ObjectiveTo analyze the long term efficacy and prognosis of ursodeoxycholic acid (UDCA) combined immunosuppressive therapy in primary biliary cirrhosis-autoimmune hepatitis overlap syndrome (PBC-AIH). Methods A total of 44 PBC-AIH cases were selected from 387 autoimmune liver diseases cases in The General Hospital of Tianjin Medical University from January 2001 to January 2011,and the medical data,treatments and efficacies were retrospective analyzed.ResultsThe serum levels of alanine aminotransferase (ALT),aspartate aminotransferase (AST),alkaline phosphatase (ALP),γ glutamyl transpeptidase (GGT) and total bilirubin (TBil) increased in different degrees in 44 PBC-AIH patients.Globulin or immunoglobulin G(IgG) increased in 84.09％(37/44) patients,immunoglobulin M(IgM) increased in 38.63％ (17/44) patients.The positive rate of antinuclear anti-body (ANA), anti-mitochondrial antibody (AMA)and anti-smooth muscle antibodies (SMA) was 97.73％,90.91％ and 11.36％,respectively. Pathological features were interface hepatitis and different degrees of intrahepatic bile ducl injuries. After UDCA combined immunosuppressant treatment,the remission rate was 61.36 ％ (27/44),the incomplete response rate was 29.55％ (13/44) and the treatment failure rate was 9.09％ (4/44).Six cases with remission withdrawal medicine,and the recurrence rate was 5/6.By the end of follow-up,the levels of ALT,AST,ALP,GGT and TBil significantly decreased in PBC-AIH patients compared with those before treatment.ALP,GGT,ALT and AST levels significantly decreased in the first 6 months while ALP and GGT showed slight upward trend at the end of follow up. The disease progression rate was 25.33％ in PBC-AIH patients (13/44) during the follow-up,and the 10 year survival rate was 93.33％ (28/30).ConclusionUDCA combined immunosuppressive therapy in PBC AIH treatment can significantly improve patients' blood biochemical indexes,delay disease progression,improve survival rate,and the remission rate is also high.However the recurrence rate is high after withdrawal of medicine.

ObjectiveTo generate a prokaryotic expression system of series predominant epitopes (UreB322 and UreB527) of Helicobacter pylori UreB protein,and to synthesize a multiple antigenic peptide (MAP) vaccine by linking both the two epitopes with a peptide carrier (Poly-Asp-Lys),and to determine the immunogenicity and immunoprotection of the MAP vaccine.MethodsLinking primer PCR was performed to generate an enterokinase(EK) site-containing series UreB322 and UreB527 epitope encoding gene for construction of its prokaryotic expression system.The expressed target recombinant fusion protein 8 ×[rEK-UreB322-EK-UreB527-EK] was hydrolyzed with EK and then rUreB322-EK and rUreB527-EK epitope peptides were extracted using a Sephadex G-25 column.rUreB322-EK,rUreB527-EK and Poly-Asp-Lys were linked using carbodiimide method to produce a MAP vaccine (MAP-rUreB322/B527).The antigenicity and immunoreactivity of each of the two epitope peptides and MAP-rUreB322/527 were determined by ELISA and Western blot assay.An animal H.pylori strain SS1-infected model in BALB/c mice was used to detect the immunoprotection of MAP-rUreB322/527.ResultsAn octuple-repeated series UreB322-UreB527 encoding gene and its prokaryotic expression system were obtained.The yield of target fusion protein 8×[rEKUreB322-EK-rUreB527-EK] was as high as 48％ of the total bacterial proteins.EK hydrolyzed the target fusion protein completely into rUreB322-EK and rUreB527-EK peptides.The linking ratio of rUreB322-EK,rUreB527-EK and Poly-Asp-Lys was as high as 92.5％.The antibody against whole cell of H.pylori and rUreB-IgG could recognize and combine with the rUreB322-EK,rUreB527-EK or MAP-rUreB322/527.The specific serum antibody level in MAP-rUreB322/527-immunized mice was significantly higher than that in rUreB-immunized mice (P＜0.05).The immunoprotective rates(83.3％ and 91.7％ ) by immunization with50 or 100 μg MAP-rUreB322/527 in the H.pyloristrain SSI-infected mice were significantly higher that those(d1.7％ and 50.0％ ) by immunization with equal rUreB(P＜0.05).ConclusionAn gene composed for encoding a repeated series predominant epitopes of H.pylori UreB protein and its prokaryotic expression system are successfully generated in this study.MAP-rUreB322/527,the multiple antigenic peptide vaccine based on the two predominant epitopes of UreB,can noticeably increase the immunoprotection in H.py/or/infected mice.

Objective To determine the modality of Leptospira interrogans invading human and murine mononuclear-macrophages and diversity of leptospiral phagocytotic vesicle formation. Methods Transmission electron microscopy was applied to observe the invasion of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai into murine mononuclear-macrophage-like cell line J774A. 1 and PMA-activated human monocyte line THP-1 and the formation of leptospiral phagocytotic vesicles. By using immunofluorescence plus either laser confocal microscopy or fluorescence spectrophotometry, the changes of intracellular leptospiral numbers in J774A. 1 and PMA-activated THP-1 cells before and after block with endocytosis inhibitors monodansylcadaverin (MDC), phenylarsine oxide (PAO) and clathrin antibody were investigated. Results The leptospires in J774A. 1 cells were located in phagocytotic vesicles while the leptospires in THP-1 cells had no package with phagocytotic vesicle membrane. Both MDC and PAO presented the effect inhibiting endocytosis of L. interrogans into J774A. 1 and THP-1 cells in dose-dependent manner. The numbers of leptospires in J774A. 1 and THP-1 cells that pre-blocked with 10 μmol/L or above MDC and 1 μmol/L or above PAO were significantly less than that in the two cells untreated with MDC and PAO (P＜0.05＝. After J774A. 1 and THP-1 cells were blocked with clathrin antibody, the numbers of intracellular leptospires were also remarkbly decreased ( P<0.05 ).Conclusion Leptospira interrogans can invade into both human and murine mononuclear-macrophages through the way of clathrin-dependent endocytosis. There is an opposite diversity of leptospiral phagocytotic vesicle formations in human and murine mononuclear-macrophages, which may result in the difference of pathogenesis in human and mice after infected with L. interrogans.

Objective To determine the effects of Che A and CheY proteins of Campylobacter jejuni regulating the bacterial chemotaxis in vitro and colonization in vivo. Methods By using pET42a plasmid and E. coli BL21DE3 as expression vector and expression strain, respectively, prokaryotic expression systems of cheA and cheY genes of C. jejuni strain NCTC11168 was constructed. Rabbits were immunized with the target recombinant expression proteins, rCheA and rCheY, to prepare the antisera. rCheA-IgG and rCheY-IgG in the antisera were separated using DEAE-52 ion exchange column. pBluescript- II -SK was applied to construct suicide plasmid which used to generate cheA gene knock-out mutant (cheA-). A chemotaxis model in vitro of C. jejuni based on DOC-HAP, the chemotactic ability of cheA' mutant as well as the effect of rCheA-IgG and closantel inhibiting the bacterial chemotaxis were demonstrated. The phosphorylation levels of CheA and CheY after DOC treatment were examined by using either rCheA-IgG or CheY-IgG capture method. The difference of colonization ability between cheA- mutant and wild-type of C. jejuni in mice were checked and then compared. Results The constructed prokaryotic expression systems could efficiently express rCheA and rCheY. The data from PCR and sequencing confirmed the cheA gene knock out from cheA- mutant chromosome. cheA- mutant lost its chemotactic ability towards DOC. Both the rCheA-IgG and closantel could inhibit the chemotaxis of wild-type of C. jejuni (P < 0.05 ). When treatment of DOC, the phosphorylation levels of CheA and CheY in wild-type of C. jejuni rapidly decreased (P < 0. 05 ). The colonization ability in murine jejunum of cheA- mutant was also lower than that of the wild-type ( P<0.05 ) . Conclusion Chemotaxis-associated two-component signaling system (Che-TCSS) of C. jejuni are composed of CheA and CheY, and the two proteins are activated by dephosphorylation. CheA in the Che-TCSS play a critical role in chemotaxis in vitro and colonization in vivo of C. jejuni, and this protein can be used as a target for developing novel anti- C. jejuni drugs.

Objective To study the application value of breast gland needle aspiration pathology on the early diagnosis of breast cancer. Methods 1324 cases of breast swelling, tumor and spilling-over patients were diagnosed and differentiation diagnosed needle aspiration pathology in position using disposable plastic injector of 5-10 volume. And it was compared with clinical and pathology tissue, CT, super-sound , breast scanning, mini-nucleus ,tuberculosis antibody ,CA153 etc. Results Breast gland needle aspirate pathology has important application value not only on diagnosis breast diseases, but also in the early diagnosis and differentiation diagnosis of breast cancer. It has especial instructive significance in differentiating whether breast proliferation and glandular fibro adenoma transfer into cancer. Puncture success rate is high up to 100%,early diagnosis rate is 16.9%,the whole diagnosis correctness is 98.6%,which is highest historically. Conclusions Breast needle aspirate pathology has the merits of little injury, being simple and quick, safe and sound, economic and practical correct results. All items of technology index are evidently higher than traditional diagnosis methods. It can’t be replaced by any other methods by now and it has high spreading value.

Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2( rSph2 ) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatogaphy. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. Interrogans strain Lai infecting J774A. 1 and IAR20 cells. Results The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern. 10μg/ml rSph2 could induce the apoptosis of J774A. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira.

Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Occupational Health ; Pneumoconiosis ; diagnostic imaging ; Radiographic Image Enhancement ; methods ; Young Adult

Objective To determine the sequence diversity in groEL gene of Helicobacter pylori isolates,and the antigenicity,immunoreactivity and immunoprotection of recombinant expressed GroEL (rGroEL) against H.pylori-infection in mice.Methods The sequences of groEL genes from H.pylori isolates were detected by PCR and sequencing.A prokaryotic expression system of H.pylori groEL gene using pET42a and E.coli BL21DE3 was generated.SDS-PAGE and Gel Image Analyzer were used to measure the yield of rGroEL and Ni-NTA affinity chromatography was applied to extract the expressed rGroEL.The antigenicity and immunoreactivity of rGroEL were examined using ELISA and Western blot assay.The membrane location of GroEL was determined by ELISA using whole cells of H.pylorias the coated antigen.The immunoprotection of rGroEL was detected in H.pylori-infected mouse model.Results All the 35 tested H.pylori isolates possess the groEL gene with 99.4％ to 100％ sequence identities.The yield of rGroEL expression occupied 56％ of the total bacterial proteins and the extracted rGroEL showed a single band in gel after SDSPAGE.rGroEL could induce the production of high-titer IgG in rabbits or mice.The lgG against whole cell of H.pylori(Hp-IgG) and rGroEL-IgG could recognize as well as combine with rGroEL protein.GroEL is a superficial protein antigen of H.pylori.The immunization with 50,100 or 200 μg rGroEL prevented 50.0％ (6/12),75.0％ (9/12) or 91.7％ (11/12) mice from infection of H.pylori strain SS1.The immunoprotective rate ( 91.7％ ) due to the immunization of 200 μg rG roEL was significantly higher than that ( 50.0％ ) of 50 μg rGroEL ( P＜0.05 ).Conclusion The GroEL protein is a sequence-conserved,immunogenic and immunoprotective superficial antigen of H.pylori,which can be used as the immunogen candidate for developing genetically engineering vaccines.

Objective To explore the relationship between DNA repair gene XRCC1 and XPD polymorphisms and individual susceptibility to prostate cancer. Methods PCR-restriction fragment length polymorphism assay was used to analyze the XRCC1 (C26304T and G28152A) and XPD A35931C polymorphisms in 358 prostate cancer patients and 312 healthy controls. Unconditional logistic regression analysis was performed to calculate odd ratio (OR) and 95% confidence interval (CD for estimating the correlation between different genotypes and prostate cancer risks. Results Forty-seven(13.1%) cases present XRCC1 28152AA genotype in prostate cancer group, while 24 cases in the control group (7. 1%), individuals with this genotype had a significantly increased risk for prostate cancer (OR 1. 924, 95%CI=1.126 - 3. 288, P=0. 017). There was no significant difference between two groups at XRCC1 C26304T and XPD A35931C sites. Combined analysis of the three sites polymorphisms showed that individuals with XRCC1 28152 AA and XPD 35931AC+CC genotype had a higher risk of prostate cancer than those with three wild genotypes (OR = 3. 087,95%CI 1. 081 - 8.813;OR = 3. 376,95%CI 1.067-10.683;OR 3. 216,95%CI=1. 439-7.188,P = 0. 004). Analysis stratified by age of onset, PSA, Gleason score and T stage revealed that XRCC1 28152AA and XPD 35931 AC+CC high-risk genotype was especially associated with early age at onset of prostate cancer (P<0. 05). Conclusions The XRCC1 and XPD genotypes may be contributed to the risk of developing prostate cancer, particularly for younger patients.

Calcinosis ; pathology ; Child ; Humans ; Inhibins ; metabolism ; Male ; S100 Proteins ; metabolism ; Sertoli Cell Tumor ; metabolism ; pathology ; surgery ; Testicular Neoplasms ; metabolism ; pathology ; surgery ; Vimentin ; metabolism