF were detected by Q-PCR and ELISA. The in vitro model of asthma was induced by stimulating A549 or BEAS-2B cells with IL-13. Each group was replicated in three wells and four groups were designed: PBS group, IL-13 treatment group, IL-13 + rhPGRN treatment group, inhibitors of p38 phosphorylation (SB203508) treatment group. The cells or supernatant were collected after 0~48 h. PGRN and IL-6 levels were determined by Q-PCR and ELISA, the level of p38 phosphorylation was tested by Western blot (WB). Results: Compared with control group, PGRN levels were decreased in lung homogenate and BALF (P＜0.05), and PGRN presented a downtrend in serum, however, the level of IL-6 in BALF was increased in asthma mice (P＜0.01). In IL-6 ko asthma mice, compared with the wild asthma mice, leukocytes, especially neutrophils in BALF were decreased (P＜0.05), but PGRN was increased (P＜0.05), lung pathological damage was significantly alleviated. In vitro experiments, compared with PBS group, PGRN level was decreased (P＜0.05), IL-6 level was increased (P＜0.01), phosphorylation of p38 was activated in IL-13 treatment group. Compared with IL-13 treatment group, in IL-13 + PGRN treatment group, IL-6 level was decreased (P＜0.05); phosphorylation of p38 was inhibited (P＜0.05); and the production of IL-6 (P＜0.05) was decreased after treatment with inhibitor of p38 phosphorylation. Conclusion: PGRN inhibited the production of IL-6 by suppressing the p38 phosphorylation to alleviate asthmatic airway inflammation.

Molecular epidemiology of pneumococcal isolates from children in China.

OBJECTIVES: To investigate the molecular epidemiology of pneumococcal isolates in Chongqing, China. METHODS: In this cross-sectional study, 51 invasive Streptococcus pneumoniae (S. pneumoniae) strains were from children with invasive pneumococcal disease (IPD) and 32 carriage strains from healthy children from January 2010 to December 2013 at the Children's Hospital of Chongqing Medical University, Chongqing, China. Multilocus sequence typing was used to identify the sequence types (STs). Capsular serotypes were determined by multiplex polymerase chain reaction. Drug susceptibility and resistance was determined by minimum inhibitory concentrations. RESULTS: In this study, 11 serotypes were identified among the 83 S. pneumoniae clinical isolates tested. Prevalent serotypes were 19A (20.4%), 6A/B (20.4%), 19F (15.7%), 14 (14.5%), and 23F (10.8%). Serotype 19F was the most frequent carriage strain, and serotype 19A was the most frequent invasive strain. The ST983 was the most prevalent ST for carriage strains, and ST320 was the most prevalent ST for invasive strains. For gene analysis, psaA (99.5%) and piaA (98.6%) were present and much conserved in all pneumococci tested. The cps2A and pcsB genes were more frequent in invasive isolates than carriage strains. Antimicrobial resistance rates of invasive pneumococcal isolates to erythromycin, penicillin, meropenem, cefotaxime, and clindamycin were higher than the carriage isolates from children. CONCLUSION: Our epidemiological evidence shows that 19A, 6A/B, 19F, 14, and 23F remain the most prevalent serotypes, which can be targeted by PCV13. Genotypes and drug resistance varied between carriage and invasive strains. The PsaA and PiaA may be good protein vaccine candidates.

In vivo characterization of Streptococcus pneumoniae genes involved in the pathogenesis of meningitis by differential fluorescence induction.

OBJECTIVE: To further understand the pathogenesis of pneumococcal meningitis, and provide some target candidates for the development of drugs. METHODS: This study was performed at the Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine (Ministry of Education), Chongqing Medical University, Chongqing, China from March 2006 to December 2007. A promoter-trap library of Streptococcus pneumoniae TIGR4, reported by green fluorescent protein was constructed, and used to infect BALB/c mice (n=15) intranasally, to set up a meningitis model. The control group (n=5) were inoculated with sterile phosphate buffered saline. The bacteria containing the promoter fusions induced only in meningitis brain tissue, not in vitro were screened by differential fluorescence induction. The obtained bacteria were prepared to re-infect the mice and re-screened, as above. The sorted bacteria were spread on trypticase soy agar with 5% sheep blood agar plates containing chloramphenicol (2.5 g/mL), and were used for DNA cloning, sequencing, and bioinformatics analysis. RESULTS: A total of 52 genes were obtained. Bioinformatics analysis revealed that these in vivo induced genes were involved in functions such as, adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism, as well as, cell wall synthesis. In addition, there were some genes encoding for some hypothetical proteins with unknown, or putative functions. CONCLUSION: Pneumococcal genes involved in meningitis identified in this study are potential targets to understand the pathogenesis of pneumococcal meningitis.

Contribution of ClpE to virulence of Streptococcus pneumoniae.

The ATP-dependent caseinolytic proteases (Clp) play a fundamental role in stress tolerance and virulence in many pathogenic bacteria. Although ClpE of Streptococcus pneumoniae is required for growth at high temperatures, little is known about the role of ClpE in pathogenesis. In this study, we observed that the virulence of the clpE mutant of S. pneumoniae strain D39 was strongly reduced in a mouse intraperitoneal infection model. The clpE mutant also showed substantially reduced adherence to the human lung epithelial carcinoma A549 cell line and human umbilical-vein-derived endothelial cells. The underlying mechanism of virulence attenuation induced by the mutation of clpE was further investigated with real-time RT-PCR and 2-dimensional protein gel analysis. The results indicate that ClpE affects pneumococcal pathogenesis by modulating the expression of some important virulence determinants and metabolism-related factors in S. pneumoniae.

[Effect of ClpE depletion on the bacterial protein expression profiles of Streptococcus pneumoniae].

OBJECTIVE: To investigate the effect of ClpE on the protein expression profiles of Streptococcus pneumoniae. METHODS: clpE-deficient Streptococcus pneumoniae strain was constructed by long flanking homology-polymerase chain reaction (LFH-PCR) and identified by PCR and sequencing. The total bacterial proteins were analyzed by two-dimensional gel electrophoresis and imaging analysis, and the differentially expressed protein spots were excised by dot-gel digestion with trypsin. Peptide mass fingerprinting (PMF) was obtained by analysis of the fragment length by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The PMF was analyzed using software to identify the proteins. RESULTS: The number of matched protein spots of the two gels was 61%. By sequence database searching, 4 out of the 17 differential protein spots were identified, namely hypoxanthine-guanine, pyrrolidone-carboxylate peptidase1, formate-tetrahydrofolate ligase, and bifunctional protein pyrR. CONCLUSION: clpE gene-deficient Streptococcus pneumoniae expresses fewer kinds of proteins at also lower levels than the wild-type bacteria, suggesting that ClpE allows the bacteria to adapt to different host environments by inducing the expression of special proteins.