Objective To construct a prokaryotic expression system for tlyA gene of Leptospira in-terrogans ( L.interrogans) strain and to investigate the effects of the expressed rTlyA protein on the hemolysis of sheep erythrocytes and the secretion of pro-inflammatory cytokines by human THP-1 cells and murine J774A.1 macrophages.Methods The fragment of tlyA gene of L.inetrrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was amplified by PCR.The PCR product was sequenced after T-A cloning.A prokary-otic expression system for the tlyA gene was constructed by using pET-42a as the expression vector and E.coli BL21DE3 strain as the host strain.The expression of rTlyA protein was detected by SDS-PAGE.Ni-NTA af-finity chromatography was performed for purification.The lytic activity of the rTlyA protein on sheep erythro-cytes was determined by plate hemolytic test and spectrophotometry.Real-time fluorescence quantitative PCR and Western blot assay were performed to respectively detect the expression of tlyA gene in THP-1 and J774A.1 cells at mRNA and protein levels after infection with L.interrogans strain Lai.ELISA was per-formed to evaluate the effects of the rTlyA protein on the secretion of several pro-inflammatory cytokines ( IL-β, IL-6 and TNF-α) by THP-1 and J774A.1 cells.Results The nucleotide and amino acid sequences of the cloned tlyA gene were 99.2% and 99.8% identical to those corresponding sequences in GenBank, re-spectively.The constructed prokaryotic expression system for tlyA gene successfully expressed the rTlyA pro-tein.The rTlyA protein at the concentration of 10μg/ml showed stronger lytic activity on sheep erythrocytes. The transcription levels of tlyA gene (P<0.05) and the secretion of TlyA protein in THP-1 and J774A.1 cells were significantly increased after infecting with L.interrogans strain Lai for 1 to 8 hours.The rTlyA pro-tein at concentrations of 0.1, 1 and 10 μg/ml significantly enhanced the secretion of IL-1β, IL-6 and TNF-αby THP-1 and J774A.1 cells (P<0.05).Conclusion The TlyA protein, encoded by the tlyA gene of L.interrogans strain, was confirmed to be a hemolysin with the ability to induce macrophages to secret IL-1β, IL-6 and TNF-α.This study suggested that the TlyA protein played an important role in inflammatory responses during leptospirosis.

Objective To investigate the prognostic value of CD 4+CD25+/high CD127 low/-and CD14+HLA-DRlow/-for evaluating the severity of acute pancreatitis .Methods The percentages of CD4+CD25+/highCD127low/-and CD14+HLA-DRlow/-and the CD64 index were measured by flow cytometry in pa-tients with acute pancreatitis ( including 43 cases of mild acute pancreatitis and 24 cases of severe acute pan-creatitis).Moreover, the levels of C-reactive protein (CRP), acute physiology and chronic health evaluationⅡ( APACHEⅡ) score and CT severity index ( CTSI ) were detected for a correlation analysis .Results The percentages of CD4+CD25+/highCD127low/-and CD14+HLA-DRlow/-and the CD64 index in patients with severe and mild acute pancreatitis were significantly higher than those in healthy subjects .Patients with se-vere acute pancreatitis showed higher percentages of CD 14+HLA-DRlow/-than patients with mild acute pan-creatitis.With the disease progression, the CD64 index and the levels of CD4+CD25+/highCD127low/-, CD14+HLA-DRlow/-and CRP were significantly dropped after an initial increase in patients with mild acute pancrea -titis, while these indexes were continuously elevated in patients with severe acute pancreatitis .The percent-age of CD14+HLA-DRlow/-was positively correlated with CD64 index, CRP level, APACHEⅡ score and CTSI.Conclusion CD14+HLA-DRlow/-level was closely related to the severity of acute pancreatitis , which could be used as immune parameter for the estimation of the clinical severity of acute pancreatitis .

Objective To investigate the prognostic value of regulatory B cells (Bregs) in patients with acute pancreatitis .Methods Flow cytometry analysis was performed to detected the percentages of CD19+IL-10+and CD19+CD24hiCD27hi Bregs in peripheral blood samples collected from patients with acute pancreatitis (36 cases with mild acute pancreatitis and 15 cases with severe acute pancreatitis ) as well as the surface costimulatory molecules including CD80 and CD86 on CD19+CD24hiCD27hi Bregs.Their correlations with lymphocytes and C-reactive protein ( CRP) were further analyzed .Results The numbers of lympho-cytes, CD19+lymphocytes, CD19+IL-10+and CD19+CD24hi CD27hi Bregs in peripheral blood samples col-lected from patients with severe and mild acute pancreatitis as well as the mean fluorescence intensities ( MFI) of CD80 and CD86 were significantly lower than those from healthy subjects .Compared with patients with mild acute pancreatitis , the numbers of lymphocytes and CD 19+lymphocytes , the absolute numbers of CD19+IL-10+and CD19+CD24hiCD27hi Bregs as well as the mean fluorescence intensities (MFI) of CD80 and CD86 in patients with severe acute pancreatitis were significantly decreased .The percentages of CD19+IL-10+and CD19+CD24hiCD27hi Bregs in patients with mild acute pancreatitis were significantly increased af-ter an initial drop , but in patients with severe acute pancreatitis those values were continuously decreased along with the disease progression .The percentage of CD19+IL-10+Bregs was positively correlated with the percentage of CD19+CD24hiCD27hi Bregs and the absolute number of CD19+lymphocytes, but was negatively correlated with CRP .Conclusion The abnormal number and function of CD 19+IL-10+and CD19+CD24 hi CD27hi Bregs might be one of the important reasons causing immune dysfunction in patients with acute pan -creatitis.

Objective To investigate the relationship between hypoxia and insulin resistance in males with metabolic syndrome ( MS ). Methods Parameters at physical, hematological, glucose metabolism, lipid metabolism, and insulin resistance were measured in 295 middle-aged men who took health examination. The trends of laboratory indexes grouped by hemoglobin quartile and MS components were compared. The correlation between hypoxia and insulin resistance was analyzed. Results With the increase of hemoglobin, the occurrence rates of MS, TG, INS, and HOMA-IR were also significantly increased ( P < 0. 05 ), while HDL-C and HOMA-IS reduced remarkably(P<0. 05). With the increase of MS components, hematological parameters(RBC, Hb, Hct), INS, and HOMA-IR were significantly increased( P<0. 05) while HOMA-IS were significantly decreased( P<0. 05). Blood lactate and HIF-1αwere significantly increased with the increase of hemoglobin as well as MS components(P<0. 05);Hematological parameters were positively correlated with blood lactate and HIF-1α, and Hypoxia related indicators including hematological parameters, Lac, HIF-1α showed a positive correlation with HOMA-IR and a negative correlation with HOMA-IS( P < 0. 05). Conclusion Chronic hypoxia does exist in metabolic syndrome, and the degree of hypoxia is associated with insulin resistance. Hematological parameters are also significantly correlated with insulin resistance which could be as a result of chronic hypoxia.

Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; complications ; Stomach Neoplasms ; complications