The correlation between the immune imbalance of T helper 17 cell/regulatory T cell and cytokines in patients with Beh?et's disease / 中华风湿病学杂志

Objective:To investigate the characteristics of lymphocyte subsets and the correlation between the immune imbalance of T helper 17 cell/regulatory T cell (Th17/Treg) and cytokines in peripheral blood of patients with Beh?et's disease(BD).Methods:From January 2018 to November 2019, 82 outpatient and inpatient with BD of the Department of Rheumatology and Immunology of the Second Hospital of Shanxi Medical University with complete data were enrolled. The disease activity was evaluated according to BD Current Activity Form(BDCAF), and 66 age and matched healthy people were selected as the control group. The absolute numbers of lymphocyte subsets and T cell subsets dominated by Th17 and CD4 +CD25 +Foxp3 + Treg in patients with BD and healthy controls were detected by Flow cytometry. The levels of interleukin (IL-2), IL-4, IL-6, IL-10, IL-17, Interferon (IFN)-γ and Tumor necrosis factor-α (TNF)-α in patients with BD were measured by Cytometric Beads Array (CBA). The correlations between the ratio of Th17/Treg with inflammatory index, the number of organ involved and the levels of cytokines were analyzed. Data were analyzed by Mann-Whitney U test, Kruskal Wallis H test, Spearman correlation analysis and multiple linear regression. Results:①The absolute numbers of Th17 cells in peripheral blood of patients with active BD [13.9(7.7, 21.1) cells/μl] and patients with stable BD [8.7(6.1, 14.0) cells/μl] were higher than those of healthy controls [6.8(4.4, 8.5) cells/μl] ( P<0.01); The ratio of Th17/Treg was significantly increased ( P<0.01). The absolute co unts of Treg cells in BD group [24.79(15.64, 37.91) cells/μl] were sign-ificantly lower than those in healthy controls [30.59(23.04, 42.08) cells/μl], the difference was statistically significant ( P=0.016). ② The ratio of Th17/Treg was positively correlated with BDCAF ( r=0.298, P=0.007) and the number of organ involved ( r=0.304, P=0.006) was negatively correlated with age ( r=-0.254, P<0.05), and not correlated with the duration of disease and ESR ( P>0.05) in patients with BD. In addition, multiple linear stepwise regression showed that the ratio of Th17/Treg was positively correlated with BDCAF ( β=0.228, P=0.036) and negatively correlated with age ( β=-0.219, P=0.043), R2=0.101. ③ The levels of IL-2, IL-6, IL-10, IFN-γ in patients with BD were stati-stically higher than those of healthy controls ( P<0.01). The ratio of Th17/Treg was positively correlated with the levels of IL-2 ( r=0.307, P<0.01) and IL-4 ( r=0.301, P<0.01) in patients with BD. Conclusion:There is immune imbalance of Th17/Treg in patients with BD, which is closely related to disease activity, the number of organ involved, and the levels of cytokines such as IL-2 and IL-4. IL-2 and IL-4 may play an important role in the immune imbal-ance of Th17/Treg in patients with BD.

The changes on expression of IL-1βand IL-13 mRNA in lung tissue and serum after drowning rats / 中国法医学杂志

Objective To observe the change in IL-1β,IL-13mRNA expression in drowning rat lungs and serum,so as to investigate the significance of IL-1β and IL-13 mechanism in the development of drowning.Methods SD rats were randomly divided into control group,drowning group.Then using TaqMan probe method to determine the expression of IL-1β and IL-13 mRNA in Right lower lobe of lung tissue and the serum of right ventricle,which were extracted respectively from each group of rats.Results (1) The lung tissue morphological changes:Typical appearance signs and anatomy of drowning group meet ante-mortem drowning feature.(2) The expression of IL-1β,IL-13 in lung tissue:compared with the control group,the expression of IL-1β and IL-13 were slightly decreased,which has no statistical significance.(3) The expression of IL-1β and IL-13 in serum:compared with the control group,the expression of IL-1β and IL-13 were significant increased,both of which has statistical significance.Conclusion (1)The expression of IL-1β and IL-13 were decreased in lung tissue may be due to drowned rats present compensatory anti-inflammatory response syndrome which causes immune incompetent performance.(2) The expression of IL-1β and IL-13 were significant increased in serum may be relate to drown stress and drowning associated acute lung injury after traumatic stress.

Predominant β-lactamase genotypes of Escherichia coli isolates and induction and inhibition mechanisms of β-lactamase gene expression / 中华流行病学杂志

Objective To understand the predominant β-lactamase genotypes and their carrying modes ofEscherichia coli isolates in Zhejiang province,and the effects of β-1actam antibiotics on inducing or histidine kinase inhibitor closantel (CLO) on inhibiting the expression of β-1actamase genes.Methods Micro-dilution method and E-test were applied to measure the resistant rate and minimal inhibitory concentration (MIC) in E.coli isolates against β-1actam antibiotics.PCR and sequence analysis of PCR products were conducted to detect the β-lactamase genotypes and their carrying modes.Real-time fluorescent quantitative RT-PCR and β-lactamase confirmation test were performed to determine the influence of 1/4 MIC penicillin and cefotaxime,and CLO on the transcription and expression of β-lactamase genes in the resistant E.coli isolates.Results Among the 462 E.coli strains isolated in Zhejiang,285 (61.7％) were resistant to penicillin,ampicillin,cefoxitin,cefotaxim and ceftazidime.In the 285 resistant isolates,the detection rate of TEM or CTX-M β-1actamase gene (83.2％ or 75.1％) was significantly higher than that of KPC,SHV or OXA β-lactamase gene (1.4％-10.2％) (P＜0.01) and the carrying rate of two or more β-1actamase genes (68.8％) was also significantly higher than that of single β-1actamase gene (31.2％) (P＜0.01),and 61.4％ of the resistant isolates carried TEM + CTX-M genes (P＜0.01).Except KPC gene,1/4 MIC of cefotaxim and penicillin induced a rapid increase of TEM-mRNA,CTX-M-mRNA,SHV-mRNA or OXA-mRNA levels (P＜0.01),but 50-500 μg/ml CLO inhibited these levels (P＜0.01).After pre-treatment with 100 μg/ml CLO,82.8％-85.6％ of the resistant isolates became sensitive to β-lactam antibiotics (P＜0.01),while the detection rate of β-lactamases was also decreased from 95.1％ to 16.1％ (P＜0.01).Conclusion TEM and CTX-M are the predominant β-lactamase genotypes in E.coli isolates in Zhejiang and TEM+CTX-M is the predominant carrying mode of β-lactamase genes.Low concentrations of β-lactam antibiotics can up-regulate the expression levels of β-lactamase genes in E.coli through bacterial two-component signaling systems,but this effect can be inhibited by CLO,a histidine kinase inhibitor.

Study of the platelet activating factor acetylhydrolase activity of a gene product encoded by LA2144 gene of Leptospira interrogans / 中华微生物学和免疫学杂志

Objective To analyze the platelet activating factor acetylhydrolase ( PAF-AH) activity of a gene product encoded by LA2144 gene of Leptospira interrogans ( L. interrogans) , to investigate the ex-pression and secretion of LA2144 protein in various cell cultures and to further understand its function in in-ducing internal hemorrhage in an animal model. Methods The DNA sample containing LA2144 gene was extracted from L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai and used as the template for gene cloning by PCR. The LA2144 gene without the signal sequence coding region was amplified by PCR and inserted into a prokaryotic expression construct for the protein expression. The expressed recombinant protein, rLep-PAF-AH, was purified by Ni-NTA affinity chromatography. Spectrophotometry was used to measure the hydrolytic activity, hydrolytic efficiency, Km and Kcat values of the rLep-PAF-AH protein in hydrolyzing PAF substrate. Real-time fluorescent quantitative RT-PCR ( qRT-PCR) and Western blot assay were performed to measure the expression of LA2144 gene at mRNA and protein levels in human umbilical vein endothelial cells (HUVEC), human monocytes (THP-1) and murine macrophages (J774A. 1) with L. interrogans strain Lai infection, respectively. Each syrian hamster was intravenously injected with 100 μg of LPS-free rLep-PAF-AH for two times. Hemorrhage in the lungs, livers and kidneys were observed in three days after the injection. Results The constructed prokaryotic expression system for LA2144 gene of L. inter-rogans strain Lai could highly express the rLep-PAF-AH upon the induction of IPTG. The purified rLep-PAF-AH showed high purity with a single protein band in gel as indicated by SDS-PAGE. The efficiency of 5 μg of rLep-PAF-AH in hydrolyzing PAF substrate was 26. 6 U/L with a Km value of 82. 79 μmol/L and a Kcat value of 0. 24 S-1 . The expression of Lep-PAF-AH at mRNA level in HUVEC, THP-1 and J774A. 1 cells were significantly elevated after co-culture with L. interrogans strain Lai for 1 or 2 hours (P<0. 05). A large amount of Lep-PAF-AH were detected in the supernatants from co-cultures of L. interrogans strain Lai with the three cell lines, but not from the culture of the spirochete in EMJH medium. The signs of hemor-rhage were observed in the lung of hamsters injected with rLep-PAF-AH, but not in tissue samples from liver and kidney. Conclusion The LA2144 gene product was characterized by a stronger PAF-AH activity. The expression of LA2144 gene at mRNA and protein levels in various cell lines were enhanced during L. interro-gans infection. Moreover, the rLep-PAF-AH could induce the pulmonary hemorrhage in hamsters. This stud-y indicated that the protein encoded by LA2144 gene was an important virulence factor causing hemorrhage in hosts during L. interrogans infection.

Comparison of three oral regimens with compound polyethylene glycol electrolyte for bowel prepara-tion / 中华消化内镜杂志

were no significant differences in the detection rate of recto-sigmoid colon,mid colon,right colon and total detection of polyps among the 3 groups (P >0.05).Conclusion 4-L split-dose PEG is better than the oth-er 2 regimens in the colon cleansing quality,so it can better reach the intestinal cleaning standards before enteroscopy,which is a more suitable regimen for bowel preparation.

Identification and immunogenicity analysis of predominant T-B combined antigenic epitopes on the ;outer membrane protein Loa22 of Leptospira interrogans strains / 中华微生物学和免疫学杂志

Objective To screen and identify the predominant T-and B-cell ( T-B) combined an-tigenic epitopes on the outer membrane protein Loa22 of pathogenic Leptospira interrogans ( L.interrogans) stains and to further analyze their immunogenicity.Methods PCR analysis was used to detect loa22 gene in L.interrogans strains belonging to eight different serogroups or serovars prevalent in China.The PCR prod-ucts were sequenced after T-A cloning.A prokaryotic expression system for loa22 gene of L.interrogans sero-group Icterohaemorrhagiae serovar Lai strain Lai was constructed.The expressed recombinant protein rLoa22 was extracted by Ni-NTA affinity chromatography.The rabbit anti-rLoa22 serum samples and IgG were pre-pared.The T-B combined antigenic epitopes on Loa22 protein were predicted by using professional bioinfor-matic softwares.Phage display in combination with Western blot assay and ELISA were performed to identify the immunogenicity of the recombinant phage PⅢ protein-displayed and artificially-synthesized T-B com-bined antigenic epitopes, respectively.MTS assay and ELISA were performed to detect the activation of T cells and the expression of IL-2, IL-4 and IFN-γinduced by T-B combined antigenic epitopes, respectively. Results All of the tested pathogenic Leptospira strains were positive for loa22 gene, sharing 85.5%-99.8%homologies in nucleotide sequences and 93.9%-99.5%homologies in amino acid sequences.The construc-ted prokaryotic expression system for loa22 gene efficiently expressed the rLoa22 protein.Among four T-B combined antigenic epitopes (Loa22-77, Loa22-90, Loa22-125 and Loa22-157), only Loa22-90 epitope presented a strong positive band in Western blot analysis.The proliferation of CD4+T cells and the expres-sion of IL-2 ( Th1 ) and IL-4 ( Th2 ) were significantly enhanced by the stimulation with Loa22-90 epitope peptide (P<0.05).Conclusion Loa22 protein is a sequence-conserved genus-specific outer membrane protein of L.interrogans.The Loa22-90 epitope is the predominant T-B combined antigenic epitope of Loa22 protein, which might be used as a candidate antigenic epitope in the development of multiple antigenic pep-tide ( MAP) vaccines against Leptospira infection.

Knockout of the ptsG gene in engineered Escherichia coli for homoethanol fermentation from sugar mixture / 生物工程学报

To realize the simultaneous fermentation of xylose and glucose, ptsG (one of the glucose-PTS genes) was deleted from the engineered ethanologenic Escherichia coli SZ470 (deltapflB, deltafrdABCD, deltaackA, deltaldhA), resulting in loss of glucose effect in the mutant SZ470P (deltaptsG). When tested in 5% mixture of glucose (2.5%) and xylose (2.5%), SZ470P simultaneously used glucose (13 g/L) and xylose (20 g/L) whereas the parent strain SZ470 sequentially used glucose (25 g/L) then xylose (5 g/L). Upon completion of the fermentation, both strains achieved similar product yield of 89%. SZ470P produced 15.01 g/L of ethanol, which was 14.32% higher than that produced by SZ470 (12.86 g/L). Deleting ptsG gene enabled the mutant strain SZ470P to simultaneously use both glucose and xylose and achieve better ethanol production.

Distribution of drug inactive enzyme genes in bacterial isolates and mechanism of its induction and inhibition / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine the distribution and the predominant gene carrying model of drug inactive enzyme genes in bacterial isolates, and the mechanism of its induction and inhibition.</p><p><b>METHODS</b>The β-lactam, aminoglycosides and macrolides inactive enzyme genes were detected by PCR and sequencing in S. aureus, E.coli, K. pneumoniae, A. baumannii and E. cloacae isolates. The expression of inactive enzyme genes were examined by real-time fluorescent quantitative RT-PCR when the bacterial isolates were treated with antibiotics or a histidine kinase blocker closantel.</p><p><b>RESULTS</b>In 63 isolates of E.coli, 4 kinds of β-lactam, 2 aminoglycosides and 1 macrolides inactive enzyme-encoding genes were detected and the predominant gene-carrying models were [TEM+CTX-M]+aac(3)-II+mphA (25.4 %) and [TEM+CTX-M]+ aac (6')-I b (20.6%). In 24 isolates of S.aureus, 2 kinds of β-lactam and 3 aminoglycosides inactive enzyme-encoding genes were detected and the predominant gene-carrying models were aph (3')(41.7%) or aac (6)-I e-aph (2)-I a (25.0%). In 28 isolates of K.pneumoniae, 4 kinds of β-lactam and 2 aminoglycosides inactive enzyme-encoding genes were detected and the predominant gene-carrying models were [TEM+SHV]+[aac(6')-I b+aac (3)-II](28.6 %) and [TEM+SHV]+[aac(6')-I b+aac (3)-II]+ mphA (17.8 %). The isolates of A.baumannii and E.cloacae also had a predominant model to carry 2 or 3 kinds of inactive enzyme-encoding genes. 1/4 MIC of penicillin, cefotaxime or streptomycin induced the up-regulation of expression of 3 β-lactam or 4 aminoglycosides inactive enzyme-encoding genes (P<0.05), and this effect was inhibited by closantel (P<0.05).</p><p><b>CONCLUSION</b>The bacterial isolates frequently carry multiple kinds of inactive enzyme-encoding genes with different predominant gene-carrying models.Low concentration antibiotics can induce the up-regulation of inactive enzyme gene expression, which can be inhibited by histidine kinase blocker.</p>

Role of fliY gene in pathogenicity-associated chemotaxis and colonization of Campylobacter jejuni / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct a knockout fliY gene mutant strain of Campylobacter jejuni for determining the role of FliY protein in flagellar movement related to bacterial motility, chemotaxis and colonization.</p><p><b>METHODS</b>The plasmid pBluescript-II-SK was used to construct the suicide plasmid; according to homologous exchange principle, the suicide plasmid was utilized to generate fliY gene knockout mutant(fliY) in Campylobacter jejuni strain NCTC11168. The fliY mutant strain was identified by PCR, sequencing and Western blotting. The chemotactic and colonizing abilities of fliY mutant were determined by colony migration test and bacterial chemotactic test in vitro, and colonization test in jejunum of mice.</p><p><b>RESULTS</b>The fliY(-)mutant strain showed a growth curve in medium similar to that of wild-type strain. PCR, sequencing and Western blotting assay confirmed that the fliY gene in fliY(-)mutant was deleted. Compared to the wild-type strain, the colonies of fliY-mutant on semisolid plate were much smaller (P <0.05), the chemotactic ability of fliY mutant towards sodium deoxycholate and bovine bile was significantly attenuated (P <0.05), and the number of fliY mutant (CFU) in jejunal tissue specimens of the infected mice was significantly decreased (P<0.05).</p><p><b>CONCLUSION</b>The function of C.jejuni fliY gene refers to controlling flagellar movement, which is involved in bacterial chemotaxis and colonization.</p>

Multidrug resistance of enteric bacilli and its relation to structure and molecular evolution of variable region in resistance-related class-I integron / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the drug resistance of enteric bacilli and its relation to the drug resistance gene cassette in the variable region and molecular evolution of class-I integron.</p><p><b>METHODS</b>K-B assay was applied to measure the drug resistance of E.coli, E.cloacae and A.baumannii isolated against twelve antibiotics. The class-I integron and drug resistance gene cassettes in the variable region of the integron were detected by PCR and sequencing of amplification products. The molecular evolution of drug resistance genes in the class-I integrons was analyzed using Clustal X and MEGA software.</p><p><b>RESULTS</b>54.2%-100% of A.baumannii isolates were resistant to the penicillin and cephem antibiotics, while E.coli and E.cloacae isolates had resistance rates of 41.6%-62.5% to cephem antibiotics. 62.5%(15/24) of E.coli, 67.9%(19/28) of E.cloacae and 83.3%(20/24) of A.baumannii isolates were positive for class-I integrons. 81.5% (44/54) of class-I integrons showed 4 different single band spectrums and the other class-I integrons displayed 3 different double band spectrums. In the drug resistance gene cassettes in variable regions of class-I integrons there were 7 types in 4 groups of drug resistance genes, including aac(6'), sad(3"), aad(2"), cat(4') and dfr (types 7, A13 and 15), which induced the resistance to aminoglycosides and sulfamido antibiotics and chloromycin. The class-I integrons in the isolates might be divided into 4 molecular evolution groups according to the diversity of dihydrofolate reductase encoding gene sequences.</p><p><b>CONCLUSION</b>The enteric bacilli have a high drug resistance and frequently carry class-I integrons with 7 drug resistance gene cassettes which present 4 different evolutionary pathways.</p>

Identification of phospholipase C in Leptospira interrogans and the mechanism of macrophage apopto-sis induced by phospholipase C / 中华微生物学和免疫学杂志

Objective To investigate the function of phosphatidylinositol phospholipase C encoded by LB361 gene of L.interrogans ( L-PI-PLC) and its mechanism in inducing macrophage apoptosis .Meth-ods The PI-PLC domains in the sequence of LB 361 gene of L.interrogans serovar Lai strain were analyzed by bioinformatics method .Prokaryotic expression system was established to express the recombinant L -PI-PLC ( rL-PI -PLC).The enzymatic activity of rL-PI-PLC in hydrolyzing phosphatidylinositol -4,5-bisphos -phate (PIP2) substrate into inositol-1,4,5-trisphosphate (IP3) was determined by IP3 fluorescence polariza-tion assay.LB361gene expressions at mRNA and protein levels as well as the secretion of LB 361gene prod -ucts were detected by real-time fluorescent quantitative RT -PCR and Western blot assay after infection of hu-man THP-1 macrophages with L.interrogans serovar Lai strain.A LB361 gene-transfected THP -1cell line was generated for evaluation of the mechanism of LB 361 gene products in elevating intracellular free Ca 2+( [Ca 2+] i) concentration and inducing the apoptosis of transfected THP -1 cells with the use of laser confocal microscopy and flow cytometry.Re sul ts The rL-PI-PLC hydrolyzed PIP2 into IP3 with a Km of 199 μmol/L and a Kcat of 8.566×10-5 S-1 .The expressions of LB361gene at mRNA and protein levels were both signifi -cantly up-regulated after infection of THP-1 cells with L.interrogans serovar Lai strain .Moreover , the exter-nal secretion of L-PI-PLC was also found during infection .The concentrations of IP 3 and [ Ca2+] i in the LB361 gene-transfected THP-1 cells were significantly increased compared to those in the non-transfected THP-1 cells, resulting in a high [Ca2+]i-dependent apoptosis of partial THP-1 cells.Conclusion PI-PLC is encoded by the LB361 gene of L.interrogans, which could induce the apoptosis of macrophages through el-evating [ Ca2+] i concentration during infection of microphages with L.interrogans.

Establishment and application of a flow cytometry-based rapid drug susceptibility test for Candida species / 中华微生物学和免疫学杂志

Objective To establish a flow cytometry-based drug susceptibility test for the rapid de-tection of antifungal susceptibility or resistance of Candida isolates.Methods The gate selection and opti-mal experimental conditions of flow cytometry-based drug susceptibility test were determined by using Candi-da albicans strain ATCC90029 as the test strain and propidium iodide ( PI) as the fluorescent dye .The es-tablished flow cytometry-based drug susceptibility test was used to detect the susceptibility or resistance to fluconazole or voriconazole of 110 isolates belonging to Candida species, and the obtained results were com-pared with those by using typical M 27-A3 constant dilution method .Results The killed and viable Candida albicans ATCC90029 strains were clearly divided into two groups on the figure of SS /log (FL3) by regulating voltages.There was a high correlation between the results of susceptibility test and the proportions of killed and viable fungi in mixture (r=0.999).The flow cytometry-based drug susceptibility test could provide the results within 30 min and its optimal concentration of fungal suspension , time of drug-fungus incubation , dyeing method and time were 1.0×106/ml, 3 h incubation and sodium deoxycholate-pretreated plus PI dye-ing for 5 min, respectively .The total coincident rates between the established test and the constant dilution method were 98.2%and 87.3%in the detection of drug susceptibility of 110 fungal isolates to fluconazole and voriconazole .Conclusion The flow cytometry-based drug susceptibility test shows advantages of rapidi-ty, accuracy and high sensitivity compared with the constant dilution method .It has a great potential for clin-ical application .

Clinical significance of dynamic hypoalbuminemia monitoring in children with severe sepsis / 中国小儿急救医学

Objective To study serum albumin levels in children with severe sepsis and to correlate serum albumin levels with patient outcome and to identify the causes inducing hypoalbuminemia and its effective countermeasures.Methods Seventy-five children admitted to PICU of Provincial Hospital Affiliated to Shandong University for severe sepsis were included in the study from Aug 2010 to Sep 2011.According to their serum albumin levels within 24 hours and on the third and the seventh day of admission to PICU,75 children were divided into hypoalbuminemia group and normal serum albumin group.Then hypoalbuminemia group was divided into instant hypoalbuminemia group and continuous hypoalbuminemia group according to the duration of hypoalbuminemia.The correlation between the occurring and duration of hypoalbuminemia with patients' prognosis,the etiopathogenisis of hypoalbuminemia and its effective countermeasures were analyzed.Results (1) Sixty-three cases (84.0％) proceeded hypoalbuminemia and their mortality was 33.3％ (21/63),while 12 cases (16.0％) showed normal serum albumin level and their mortality was 0.(2) In 63 patients with hypoalbuminemia,26 cases showed continuous hypoalbuminemia and their mortality was 46.0％,while 37 cases proceeded instant hypoalbuminemia and their mortality was 15.4％.There was significant difference (x2 =5.116,P ＜ 0.05) between their mortality.(3) In the 63 cases with hypoalbuminemia,32 cases presented with hepatic injury and their mortality was 37.5％,13 cases proceeded capillary leakage and their mortality was 23.1％,and 18 cases displayed hepatic injury complicated with capillary leakage and their mortality was 33.3％.There was significant difference (x2 =7.812,P ＜ 0.05) between the mortality with different causes.Conclusion Hypoalbuminemia influenced the prognosis of children with severe sepsis,the longer duration correlated with the worse prognosis.Hepatic injury and capillary leakage were two main causes inducing hypoalbuminemia.Active treatment of hypoproteinemia aimed directly at different causes could improve their prognosis significantly.

Functional analysis of Leptospira interrogans sphingomyelinase hemolysin genes and their transcriptional level alterations in the infected host cells / 中华微生物学和免疫学杂志

Objective To determine the hemolytic activity of products of sphingomyelinase hemolysin encoding genes of Leptospira interrogaas, and the transcriptional level alterations in the infected host cells. Methods By using genomic DNAs of pathogenic L. interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai and serogroup Pomona serovar Pomona strain Luo, and non-pathogenic L. biflexa serogroup Sama-ranga serovar Patoc strain Patoc Ⅰ as templates, PCRs were performed to amplify entire sph1-sph4 genes. The amplified products were sequenced after T-A cloning. Prokaryotic expression systems of sph1-sph4 genes were re-spectively constructed, and the expressions of target recombinant proteins rSph1-rSph4 were examined by SDS-PAGE. Ni-NTA affinity chromatographic column was used to extract the expressed rSph1-rSph4. Hemolytic ac-tivities of rSph1-rSph4 on sheep blood agar plate were identified. Transcription alterations of sphl-sph4 genes in L. interrogans strain Lai after infected J774A. 1 cells were measured by real-time fluorescence quantitative RT-PCR. Results From genomic DNAs of both L. interrogans strain Lai and Luo, but not from that of L. biflexa strain Patoc Ⅰ , the target fragments of sph1-sph4 genes could be amplified. All the cloned sph1-sph4 genes had 100% nucleotide sequence identities compared to the corresponding reported sequences. The constructed pro-karyotic expression systems were able to efficiently express the target recombinant proteins rSph1-rSph4, respec-tively. All the rSph1-rSph4 had hemolytic activities, and among the four products rSph2 displayed the strongest hemolytic activity. After L. interrogaas strain Lai infecting J774A. 1 cells, the transcriptional levels of sph1-sph4 genes were remarkably up-regulated, especially for mRNA levels of sph2 and sph4 genes. Conclusion sph1- sph4 genes exist only in pathogenic L. interrogans species, and their products have hemolytic activity. The up-regulation of sph1-sph4 gene transcriptional levels in L. interrogans strain Lai after infected cells implies that the sphingomyelinase hemolysins may play important roles in the process of L. interrogans infection in hosts.

Prokaryotic expression of Campylobacter jejuni mcp1/2/3 genes and relationship among the expressed products and bacterial chemotactic behavior / 中华微生物学和免疫学杂志

Objective To clone mcp1, mcp2 and mop3 genes that encoding methyl-accepting chemotaxis proteins(MCP) of Campy/obacter jejuni for construction of their prokaryotic expression systems, and to establish chemotactic model in vitro of the microbe for determining chemotaxis-inducing substances and to understand relationship among MCPs and chemotactic inducers. Methods The segments of mep1, mcp2 and mcp3 genes were amplified by PCR and then sequenced after T-A cloning. Prokaryofic expression systems of the genes were subsequently constructed. SDS-PAGE pins Bin-Rod Gel Image Analyzer were used to examine the expression of target recombinant proteins rMCP1, rMCP2 and rMCP3, and Ni-NTA affinity chromatography was performed to purify the rMCPs. Rabbits were immunized with each of the three rMCPs to obtain antisera. Immunodiffusion assay was performed to measure the titers of antisera. IgG in each of the antisera were extracted by ammonium sulfate precipitation and DEAE-32 ion exchange chromatography, and IgG F(ab')2 were then prepared by pepsin enzymolysis and Sephadex G-100 chromatography. Chemotactic model in vitro of C. jejuni based on HAP( hard-agar plus) method was established to determine the chemo-taxis-inducing effect of eight candidate substances. Chemotaxis inhibition test based on IgG F(ab')2 bloc-king was applied to determine the function and diversity of MCPs. Results The segments with expected si-zes amplified from mcp1, racp2 and mcp3 genes were obtained by PCRs, and their nucleotide and putative amino acid sequences were completely same as the reported. The constructed prokaryotic systems could effi-ciently express rMCPs with the yields about 10% of the total bacterial proteins. Immunization with rMCP1, MCP2 and rMCP3 enables the rabbits to produce specific antibody. All the antisera had 1: 4 immunodiffusion titers. Both bovine bile and sodium deoxycholate(DOC) were able to induce ehemotactie movement of C. je-juni in a dosage-dependent manner ( P < 0.05 ). When MCP1 and MCP2 were blocked with their IgG F(ab')2, the ehemotaetic ability of C. jejurd were remarkably decreased (P <0. 05). However, MCP3 blocking did not affect the chemotaxis ( P > 0.05 ). Conclusion The C. jejuni MCPs are successfully ex-pressed in this study. Bovine bile and DOC are the inducers for chemotaxis of C. jejuni, and MCP1 and MCP2 are involved in the process of ehemotaxis iadueed by DOC.

Apoptosis-inducing effect and intracellular location of Vibrio vulnificus cytolysin to human umbilical vein endothelial cell in vitro / 中华微生物学和免疫学杂志

Objective To determine the effect of Vibrio vulnificus cytolysin (VVC) inducing ap-optosis in human umbilical vein endothelial cell(HUVEC) and its possible mechanism. Methods The en-tire vvhA gene that encoding VVC from V. vulnificus strain GTC333 was amplified by PCR and sequenced af-ter T-A cloning. E. coli BL21DE3pET-42a-vvhA, a prokaryotic expression system of the vvhA gene, was then con-structed. Ni-NTA affinity chromatography was applied to purify the target recombinant protein rVVC, and SDS-PAGE plus Bio-Rad Agarose Image Analyzor were used to measure the output of rVVC and to determine the purity of rVVC extract. The activity of rVVC dissolving rabbit erythrocytes was detected by hemolysis test. DPNH chromotometry and TphBNa chromotometry were performed to examine the contents of LDH and K+ in the supernatants of rVVC-treated HUVEC cultures, respectively. The effect of rVVC inducing apepto-sis of HUVEC was detected by flow cytometry, rVVC was labeled with FITC and the location of FITC-labe-ling rVVC in HUVEC was observed by laser canfocal microscopy. Results The cloned whA gene had 96.09% and 98.26% similarities of nucleotide and amino acid sequences compared to the corresponding se-quences in GenBank. rVVC, with a dosage of 1 μg/ml, could dissolve rabbit erythrocytes (P<0.01). 10 μg/ml rVVC was able to promote the increases of K+ content (P<0.01) but no change of LDH content could be found in the cell supernatants. HUVEC was apoptotic after the cell was treated with 1～100 μg/ml of rVVC for 2 h. In the 5～240 min duration of co-incubation of FITC-labeling rVVC and HUVEC, the rV-VC gradually moved from surface to inner side of the membrane and then entered the cytoplasms. When FITC-labeling rVVC treated HUVEC for 30 min, most of the rVVC was found to be intracellular location. Conclusion rVVC has cytolytic activity. VVC has an ability to enter HUVEC and causes injury of HUVEC via inducing apoptosis, which may be the major pathogenic mechanism of VVC.

Reasons for the decline of autopsy rate after accidental death / 中国法医学杂志

To investigate and analyze the factors which influence the public attitudes towards autopsy after accidental deaths.The study has carried on an investigation among 386 individuals in random,with a questionnaire named "The cognition of the public whether to carry on the autopsy after accidental deaths".Using the statistical methods of the Logistic Regression analysis and optimum regression analysis to analyze the recycling questionnaires.Through the diagnosis,we found there are four main factors which influence public opinion upon the autopsy after accidental deaths,the knowledge of autopsy,the believing in autopsy,years of schooling and family financial circumstances.