Changes of ECM and CAM gene expression profile in the cirrhotic liver after HCV infection: Analysis by cDNA expression array

AIM: We aimed to observe the expression of extracellular matrix (ECM) and cellular adhesion molecules (CAM) in cirrhotic liver tissues after hepatitis C virus (HCV) infection. METHODS: Twelve patients with post HCV inflammatory liver cirrhosis were selected to evaluate their liver function and other virological, pathological parameters. Then three specimens of cirrhotic patients whose health assessment results and laboratory data were similar and three normal liver specimens explanted from liver grafts prepared for liver transplantation were chosen for investigating gene expression of ECM and CAM using cDNA expression array. RESULTS: The cDNA array assay revealed 36.7% (36/96)of genes with changes, in which 26.3% (26/96) was up regulated and 10.1% (10/96) was down-regulated. Integrin (ITGA), collagen (COL), ADAMTS were identified as the characteristic changes of ECM and CAM gene expression levels. ITGA were demonstrated β1 and β2 sub-section changed in liver cirrhosis.CONCLUSION: ECM and CAM play an important role inthe progression of liver cirrhosis after HCV infection. The capital mechanism is related to the inflammatory cellsinfiltration, the activation and transformation of ECM producing cells and the imbalance between production and elimination of ECM.

EXPRESSION OF MIDKINE IN BENIGN, PREMALIGNANT AND MALIGNANT VULVAR TUMORS

OBJECTIVE: To clarify the role of midkine (MK) in vulvar carcinogenesis though examination of its expression in vulvar lesions including vulvar condyloma acuminata (VCA), vulvar intraepithelial neoplasia (VIN) and vulvar squamous cell carcinomas (VSCC), and to analyze the relationship between MK expression and human papilloma virus (HPV) infection. METHODS: Thirty VSCC, 15 VIN and 10 VCA patients were studied by streptavidin-biotin-immunoperoxidase method. MK expression was compared with clinicopathologic features of vulvar tumors. RESULTS: MK was expressed in 26 of 30 VSCC (87%), 3 of 5 VIN III and all VCA samples, whereas no MK expression was detected in the VIN I-II samples or in normal epithelium. The difference of MK expression between VIN III and VSCC was statistically significant (P

Role of AFP mRNA expression in peripheral blood as a predictor for postsurgical recurrence of hepatocellular carcinoma: A systematic review and meta-analysis

AIM:To identify the role of alpha-fetoprotein (AFP) mRNA expression in peripheral blood one week after surgery as a predictor for recurrence of hepatocellular carcinoma (HCC). METHODS: Published studies fulfilling the selection criteria were identified by searching several databases online. After a methodology assessment using a quality scale designed by European Lung Cancer Working Party, data in each research were aggregated by means of meta-analysis. RESULTS: Altogether 368 cases were included in the 9 selected studies, which fulfilled the selection criteria. The quality scores ranged from 35% to 84% with a median score of 55%. The 'design' subscore had the lowest median value (38%). By aggregating the data, a high x2 value (77.576) was presented. The fail-safe number was 136 and 64 for P= 0.05 and 0.01, respectively. CONCLUSION: AFP mRNA expression in peripheral blood 1 wk after surgery correlated with the recurrence of HCC and was a good predictor for tumor recurrence.

Association between TRAIL expression on peripheral blood lymphocytes and liver damage in chronic hepatitis B

AIM:To explore a novel mechanism for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), upregulation of CD4+ and CD8+T lymphocytes participating in the patho-physiological process of chronic hepatitis B (CHB). METHODS: The levels of serum soluble TRAIL (sTRAIL), serum IFN-γ and membrane-bound TRAIL expression on peripheral leucocytes from 58 CHB patients were examined by ELISA and flow cytometry respectively. The levels of TRAIL were compared with the baseline levels of 17 healthy controls, and correlation analysis was performed between ALT, TBIL, PT, morphological change in hepatic tissues, and serum IFN-γ. RESULTS: The results showed that TRAIL levels on membranes of CD4+, CD8+ T cells in CHB patients were much higher than those in healthy controls (P<0.001), and were correlated with serum TBIL (r=0.354, P= 0.008 for CD4+ and r= 0.522, P= 0.000 for CD8+, respectively), ALT (r= 0.393, P= 0.003 for CD8+), PT (r = 0.385, P = 0.004 for CD8+) and serum IFN-y level (r = 0.302, P= 0.011 for CD4+ and r= 0.307, P= 0.009 for CD8+). On the contrary to membrane-bound TRAIL expression, serum level of sTRAIL was not correlated with that of TBIL and PT, though it was higher than that of the normal population and was positively correlated with serum HBeAg expression (r= 0.695, P = 0.001). CONCLUSION: The expression level of TRAIL on the membrane of lymphocytes was upregulated and associated with the liver injury in CHB patients. These findings suggest that upregulation of TRAIL expression may be induced by virus antigen and inflammatory cytokine IFN-γ.

Differential expression of Bcl-2 and Bax during gastric ischemia-reperfusion of rats

AIM: To investigate expression of Bcl-2 and Bax in gastric ischemia-reperfusion (GI-R) and involvement of extracellular signal-regulated kinase (ERK) 1/2 activation. METHODS: The GI-R model was established by ligature of the celiac artery for 30 min and reperfusion in SpragueDawley rats. Rats were assigned to groups in accordance with their evaluation period: control, 0, 0.5, 1, 3, 6, 24, 48, and 72 h. Expression and distribution of Bcl-2 and Bax proteins were analyzed by immunohistochemistry and western blotting in gastric tissue samples after sacrifice. RESULTS: Compared with controls, the percentage of positive cells and protein levels of Bcl-2 decreased inthe early phases of reperfusion, reached its minimum at 1 h (P < 0.05); it then increased, reaching its peak at 24 h of reperfusion (P < 0.05). The pattern of Bax expression was opposite to that of Bcl-2. Bax expression increased after reperfusion, with its peak at 1 h of reperfusion (P < 0.05), and then it decreased gradually to a minimum at 24 h after reperfusion (P < 0.05). On the other hand, inhibition of activation of ERK1/2 induced by PD98059, a specific upstream MEK inhibitor, had significant effects on Bcl-2 and Bax in GI-R. Compared with GI-R treatment only at 3 h of reperfusion, PD98059 reduced the number of Bcl-2 positive cells (0.58% of R3h group, P < 0.05) and Bcl-2 protein level (74% of R3h group, P < 0.05) but increased the number of Bax-positive cells (1.33-fold vs R3h group, P < 0.05) and Bax protein level (1.35-fold of R3h group, P < 0.05). CONCLUSION: These results indicated that the Bcl-2 and Bax played a pivotal role in the gastric mucosal I-R injury and repair by activation of ERK1/2.

Gene expression profiles in peripheral blood mononuclear cells of SARS patients

AIM： To investigate the role of inflammatory and anti-vira genes in the pathogenesis of SARS. METHODS： cDNA microarrays were used to screen the gene expression profiles of peripheral blood mononuclear cells （PBMCs） in two SARS patients （one in the acute severe phase and the other in the convalescent phase） and a healthy donor. In addition, real-time qualitative PCR was also performed to verify the reproducibility of the microarray results. The data were further analyzed. RESULTS： Many inflammatory and anti-viral genes were differentially expressed in SARS patients. Compared to the healthy control or the convalescent case, plenty of pro-inflammatory cytokines such as IL-1, TNF-α, IL-8, and MAPK signaling pathway were significantly upregulated in the acute severe case. However, anti-inflammatory agents such as IL-4 receptor, IL-13 receptor, IL-1Ra, and TNF-α-induced proteins 3 and 6 also increased dramatically in the acute severe case. On the contrary, a lot of IFN-stimulated genes like PKR, GBP-1 and 2, CXCL-10 and 11, and JAK/STAT signal pathway were downregulated in the acute severe case compared to the convalescent case. CONCLUSION： Gene expression in SAPS patients mirrors a host state of inflammation and anti-viral immunity at the transcription level, and understanding of gene expression profiles may make contribution to further studies of the SAPS pathogenesis.

Effects of resistin-like molecule β over-expression on gastric cancer cells in vitro

AIM: To investigate the effects of resistin-like molecule β (RELMβ) over-expression on the invasion, metastasis and angiogenesis of gastric cancer cells. METHODS: Human RELMβ encoding expression vec tor was constructed and transfected into the RELMβ lowly-expressed gastric cancer cell lines SGC7901 and MKN-45. Gene expression was measured by Western blotting, reverse transcription polymerase chain reaction (PCR) and real-time quantitative PCR. Cell proliferation was measured by 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry, colony formation and 5-ethynyl-20-deoxyuridine incorporation assays. The in vitro migration, invasion and metastasis of cancer cells were measured by cell adhesion assay, scratch assay and matrigel invasion assay. The angiogenic capabilities of cancer cells were measured by tube formation of endothelial cells. RESULTS: Transfection of RELMβ vector into SGC-7901 and MKN-45 cells resulted in over-expression of RELMβ, which did not infl uence the cellular proliferation. However, over-expression of RELMβ suppressed the in vitro adhesion, invasion and metastasis of cancer cells, accompanied by decreased expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. Moreover, transfection of RELMβ attenuated the expression of vascular endothelial growth factor and in vitro angiogenic capabilities of cancer cells. CONCLUSION: Over-expression of RELMβ abolishes the invasion, metastasis and angiogenesis of gastric cancer cells in vitro, suggesting its potentials as a novel therapeutic target for gastric cancer.

Differentiation of mouse embryonic stem cells into insulin-secreting cells in vitro

Regenerative medicine, including cell-replacement strategies, may have an important role in the treatment of type 1 diabetes which is associated with decreased islet cell mass. To date, significant progress has been made in generating insulin-secreting 13 cells from pluripotent mouse embryonic stem cells （ESCs）.The aim of this study is to explore the potential of regulating the differentiation of ESCs into pancreatic endocrine cells capable of synthesizing the pancreatic hormones including insulin, glucagon, somatostatin and pancreatic polypeptide under proper conditions. Undifferentiated ES cell line was stably transfected with mouse RIP-YFP plasmid construction in serum-free medium using LipofectamineTM 2000 Reagents. We tested pancreatic specific gene expression and characterized these ESC-derived pancreatic endocrine cells. Most of these insulin-secreting cells co-expressed many of the phenotypic markers characteristic of 13 cells such as insulinl, insulin2, Isletl, MafA, insulinoma-associated antigen 1 （IA1） and so on, indicating a similar gene expression pattern to adult islet 13 cells in vivo. Characterization of this population revealed that it consisted predominantly of pancreatic endocrine cells that were able to undergo pancreatic specification under the appropriate conditions. We also demonstrated that zinc supplementation mediated up-regulation of insulin-secreting cells as an effective inducer promoted the development of ESC-derived diabetes therapy. In conclusion, this work not only established an efficient pancreatic differentiation strategy from ESCs to pancreatic endocrine lineage in vitro, but also leaded to the development of new strategies to derive transplantable islet-replacement 13 cells from embryonic stem cells for the future applications of a stem cell based therapy of diabetes.

Expression and purification of the recombinant outermembrane protein Tp0453 of Treponema pallidum and its characterization of immuno-competence

To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was amplified from the complete genome of T.pallidum by PCR, subcloned into expression vector pQE32 to generate the recombinant plasmid pQE32/Tp0453, then expressed in E.coli M15 and analyzed by SDS/PAGE and Western blotting. The fusion protein expressed was purified with Ni-NTA affinity chromatography. Its immuno-reactivity was assayed by indirect ELISA, and the immunogenicity was determined by immunization with this fusion protein in New Zealand rabbits. In the present study, a fusion protein of molecular weight about 32 kDa was obtained. As demonstrated by Western blotting, the recombinant protein could react specifically with positive IgG sera of patients with syphilis, and the antibodies against T.pallidum in human sera were successfully detected by indirect ELISA. Both the sensitivity and specificity of ELISA based on the Tp0453 fusion protein as were 100% (30/30) when detected with control sera. In comparison with the results of IgG ELISA with those of TPPA. It was found that the sensitivity of ELISA was 96.8% and the specificity was 100%. The difference of ELISA and TPPA was not significant, and the concordance of results between ELISA and TPPA was 98.2%. In addition, specific humoral responses could be elicited by immunization with the recombinant fusion protein in New Zealand rabbits with a specific antibody titer of 1∶1280 after 3 successive doses of immunization. These results demonstrate that the expressed recombinant fusion protein shows excellent immuno-competence and provide foundation to develop a quick diagnostic kid applied to detect the presence of T.pallidum infections.

Expression and Purification of the Major Outer Membrane Protein of Chlamydia Trachomatis in Prokaryotic Cell

To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C.trachomatis) and to express the fusion protein in E.coli BL21, the MOMP gene was amplified by polymerase chain reaction (PCR) from genome of C.trachomatis serovar D. The fragment was cloned into the prokaryotic expression vector pET-22b(+) after digestion with BamHⅠ and NotⅠ and transformed into E.coli XL1-Blue. Recombinants were selected by enzyme digestion and sequencing and the recombinant plasmid with MOMP gene was then transformed into E.coli BL21 with IPTG to express the target gene. The expression recombinant proteins were purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Western blot. It was found that a 1.2?kb MOMP gene was isolated. The DNA sequence of MOMP was found to be just the same as the sequence published by GenBank. A recombinant plasmid containing MOMP gene was constructed to express the fusion proteins in E.coli. SDS-PAGE analysis showed that the relative molecular weight of the recombinant protein was about 47?kDa that was consistent with the theoretical predicted value, and the specificity of the expressed protein was conformed by Western blot. It concluded that the MOMP gene could be expressed in the prokaryotic system, by which it provided the foundation for the future studies on the biological activities of C.trachomatis and for the development of vaccine against this pathogen.

Correlation between circulating myeloid-derived suppressor cells and Th17 cells in esophageal cancer

AIM: To perform a comprehensive investigation into the potential correlation between circulating myeloidderived suppressor cells (MDSCs) and Th17 cells in esophageal cancer (ECA). METHODS: A total of 31 patients newly diagnosed with ECA and 26 healthy subjects were included in the current study. The frequencies of MDSCs and Th17 cells in peripheral blood were determined by flow cytometry. The mRNA expression of cytokines, arginase 1 (Arg1) and inducible NO synthase (iNOS) in peripheral blood mononuclear cells (PBMCs) and plasma Arg1 were assessed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: There was an increased prevalence of MDSCs in the peripheral blood from ECA patients (15.21% ± 2.25%) when compared with healthy control (HC) (1.10% ± 0.12%, P < 0.0001). The plasma levels of Arg1 in ECA patients were significantly higher than those in HC (28.28 ± 4.10 ng/mL vs 9.57 ± 1.51 ng/ mL, P=0.0003). iNOS mRNA levels in the peripheral blood of ECA patients also showed a threefold increase compared with HC (P=0.0162). The frequencies of Th17 cells (CD4 + IL-17A + ) were significantly elevated in ECA patients versus HC (3.50% ± 0.33% vs 1.82% ± 0.19%, P=0.0001). Increased mRNA expression of IL-17 and ROR-γt was also observed in ECA patients compared with HC (P=0.0041 and P=0.0004, respectively), while the mRNA expression of IL-6 and tumor necrosis factor-α (TNF-α) showed significant decreases (P=0.0049 and P < 0.0001, respectively). No obvious correlations were found between the frequencies of MDSCs and Th17 cells in the peripheral blood from ECA patients(r=-0.1725, P=0.3534). Arg1 mRNA levels were positively correlated with levels of IL-6 (r=0.6404, P=0.0031) and TNF-α (r=0.7646, P=0.0001). Similarly, iNOS mRNA levels were also positively correlated with levels of IL-6 (r=0.6782, P=0.0007) and TNF-α (r=0.7633, P < 0.0001). CONCLUSION: This study reveals the relationship between circulating MDSCs and Th17 cells, which may lead to new immunotherapy approaches for ECA based on the associated metabolites and cytokines.

Differences in platelet endothelial cell adhesion molecule-1 expression between peripheral circulation and pancreatic microcirculation in cerulein-induced acute edematous pancreatitis

AIM: To investigate the changes of platelet endothelial cell adhesion molecule-1 (PECAM-1) expression on polymorphonuclear leukocytes (PMNs) in peripheral circulation and pancreatic microcirculation in cerulein-induced acute edematous pancreatitis (AEP).METHODS: Fifty Wistar rats were randomly divided into control group (n=10) and AEP group (n=40). A model of AEP was established by subcutaneous injection of cerulein 5.5 and 7.5 μg/kg at 0 and 1 h after the beginning of experiment respectively. PECAM-1 expression on PMNs from splenic vein and inferior vena cava was determined by RT-PCR at mRNA level and determined by flow cytometry at protein level.RESULTS: In experimental rats, an increased PECAM-1mRNA expression was seen from 4 to 8 h of AEP in peripheral circulation (0.77±0.25%, 0.76±0.28%, 0.89±0.30%,1.00±0.21% ), while in pancreatic microcirculation,expression decreased from 2 h and reached the lowest level at 6 h of AEP (0.78±0.29%, 0.75±0.26%, 0.62±0.28%,0.66±0.20%). There were significant differences at 8-h time point of AEP between peripheral circulation and pancreatic microcirculation (1.00±0.21% vs0.66±0.20%, P＜0.05).Meanwhile,the difference at protein level was also found.CONCLUSION: A reverse expression of PECAM-1 on PMNs was found between peripheral circulation and pancreatic microcirculation, suggesting that inhibition of PECAM-1expression may improve the pathological change of AEP.

Relationship between FHIT Expression and HPV Status in Vulvar Squamous Cell Carcinomas

OBJECTIVE To investigate the relationship between fragilehistidine triad (FHIT) depletion and human papillomavirus (HPV)status in the vulvar squamous cell carcinomas (VSCC).METHODS Immunohistochemical method was used to detectthe expression of FHIT protein in 42 cases of VSCC and 10 casesof normal vulvar tissues. PCR was used to detect HPV infectionstatus. We analyzed the relationship of the expression of FHITprotein between the 2 groups: HPV positive and HPV negative, aswell as the clinically pathological characteristics.RESULTS The expression of FHIT was positive in all normal vul-var tissues, and 71.4% was depletion in VSCC (P < 0.01). Abnormalexpression of FHIT was significantly correlated with pathologicalgrade (P < 0.05). There was a significant difference between FHITdepletion and HPV infection based on the statistial analysis (P <0.05).CONCLUSION The FHIT depletion is related to occurrenceand development of VSCC, and the abnormal expression of FHITsignificantly correlates with HPV infection.

Diagnostic and monitoring values of circulating AFPmRNA and HS-GGT for hepatocellular carcinoma

Objective: Early finding of hepatocellular carcinoma(HCC) and monitoring of its metastasis are of the utmost importance. The objectives of this study were to investigate the values of circulating hepatoma-specific gamma-glutamyl transferase(HS-GGT) fraction and α-fetoprotein mRNA(AFP-mRNA) from peripheral blood mononuclear cells(PBMCs) in diagnosis or monitoring metastasis of HCC. Methods: Total RNA was extracted from hepatomas or PBMCs of patients, synthesized to AFP-cDNA through random primers and reverse transcriptase, amplified by using a nested PCR assay, and confirmed by sequencing. Simultaneity, the HS-GGT activities were quantitatively determined in sera of patients. The comprehensive assessments of two markers were investigated in HCC patients. Results: The amplified fragments of AFP-mRNA were identical to original designed ones(159 bp) and confirmed by sequencing. The lowest sensitivity was 2 ng/L of total RNA. The incidences of AFP-mRNA and HS-GGT were 60.4%(113 of 187) and 84.5%(158 of 187) in the HCC group, and their significantly higher(P < 0.01) than that in any of other groups. The high frequencies of HS-GGT and AFP-mRNA were 78.2% and 56.4% in the HCC patients with AFP < 50 ng/mL. All positive of AFP-mRNA was found in HCC patients with extrahepatic metastasis. No significant correlation was found between tumor size and AFP-mRNA or HS-GGT. The total incidence of combined AFP-mRNA and HS-GGT detection was 93.6 % for HCC diagnosis. Conclusion: HS-GGT and AFP-mRNA are specific markers for HCC diagnosis or differentiation, especially the former for early finding and the later for monitoring metastasis of HCC.

Anti-angiogenesis effect of generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide on breast cancer in vitro

Objective:To study the effects of the generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide(G4PAMAM/VEGFASODN) compound on the expressions of vascular endothelial growth factor(VEGF) and its mRNA of breast cancer cells and on the inhibition of vascular endothelial cells. Methods:We examined the morphology of G4PAMAM/VEGFASODN compound and its pH stability,in vitro transfection efficiency and toxicity,and the expressions of VEGF and its mRNA. Methyl thiazolyl tetrazolium assay was used to detect the inhibitory function of the compound on vascular endothelial cells. Results:The compound was about 10 nm in diameter and was homogeneously netlike. From pH 5 to 10,it showed quite a buffered ability. The 48-h transfection rate in the charge ratio of 1:40 was 98.76%,significantly higher than that of the liposome group(P<0.05). None of the transfection products showed obvious toxicity on the cells. The expressions of both VEGF protein and its mRNA after G4PAMAM/VEGFASODN transfection decreased markedly. Conclusion:With a low toxicity,high safety,and high transfection rate,G4PAMAM/VEGFASODN could be a promising gene vector. Specifically,it inhibits VEGF gene expression efficiently,laying a basis for further in vivo animal studies.

Restriction of exogenous DNA expression by SAMHD1

SAMHD1(Sterile Alpha Motif and Histidine-aspartate Domain containing protein 1) has been documented as a host factor that restricts HIV-1 and some DNA viruses. In this work, we attempted to explore possible effects of SAMHD1 on exogenous DNA and show that SAMHD1 exerts a general inhibition on the expression of exogenous DNA in vitro and in mice. This inhibition is achieved through repressing transcription of exogenous DNA. Intriguingly, unlike SAMHD1’s restriction of HIV-1, such restriction does not require the dNTPase or RNase activities, or T592 phosphorylation of SAMHD1. Mechanistically,SAMHD1 enhances the expression of interferon regulatory factor-1(IRF1), while IRF1 upregulation was demonstrated to inhibit exogenous DNA expression in a similar fashion as SAMHD1. IFNk1, whose induction has been associated with IRF1 activation, is dispensable for SAMHD1/IRF1-mediated restriction of exogenous DNA, and neither type Ⅰ nor Ⅱ interferons appear to be involved. We also demonstrate that SAMHD1/IRF1-mediated restriction can effectively inhibit hepatitis B virus(HBV) antigen expression and progeny virus production in mouse models. In conclusion, these data support restriction of exogenous DNA as a novel function of SAMHD1.

Comparing gene expression profiles of Kashin-Beck and Keshan diseases occurring within the same endemic areas of China

In this study, differentially expressed genes in peripheral blood from patients with Kashin-Beck disease and Keshan disease were compared to further investigate the etiology and pathogenesis of both diseases, which occur in a common endemic area of China. Twenty Kashin-Beck disease patients and 12 healthy controls, and 16 Keshan disease patients and 16 healthy controls, were grouped into four pairs. Patients and controls were selected from common endemic areas for the two diseases. Total RNA was isolated from peripheral blood mononuclear cells from all patients and controls, and gene expression profiles analyzed by oligonucleotide microarrays. Sixteen genes differentially expressed in both Kashin-Beck disease and Keshan disease (versus controls) were identified, and comprised nine genes showing synchronous and seven asynchronous expression. The Comparative Toxicogenomics Database shows that expression and biological function of these genes can be affected by multiple environmental factors, including mycotoxin and selenium content, potential environmental risk factors for the two diseases. Thus, these shared differentially expressed genes may contribute to the distinct organ lesions, caused by common environmental risk factors of Kashin-Beck disease and Keshan disease.