Leptospira interrogans infection leads to IL-1ß and IL-18 secretion from a human macrophage cell line through reactive oxygen species and cathepsin B mediated-NLRP3 inflammasome activation.

Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Although there is a large diversity of clinical signs and symptoms, a severe inflammatory response is common to all leptospirosis patients. The mechanism of IL-1ß secretion during Leptospira infection has been previously studied in mouse macrophages. However, the outcome of Leptospira infection is very different in human and murine macrophages, and the mechanisms responsible for IL-1ß secretion in human macrophages had not been investigated. This study therefore examines the effects of Leptospira interrogans infection on inflammasome activation and proinflammatory cytokine expression in human macrophages. Increased mRNA and protein expression of NLRP3 was observed by real time RT-PCR and flow cytometry at 1 h after co-cultivation. Enzyme-linked immunosorbent assay (ELISA) determination showed that IL-1ß and IL-18 are released in the culture supernatants at 1 h after cultivation. The inhibition assay showed that glybenclamide (a K+ efflux inhibitor that blocks NLRP3 inflammasome activation) and N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) and NLRP3 depletion with siRNAs reduced the levels of IL-1ß and IL-18 release. Moreover, the levels of IL-1ß and IL-18 production decreased in CA-074 (a cathepsin B inhibitor) and NAC (an anti-oxidant) pretreated human macrophages, compared to untreated controls. This study suggests that L. interrogans infection leads to reactive oxygen species (ROS)- and cathepsin B-dependent NLRP3 inflammasome activation, which subsequently mediates caspase-1 activation and IL-1ß and IL-18 release.

The role of reactive oxygen intermediates in the intracellular fate of Leptospira interrogans in the macrophages of different hosts.

BACKGROUND: Pathogenic species of Leptospira cause leptospirosis, a global zoonotic disease. Our previous work showed that leptospires survive and replicate in human macrophages but are killed in murine macrophages. However, the mechanism responsible for the different intracellular fates of leptospires within the macrophages of different hosts remains unclear. RESULTS: The present study demonstrates that infection with Leptospira interrogans caused significant up-regulation of reactive oxygen species (ROS) and superoxide in J774A.1 cells but did so to a lesser extent in THP-1 cells. The up-regulation of ROS and superoxide was significantly inhibited by the NADPH oxidase inhibitor apocynin. The damaged leptospires and remnants of leptospires within membrane-bound vacuoles were significantly inhibited by apocynin in J774A.1 cells but were less inhibited in THP-1 cells. In addition, apocynin significantly prevented damage to leptospires and the co-localization of L. interrogans with lysosomes in J774A.1 cells but did so to a lesser extent in THP-1 cells. Furthermore, the relative fluorescence intensity levels of intracellular leptospires and the viability of the intracellular leptospires increased in apocynin pretreated J774A.1 and THP-1 cells after 2 h of infection. CONCLUSIONS: The present study, based on our previous findings, further demonstrated that ROS contributed substantially to the bactericidal ability of mouse macrophages to kill intracellular leptospires. However, ROS did not contribute as much in human macrophages, which partially explains the different intracellular fates of L. interrogans in human and mouse macrophages.

Simultaneous virus identification and characterization of severe unexplained pneumonia cases using a metagenomics sequencing technique.

Many viruses can cause respiratory diseases in humans. Although great advances have been achieved in methods of diagnosis, it remains challenging to identify pathogens in unexplained pneumonia (UP) cases. In this study, we applied next-generation sequencing (NGS) technology and a metagenomic approach to detect and characterize respiratory viruses in UP cases from Guizhou Province, China. A total of 33 oropharyngeal swabs were obtained from hospitalized UP patients and subjected to NGS. An unbiased metagenomic analysis pipeline identified 13 virus species in 16 samples. Human rhinovirus C was the virus most frequently detected and was identified in seven samples. Human measles virus, adenovirus B 55 and coxsackievirus A10 were also identified. Metagenomic sequencing also provided virus genomic sequences, which enabled genotype characterization and phylogenetic analysis. For cases of multiple infection, metagenomic sequencing afforded information regarding the quantity of each virus in the sample, which could be used to evaluate each viruses' role in the disease. Our study highlights the potential of metagenomic sequencing for pathogen identification in UP cases.

Screening of pregnant women for hepatitis B virus surface antigen (HBsAg) and subsequent management, Qiandongnan prefecture, Guizhou, China, 2010.

BACKGROUND: In China, in 2010, a high proportion of pregnant women were tested for hepatitis B surface antigen (HBsAg). However, the preventive actions taken following screening were unclear. We followed up infants who were born to HBsAg positive mothers to describe the management that took place after screening. METHODS: We selected eight counties with a probability proportional to population size in the Qiandongnan prefecture, Guizhou province. In each county, we selected a hospital at random. In each hospital, we (a) reviewed records to estimate the proportion of pregnant women who had been screened for HBsAg in 2010 and (b) sampled 10 screened women at random to assess management after one year in 2011. We calculated proportions and confidence intervals (CI) using standard formulae. RESULTS: Among the 7232 women who delivered in 2010 in the 8 hospitals, 98% (95% CI: 97%-99%) had been tested for HBsAg. Among 82 HBsAg women sampled for follow-up, 45 (55%; 95% CI: 44%-65%) knew they had been tested during pregnancy and 60 (73%; 95% CI: 63%-82%) knew they were HBsAg positive. The 82 infants had received three doses of hepatitis B vaccines and 79 (96%; 95% CI: 90%-99%) had received the first dose within 24h. However, only 11 infants (13%; 95% CI: 9%-25%) had received HBIG in addition to hepatitis B vaccine and 16 (20%; 95% CI: 12%-29%) had been tested for HBsAg upon completion of the vaccine series as part of their routine management. CONCLUSIONS: HBsAg testing of pregnant women was common in Qiandongnan, Guizhou, but post-screening management was limited. There is a need to ensure continuity of care through engaging women in HBsAg testing and following up infants with comprehensive management, including immunoprophylaxis and serological testing.

Association of bone morphogenetic protein 4 gene polymorphisms with nonsyndromic cleft lip with or without cleft palate in Chinese children.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is one of the most common congenital anomalies in humans. The pathogenesis of nsCL/P involves both genetic and environmental factors. On the basis of linkage data suggesting that 14q21-24 is one of the chromosomal regions that affects nsCL/P and data locating the BMP4 gene to 14q22-23, we performed a case-control study to evaluate whether BMP4 538T/C polymorphism, resulting in an amino acid change of Val/Ala (V152A) in the polypeptide, is associated with nsCL/P in a Chinese children population. Genotypes of 184 patients with nsCL/P and 205 controls were detected using a PCR-RFLP strategy. The results showed significant differences in the genotype and allele distribution of 538T/C polymorphisms of the BMP4 gene among the cases and controls. The 538C allele carriers were associated with a significantly increased risk of nsCL/P as compared with the noncarriers (odds ratio = 1.52; 95% confidence interval, 1.13-2.03; p = 0.005). Hence, our results support the hypothesis that this polymorphism contributes to risk of nsCL/P, which suggests that BMP4 538T/C polymorphisms could be used as genetic susceptibility markers of nsCL/P.