Role of platelet-activating factor receptor in adhesion and invasion of Aggregatibacter actinomycetemcomitans in human umbilical vein endothelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of platelet-activating factor receptor (PAFR) in adhesion and invasion of phospho- rylcholine (PC)-positive Aggregatibacter actinomycetemcomitans in cultured human umbilical vein endothelial cells (HUVEC).</p><p><b>METHDOS</b>Cultured HUVECs were pretreated with the PAFR antagonist CV3988 or anti-human PAFR monoclonal antibody for 30 min before infection with PC-positive or -negative A. actinomycetemcomitans strains. The bacterial adhesion and invasion and cytotoxicity in the cells were examined using MTT assay.</p><p><b>RESULTS</b>Pretreatment with PAFR antagonists at 100, 200 and 500 nmol/L significantly reduced the adhesion rate (36.29∓3.52)%, (19.04∓3.35)% and (7.69∓3.19%), respectively] and invasion rate [(12.12∓1.58)%, (7.08∓0.29)% and (2.60∓2.26)%, respectively] of PC-positive A.actinomycetemcomitans in HUVECs. Similarly, pretreatment with anti-PAFR antibody also significantly reduced A.actinomycetemcomitans adhesion and invasion in HUVECs [(50.05∓5.28)% and (39.09∓6.50)%, respectively]. Pretreatment with PAFR antagonist (200 and 500 nmol/L) and anti-PAFR antibody (25 µg/mL) significantly increased the viability of HUVECs incubated with PC-positive A.actinomycetemcomitans from (25.39∓9.33)% to (91.12∓3.14)%, (94.12∓2.15)% and (65.5∓1.87)%, respectively, but such pretreatments did not increase the viability of cells incubated with PC-negative A.actinomycetemcomitans.</p><p><b>CONCLUSIONS</b>PAFR plays an important role in the adhesion, invasion, and cytotoxicity of PC-positive A.actinomycetemcomitans in cultured HUVECs.</p>

Pathogenesis of uropathogenic Escherichia coli: role of outer membrane protein T and the mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the role of outer membrane protein T (OmpT) in the pathogenesis of uropathogenic Escherichia.coli.</p><p><b>METHODS</b>In cultured human bladder epithelial cell line 5637, we examined the adhesion ability of wild-type (CFT073), ompT gene knockout (COTD), and revertant (pST) strains of E.coli to the cells and the extracellular matrix (ECM). The expressions of the adhesion gene iha and virulence gene iroN were detected by real-time PCR. Murine models of urinary tract infection with the 3 strains were established to evaluate the bacterial burden of the bladder and kidney tissue and bacterial counts in blood. We also detected the expressions of interleukin-6 (IL-6) and IL-8 in the bladder and kidney tissues of the mice.</p><p><b>RESULT</b>The COTD strain showed a significantly lower cell adhesion rate than CFT073 strain [(4.62∓0.39)% vs (8.81∓1.13)%, P<0.05] with also a lower ECM-adhesion rate [(4.95∓0.59)% vs (8.85∓0.79)%, P<0.05]. The mRNA expressions of iha and iroN in CFT073 strain were 2.1 and 3.8 times that of COTD strain. In the mouse model, the mean bacterial load of CFT073 strain in the bladder tissue was 6.36∓0.06, significantly greater than that of COTD (6.01∓0.07) and revertant (6.29∓0.06) strains (P<0.05); the bacterial load of CFT073 strain in the kidney tissue was also significantly higher than that of COTD strain (6.25∓0.05 vs 5.87∓0.06, P<0.05). In mice infected with the wild-type, knockout, and revertant strains, the detection rates of IL-6, which were identical to those of IL-8, in the inflammatory bladder and kidney tissues were 60%, 12.5%, and 50%, respectively.</p><p><b>CONCLUSIONS</b>OmpT may regulate the expression of the adhesion gene iha and the transferrin gene iroN to affect the adhesion of uropathogenic E.coli to host cells.</p>

Role of MUC2 gene in the regulation of rat intestinal barrier function by probiotics / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate MUC2 expression in rat colons induced by probiotics and its effects on the inhibition of E.coli K1 (E44) penetration of the intestinal barrier by probiotics.</p><p><b>METHODS</b>SD rats were subjected to intragastric administration of probiotics, E44, or probiotics +E44 on a daily basis for 7 days, and MUC2 expression in the colons was determined by RT-PCR. MUC2-targeted shRNA (shRNA MUC2) and scrambled shRNA plasmids (shRNA NC) were respectively transfected into Lovo cells, and the efficiency of MUC2 knockdown was determined using qRT-PCR. Competitive exclusion assay was used to evaluate the effects of the probiotics against E44 adhesion and invasion.</p><p><b>RESULTS</b>Intestinal MUC2 mRNA expression was up-regulated in the rats after intragastric administration of probiotics, while E44 administration caused significantly lowered MUC2 expression. MUC2 expression was down-regulated (by 66.7%) by transfection with shRNA MUC2 in Lovo cells as compared with the negative control and mock control cells. The inhibition of E44 adherence and invasion by probiotics was significantly attenuated in transfected Lovo cell culture (in which the relative adhesion and invasion rates of E44 were 56.64% and 66.64%, respectively) as compared with those in the control group.</p><p><b>CONCLUSION</b>The up-regulation of MUC2 in rat colons can be one of the mechanisms of the probiotics in antagonizing the translocation of the pathogenic bacteria. Silencing MUC2 expression causes attenuated inhibitory effect of the probiotics on E. coli K1 penetration across human intestinal epithelial cells.</p>

Construction of dengue virus-specific full-length fully human antibody libraries by mammalian display technology / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique.</p><p><b>METHODS</b>Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry.</p><p><b>RESULTS</b>Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)].</p><p><b>CONCLUSION</b>Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).</p>

Study of RASSF1A expression and promoter demethylation in Hep-2 cell line / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the effect of 5-Aza-dC and TSA to tumor suppressor gene RASSF1A expression and methylation level in Hep-2 cell line.@*METHOD@#Hep-2 cell line were cultured in vitro and handled with 5-Aza-dC and TSA. Detected RASSF1A expression and methylation level were detected before and after drug intervention using Realtime PCR and MSP.@*RESULT@#(1) Before intervention with drug, tumor suppressor gene RASSF1A was weakly expressed and methylated in Hep-2 cell line. (2) With the effect of 5-Aza-dC and TSA, the methylation of RASSF1A gene was reversed. And the effect of combination of 5-Aza-dC and TSA was similar with 5-Aza-dC alone. There was no obvious effect using TSA alone. (3) With the effect of 5-Aza-dC and TSA, the expression of RASSF1A was improved. And the effect of 5-Aza-dC was stronger than TSA. Synergetic effect was found when using 5-Aza-dC and TSA simultaneously.@*CONCLUSION@#In Hep-2 cell line, Promoter methylation of tumor suppressor RASSF1A may play a very important role in loss of gene expression, but it is not the only cause. 5-Aza-dC and TSA can improve RASSF1A expression by reversing DNA methylation and histone deacetylation.