Yuanlin Xi

Serum inflammatory cytokine levels correlate with Hand-Foot-Mouth disease severity: A nested serial case-control study

Background Hand-food-mouth disease (HFMD) cases can be fatal. These cases develop rapidly, and it is important to predict the severity of HFMD from mild to fatal and to identify risk factors for mild HFMD. The objective of this study was to correlate the levels of serum inflammatory cytokines with HFMD severity. Methods This study was designed as a nested serial case-control study. The data collected included general information, clinical symptoms and signs, laboratory findings and serum cytokine levels. Results The levels of IL-4, IL-6, IL-10, TNF-α and IFN-γ in patients with severe HFMD were significantly higher than in mild patients during the 2nd to 5th day after disease onset. The levels of IL-4, IL-6, IL-10 and IFN-γ increased from the 2nd day to the 4th day and later decreased. The levels of TNF-α were high on the first two days and subsequently decreased. The changes of IL-10, TNF-α and IFN-γ in the controls were similar for all cases. The levels of IL-4, IL-6 and IL-17 in the controls were not significantly different with the progression of HFMD. Conclusions Our findings indicate that the IL-4, IL-6, IL-10, TNF-α and IFN-γ levels correlate with HFMD severity.

A Higher Correlation of HCV Core Antigen with CD4+ T Cell Counts Compared with HCV RNA in HCV/HIV-1 Coinfected Patients

Development of HCV infection is typically followed by chronic hepatitis C (CHC) in most patients, while spontaneous HCV viral clearance (SVC) occurs in only a minority of subjects. Compared with the widespread application of HCV RNA testing by quantitative RT-PCR technique, HCV core antigen detection may be an alternative indicator in the diagnosis of hepatitis C virus infections and in monitoring the status of infectious individuals. However, the correlation and differences between these two indicators in HCV infection need more investigation, especially in patients coinfected by HIV-1. In this study, a total of 354 anti-HCV and/or anti-HIV serum positive residents from a village of central China were enrolled. Besides HCV-related hepatopathic variables including clinical status, ALT, AST, anti-HCV Abs, as well as the altered CD4+/CD8+ T cell counts, HCV core antigen and HCV viral load were also measured. The concentration of serum HCV core antigen was highly correlated with level of HCV RNA in CHC patients with or without HIV-1 coinfection. Of note, HCV core antigen concentration was negatively correlated with CD4+ T cell count, while no correlation was found between HCV RNA level and CD4+ T cell count. Our findings suggested that quantitative detection of plasma HCV core antigen may be an alternative indicator of HCV RNA qPCR assay when evaluating the association between HCV replication and host immune status in HCV/HIV-1 coinfected patients.

Detection and Analysis of CRISPRs of Shigella

The recently discovered CRISPRs (Clustered regularly interspaced short palindromic repeats) and Cas (CRISPR-associated) proteins are a novel genetic barrier that limits horizontal gene transfer in prokaryotes and the CRISPR loci provide a historical view of the exposure of prokaryotes to a variety of foreign genetic elements. The aim of study was to investigate the occurrence and distribution of the CRISPRs in Shigella. A collection of 61 strains of Shigella were screened for the existence of CRISPRs. Three CRISPR loci were identified among 61 shigella strains. CRISPR1/cas loci are detected in 49 strains of shigella. Yet, IS elements were detected in cas gene in some strains. In the remaining 12 Shigella flexneri strains, the CRISPR1/cas locus is deleted and only a cas3’ pseudo gene and a repeat sequence are present. The presence of CRISPR2 is frequently accompanied by the emergence of CRISPR1. CRISPR3 loci were present in almost all strains (52/61). The length of CRISPR arrays varied from 1 to 9 spacers. Sequence analysis of the CRISPR arrays revealed that few spacers had matches in the GenBank databases. However, one spacer in CRISPR3 loci matches the cognate cas3 genes and no cas gene was present around CRISPR3 region. Analysis of CRISPR sequences show that CRISPR have little change which makes CRISPR poor genotyping markers. The present study is the first attempt to determine and analyze CRISPRs of shigella isolated from clinical patients.

Pulmonary edema following central nervous system lesions induced by a non- mouse-adapted EV71 strain in neonatal BALB/c mice

BackgroundEnterovirus (EV) infection has been a serious health issue in Asia-Pacific region. It has been indicated that the occurrence of fatal hand foot and mouth disease (HFMD) cases following EV71 infection is mainly attributed to pulmonary edema. However, the development of pulmonary disorders after EV71 infection remains largely unknown. To establish an EV71-infected animal model and further explore the underlying association of central nervous system (CNS) invasion with pulmonary edema, we isolated a clinical source EV71 strain (ZZ1350) from a severe case in Henan Province.MethodsWe evaluated the cytotoxicity of ZZ1350 strain and the susceptibility in 3-day-old BALB/c mice with intraperitoneal, intracerebral and intramuscular inoculation. Various histopathological and immunohistochemical techniques were applied to determine the target organs or tissue damage after infection. Correlation analysis was used to identify the relationship between CNS injury and pulmonary disorders.ResultsOur experimental results suggested that ZZ1350 (C4 subtype) had high cytotoxicity against African green monkey kidney (Vero) cells and human rhabdomyosarcoma (RD) cells and neonatal BALB/c mice were highly susceptible to the infection with ZZ1350 through three different inoculation routes (2 × 106 pfu/mouse) exhibiting severe neurological and respiratory symptoms that were similar to clinical observation. Viral replication was found in brain, spinal cord, skeletal muscle, lung, spleen, liver, heart of infected mice and these sections also showed histopathological changes. We found that brain histology score was positive correlated with lung histology score in total experimental mice and mice under the three inoculation routes (P < 0.05). At the same time, there were positive correlations between spinal cord score and lung score in total experimental mice and mice with intracerebral inoculation (P < 0.05).ConclusionsZZ1350 strain is effective to establish animal model of EV71 infection with severe neurological and respiratory symptoms. The development of pulmonary disorders after EV71 infection is associated with severity of CNS damage.

Bioinformatics analyses of Shigella CRISPR structure and spacer classification

Clustered regularly interspaced short palindromic repeats (CRISPR) are inheritable genetic elements of a variety of archaea and bacteria and indicative of the bacterial ecological adaptation, conferring acquired immunity against invading foreign nucleic acids. Shigella is an important pathogen for anthroponosis. This study aimed to analyze the features of Shigella CRISPR structure and classify the spacers through bioinformatics approach. Among 107 Shigella, 434 CRISPR structure loci were identified with two to seven loci in different strains. CRISPR-Q1, CRISPR-Q4 and CRISPR-Q5 were widely distributed in Shigella strains. Comparison of the first and last repeats of CRISPR1, CRISPR2 and CRISPR3 revealed several base variants and different stem-loop structures. A total of 259 cas genes were found among these 107 Shigella strains. The cas gene deletions were discovered in 88 strains. However, there is one strain that does not contain cas gene. Intact clusters of cas genes were found in 19 strains. From comprehensive analysis of sequence signature and BLAST and CRISPRTarget score, the 708 spacers were classified into three subtypes: Type I, Type II and Type III. Of them, Type I spacer referred to those linked with one gene segment, Type II spacer linked with two or more different gene segments, and Type III spacer undefined. This study examined the diversity of CRISPR/cas system in Shigella strains, demonstrated the main features of CRISPR structure and spacer classification, which provided critical information for elucidation of the mechanisms of spacer formation and exploration of the role the spacers play in the function of the CRISPR/cas system.

Production and delivery of Helicobacter pylori NapA in Lactococcus lactis and its protective efficacy and immune modulatory activity

Helicobacter pylori neutrophil-activating protein A subunit (NapA) has been identified as a virulence factor, a protective antigen and a potent immunomodulator. NapA shows unique application potentials for anti-H. pylori vaccines and treatment strategies of certain allergic diseases and carcinomas. However, appropriate production and utilization modes of NapA still remain uncertain to date. This work has established a novel efficient production and utilization mode of NapA by using L. lactis as an expression host and delivery vector, and demonstrated immune protective efficacy and immune modulatory activity of the engineered L. lactis by oral vaccination of mice. It was observed for the first time that H. pylori NapA promotes both polarized Th17 and Th1 responses, which may greatly affect the clinical application of NapA. This report offers a promising anti-H. pylori oral vaccine candidate and a potent mucosal immune modulatory agent. Meanwhile, it uncovers a way to produce and deliver the oral vaccine and immunomodulator by fermentation of food like milk, which might have striking effects on control of H. pylori infection, gastrointestinal cancers, and Th2 bias allergic diseases, including many food allergies.

An engineered food-grade Lactococcus lactis strain for production and delivery of heat-labile enterotoxin B subunit to mucosal sites

BackgroundRecent researches have been focusing on mucosal immune adjuvants, which play the key roles in mucosal immunization and have become the limitation for non-injected vaccine development. Escherichia coli heat-labile enterotoxin B subunit (LTB) was regarded as a promising mucosal adjuvant for its nontoxicity and potent activity. LTB preparation issues have always been recurring, in part owing to that the recombinant LTB expressed by E. coli does not act as its native form.ResultsWe constructed an engineered Lactococcus lactis strain using a food-grade expression system. The LTB secreted by the engineered strain was detected in the culture supernatant, constituting 10.3% of the supernatant proteins, and recognized by mouse anti-LTB antibodies. The engineered strain, co-administered orally to SPF BALB/c mice with a H. pylori vaccine candidate expressing Lpp20 antigen, could significantly enhance the Lpp20-induced mucosal SIgA antibody responses against H. pylori.ConclusionsThis is the first report that LTB was efficiently produced and delivered via using a food-grade lactococcal expression system, which offers a novel production and utilization mode of this crucial mucosal adjuvant. The engineered L. lactis strain secreting LTB has considerable potential for oral vaccine formulation owing to its outstanding safety, adjuvant activity and high-level production.

Involvement of the renin-angiotensin system in the progression of severe hand-foot-and-mouth disease

Background Hand-foot-and-mouth disease (HFMD) is generally considered as a mild exanthematous disease to infants and young children worldwide. HFMD cases are usually mild and self-limiting but for few cases leads to complicated severe clinical outcomes, and even death. Previous studies have indicated that serum Ang II levels in patients with H7N9 infection were related to the severity of infection. However, the mechanisms underlying the pathogenesis of severe HFMD remain unclear. This study was undertaken to clarify the role of the renin-angiotensin system (RAS) in the progression of severe HFMD. Methods In the present study, 162 children including HFMD patients and healthy controls were recruited. The data was analyzed by time-series fashion. Concentrations of angiotensin II (Ang II) and noradrenaline (NA) in serum of patients were measured with ELISA. We established a mouse model for enterovirus 71 (EV71) infection and determined concentrations of Ang II, NA in tissue lysates at 3, 5 and 7 days post infection (dpi). Results The concentrations of Ang II and NA in serum of the HFMD patients with mild or severe symptoms were significantly higher than that in healthy controls. Additionally, the concentrations of Ang II and NA in serum of severe cases were significantly higher than those mild cases and the increased concentrations of Ang II and NA showed the same time trend during the progression of HFMD in the severe cases. Furthermore, the concentrations of Ang II and NA in target organs of EV71-infected mice including brains, skeletal muscle, and lungs were increased with the progression of EV71 infection in mice. Histopathological alterations were observed in the brains, skeletal muscle and lungs of EV71-infected mice. Conclusion Our study suggested that activation of the RAS is implicated in the pathogenesis of severe HFMD.

CRISPR/Cas系统与志贺菌毒力和耐药的关系及插入序列IS600对 cse2 表达水平的影响

目的 了解志贺菌中成簇的规律间隔短回文重复序列（Clustered regularly interspaced short palindromic repeats，CRISPR）的分布及其与毒力和耐药的关系，并分析志贺菌中插入序列IS600对CRISPR相关蛋白基因cse2 mRNA表达水平的影响。 方法 利用课题组前期设计的引物PCR扩增志贺菌的3个CRISPR位点、CRISPR相关蛋白基因cse2、耐药基因和毒力基因；改良Kirby-Bauer （K-B）纸片法进行药敏试验；台盼蓝计数试验检测细菌毒力；Real-time PCR检测志贺菌中cse2基因mRNA表达水平。分别分析志贺菌中CRISPR/Cas系统与耐药基因、耐药表型、毒力基因、毒力表型的关系；了解IS600对CRISPR相关蛋白基因cse2 mRNA表达水平的影响。 结果 志贺菌中CRISPR1位点阴性细菌的毒力强；插入序列IS600使cse2 mRNA表达水平降低。 结论 志贺菌中存在CRISPR1、2、3位点；CRISPR1位点与毒力有关；插入序列IS600对cse2 mRNA表达水平有影响。