Child ; Humans ; Vesico-Ureteral Reflux ; therapy

Porphyromonas gingivalis is a specific causative agent of human chronic periodontitis. This anaerobe produce collagenase PrtC encoded by prtC. To constructed the prokaryotic expression system for the prtC gene from P.gingivalis so as to be used to explore antigenicity and immunoreactivity of prtC gene product as well as the relationship between the local level of PrtC and the periodontitis damage, the entire length of prtC genes fragment from the ATCC-33277 and 47A-1 strains of P.gingivalis was amplified by PCR. After T-A cloning and sequencing, the prokaryotic expression system for prtC was constructed by using pET32a plasmid and E.coli BL21DE3 strain. The expression of the target recombinant PrtC protein was induce with different concentrations of IPTG. Western blot assay was used to detect the antigenicity and immunoreactivity of PrtC protein, and ELISA assay was used to detect the PrtC level in the subgingival plaque samples of patients with chronic periodontitis. The experimental results showed that the nucleotide sequences of the prtC genes from ATCC-33277 and 47A-1 strains of P.gingivalis were entirely identical, and the sequence similarities of nucleotides and amino acids were 98.46% and 99.07% respectively. Under the induction of different concentration of IPTG, the output of recombinant expressed product PrtC may reach up to 50% of the total bacterial proteins. It was also proved that the recombinant PrtC could bind with antibody against the whole cell of P.gingivalis, and could induce the production of specific antibodies in rabbit. In 91.39% of the subgingival plaque samples, the PrtC could be detectable, in which the level of positive rates of detection was higher in severe cases of chronic periodontitis than that in the mild cases. So far, a prokaryotic expression system for the prtC gene from P.gingivalis with high expression efficiency was successfully constructed in the present study, and the expressed product PrtC possesses well antigenicity and immunoreactivity, suggesting the possibility to be used as the candidate antigen for developing the serological kit and P.gingivalis vaccine.

Objective To analyze the histopathology of patients with atypical squamous cells of undetermined significance (ASCUS),and provide evidence for further classification and clinical treatment of ASCUS.Methods The histopathology of cervical biopsy specimens from 249 patients with ASCUS diagnosed by liquid-based cervical cytology examination was analyzed retrospectively. Results Among the 249 ASCUS patients, the proportion of patients with inflammation was 34.14% (85/249), morphological change by human papillomaviral infection was 19.28%(48/249), CIN I was 32.53%(81/249),CIN II was 8.84%(22/249),CIN III was 3.21%(8/249), infiltrating squamous cell carcinoma was 1.20%(3/249),and endometrioid adenocarcinoma was 0.80%(2/249) . Conclusion It is very important to to further definitude the diagnosis of ASCUS, because a certain proportion of cervical cancer and precancerous leisions woud be confirmed.

Objective To generate the spaO-ompA fusion gene of Salmonella paratyphi A and its prokaryotic expression system,and to determine the immunoprotection of the recombinant expression product rSpaO-OmpA.Methods A flexible peptide sequence was used to link spaO and ompA genes and a prokaryotic expression system of spaO-ompA fusion gene was subsequently generated.SDS-PAGE and Bio-Rad Agarose Image Analyzer were applied to examine the expression as well as the yield of the target recombinant protein rSpaO-OmpA.The antigenicity and immunoreactivity of rSpaO-OmpA were determined using immunodiffusion test,Western Blot assay and micro-Widal's test.By a mouse infection model,the immunoprotection of rSpaO-OmpA against the lethal challenge of S.paratyphi A was determined.In the animal protective test,the recombinant expressed SpaO (rSpaO) and OmpA ( rOmpA ) were used as the controls.Results The generated spaO-ompA fusion gene had 100％ nucleotide and amino acid sequence identities compared to the single spaO or ompA gene.The constructed prokaryotic expression system IPTG E.coli BL21DE3pET42a-spaO-ompA expressed the recombinant protein rSpaO-OmpA.rSpaO-OmpA combined with the antiserum against wholecell of S.paratyphi A to present positive hybridization signal and induced specific antibody in the immunized rabbits.Immunization with 100 or 200 μg rSpaO-OmpA contributed 66.7％ (8/12) or 83.3％ (10/12) immunoprotective rates in mice when the animals were attacked with S.paratyphi A.The immunoprotective rates produced by rSpaO-OmpA were significantly higher than that of equal rSpaO or rOmpA( P＜0.05 ).The sera from rSpaO-OmpA immunized mice presented 1∶5-1∶40 agglutination titers to the H antigens of different S.paratyphi species,and 1∶1-1∶16 immunodiffusion titers to rSpaO,rOmpA and rSpaO-OmpA proteins,respectively.Conclusion The artificially fusion antigen,rSpaO-OmpA,has more powerful immunogenicity and immunoprotection that the equal rSpaO or rOmpA.

Objective This paper is mainly to discuss accuracy and clinical application value of MDCT double-period enhanced scanning with low -tension water enteroclysis for colon cancer preoperative TNM stag-ing.Methods Sixty-two colon cancer patients with complete images and pathological data were selected in our hospital from January 2012 to May 2013 .We retrospectively analyzed CT image changes of the tumor location ,the extent of tumor invasion,the surrounding fat space,lymph node metastasis and distant metastasis.We compared them with postoperative pathology to prove the accuracy of MDCT double -period enhanced scanning with low -tension water enteroclysis.Results The results showed that its accuracy rate reached to 90.32%(56/62)in co-lon cancer preoperative Stage T,80.64%(50/62)in Stage N,and 100%(62/62)in Stage M respectively.Con-clusions MDCT double-period enhanced scanning with low -tension water enteroclysis can accurately display the site of colon cancer and determine the scope of tumor invasion ,lymph node metastasis and distant metastasis , and give more precise diagnosis of colon cancer and preoperative staging assessments .In conclusion , it can be used as the preferable method of preoperative examination in the colon cancer .

Objective To investigate the effect of isoflurane preconditioning on the nuclear factor kappa B (NF-kB)activity of leukocytes in patients undergoing cardiac valve replacement with cardiopulmonary bypass (CPB).Methods Twenty ASAⅡorⅢpatients of both sexes aged 20-60 yrs undergoing cardiac valve replacement under CPB were randomly divided into 2 groups(n=10 each):control group(C)and isoflurane preconditioning group(I).Anesthesia was induced with midazolam 0.08-0.12 mg?kg~(-1),fentanyl 5-10?g?kg~(-1) and vecuronium 0.1 mg?kg~(-1).The patients were mechanically ventilated(FiO_2=100%)after tracheal intubation.Anesthesia was maintained with intermittent i.v.boluses of fentanyl and midazolam in group C or with 2 MAC isoflurane and intermittent i.v.boluses of fentanyl and midazolam in group I before CPB.Systolic BP was kept between 90-120 mm Hg,diastolic BP between 50-80 mm Hg and HR between 60-90 bpm in both groups. Isoflurane was discontinued at the initiation of CPB.Arterial blood samples were taken after tracheal intubation and before inhalation of isoflurane(T_0)at 30 min(T_1),1 h(T_2)and 2 h(T_3)after aortic unclamping for determination of NF-kB activity of leukocytes using electrophoretic mobility shift assay(EMSA).The amount of fentanyl,midazolam,dopamine and sodium nitroprusside(SNP)consumed during operation and the rate of recovery of spontaneous heart beat in both groups were recorded.Results The NF-kB activity was significantly increased after aortic unclamping in C group but there was no significant difference in NF-kB activity before CPB (T_0)and after aortic unclamping(T_(1-3))in I group.The NF-kB activity was significant lower at T_(1-3) in group I than in group C.The total amount of fentanyl consumed was 40-60?g?kg~(-1) in C group and 20-30?g?kg~(-1) in group I. Significantly less amount of dopamine was used in group I than in group C.There was no significant difference in SNP consumption between the 2 groups.The rate of recovery of spontaneous heart beat was significantly higher in group I than in group C(P＜0.01).The amount of dopamine consumed was positively correlated with the highest level of NF-kB activity in both group[r=0.962 in group C;r=0.908 in group I(P＜0.01)].Conclusion Isoflurane preconditioning can attenuate the NF-kB activity of leulocytes in patients undergoing cardiac valve replacement under CPB.

0.05),but that MFI and/or PPC of CAR in the 2 types cells markedly increased compared with lymphocytes in the same group(Pa

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.

Objective To determine the role of flagellar motor protein MotA in the pathogenesisassociated chemotaxis and colonization of Campylobacter jejuni. Methods The motA gene as well as Kan~r gene and plus-motA gene segments for motA gene knock-out were amplified by PCR and the target amplification fragments were sequenced after cloning. A suicide plasmid(pBlueskrit-Ⅱ-SK~(motA-kan)) and a motA gene knock-out mutant (motA~-) were constructed based on homologious recombination. By using semisolid plate migration test, hard agar plus (HAP)-based chemotactic test towards sodium deoxycholate (SDC) in vitro, and jejunal colonization test in BALB/c-ByJ mice were performed to determine the differences of flagellar motility, chemotaxis towards SDC and colonization in murine jejunum between motA~- mutant and wild-type strain. Results The nucleotide and amino acid sequences of the cloned motA gene were 100% identical to the reported corresponding sequences. The results of PCR, sequencing and continuous passage culture in antibiotics-contained medium demonstrated that both suicide plasmid and motA~- mutant were successfully generated. The diameters of clonies on semisolid plate and 0.2 mol/L SDC-induced chemotactic tings in HAP as well as the bacterial numbers adhering to the surface of murine jejunal mucosa and in jejunal content of motA~- mutant were significantly less than those of wild-type strain(P<0.05). Conclusion A motA gene knock-out mutant of C. jejuni was successfully constructed in this study, motA is an essential gene for flagellax motility, pathogenesis-associated chemotaxis and colonization of C. jejuni.

Objective To generate a prokaryotic expression system of Salmonella paratyphi A nmpC gene that encoding an outer membrane protein(OMP),and to determine immunogenicity and immonuprotection of the recombinant expressed product rNmpC and carrying and expression frequencies of the nmpC genes in isolates of S.paratyphi A.Methods A nmpC gene clone was obtained from a clinical S.paratyphi A strain JH01 by PCR and T-A cloning method,and then a prokaryotic expression system of the gene clone was generated.SDS-PAGE and Bio-Rad Agarose Image Pattern Analysis System were applied to examine the expression and yield of rNmpC.Antigenicity and immunoreactivity of rNmpC were determined by immunodiffusion test,Western blot assay and micro-Widal's test.The carrying and expression rates of nmpC genes in 98 S.paratyphi A isolates were detected by PCR and ELISA.By a mouse infection model,the immunoprotective effect of rNmpC against the lethal challenge of S.paratyphi A was determined.Results All the cloned nmpC genes had 100% nucleotide and putative amino acid sequence identities compared to the reported sequencing data.The expression yield of rNmpC was approximately 30% of the total bacterial proteins.rNmpC could efficiently induce rabbits to produce specific antibody and present positive Western hybridization signals with S.paratyphi A antiserum.All the tested S.paratyphi A strains have nmpC gene as well as express NmpC protein,but no nmpC gene could be detectable in S.typhi,S paratyphi B and S paratyphi C.Immunization with 100 μg and 200 μg rNmpC contributed 41.7%(5/12) and 66.7%(8/12) immunoprotective rates in mice,respectively.The sera from rNmpC immunized mice and survival mice in the NmpC is an unique OMP antigen of S.paratyphi A with conserved sequence,extensive distribution and natural expression.This OMP can be used as one the candidate antigens for developing multiple-valence genetic engineering vaccine of S.paratyphi A based on its fine immunogenicity and certain immunoprotection.