广州市荔湾区2007~2012年霍乱监测结果分析

目的分析广州市荔湾区2007~2012年霍乱监测情况,为预防控制提供依据。方法根据《广州市霍乱监测方案》以及《霍乱防治手册》(第5版),对腹泻病人和重点人群粪便、水产品、食品及外环境水体进行采样监测、分离和鉴定。结果 2007~2012年荔湾区共监测霍乱标本20 687份,阳性19份,总阳性率0.09%。其中检索腹泻病人16 319份,阳性7份,阳性率0.04%;重点人群446份,无阳性;水产品等食品2 104份,阳性8份,其中以蛙类为主,阳性率0.38%;外环境监测点共监测1 818份,阳性4份,阳性率0.22%。在19份阳性标本中,血清学分型O1群稻叶型11株、小川型6株,O139群2株。霍乱主要毒力基因检测结果均为阴性。结论荔湾区外环境水体、水产品存在霍乱弧菌,应加强蛙类等水产品监测,及时了解其污染状况,以便采取有针对性的措施,预防控制霍乱的发生和流行。     

Evaluation of recombinant protein r140, a polypeptide segment of tegumental glycoprotein Sm25, as a defined antigen vaccine against Schistosoma mansoni

To investigate the role of tegumental glycoprotein Sm25 in protective immunity against schistosomiasis, codons 43-182 of its gene (GP22) were amplified by PCR and cloned in the pET 15b bacterial expression system. Recombinant protein r140 was inducibly expressed in the presence of rifampicin and purified by Ni-affinity chromatography. In different vaccination trials, Balb/c mice and Fischer rats repeatedly immunized with r140 in combination with one of several adjuvants (alum, cholera toxin or complexed into proteosomes) produced high titre anti-r140 responses. These antibodies detected an N-glycanase sensitive, 25 kDa antigen in a detergent solubilized worm fraction using Western immunoblotting. The choice of adjuvant affected the isotype distribution of the specific anti-r140 antibodies. Despite the presence of high antibody titres and isotypes which have been shown to correlate with protective immunity, protection against subsequent cercarial challenge was not observed. In addition, no appreciable effects on worm sex ratios or liver egg yields were detected in mice. Studies involving biotin labelling of membrane proteins in live worms showed that the majority of anti-r140 reactive molecules present in adult schistosomes are biotinylated after permeabilization of the parasite surface. Several possibilities to account for the lack of protective immunity are analysed .     

Nitrergic neurons in the medial amygdala project to the hypothalamic paraventricular nucleus of the rat

We investigated nitric oxide (NO)-producing neurons in the amygdala which project to the hypothalamic paraventricular nucleus (PVN) of the rat using retrograde tracing and NADPH-diaphorase histochemistry. Numerous NADPH-diaphorase positive neurons with moderate staining were observed mainly in the medial amygdaloid nucleus. We confirmed that these NADPH-diaphorase positive neurons are identical to NO synthase (NOS)-immunoreactive neurons by double staining with NADPH-diaphorase histochemistry and NOS immunocytochemistry. Most neurons containing cholera toxin B subunit (CTb) – which was retrogradely transported from the PVN – were observed in the medial amygdaloid nucleus. In other amygdaloid nuclei, they were observed much less in the central nucleus, basomedial and anterior cortical nucleus. Double labeled neurons by NADPH-diaphorase and CTb were also identified mostly in the medial nucleus. Approximately 40% of the neurons projecting to the PVN were nitrergic neurons and 16% of NADPH-diaphorase positive neurons in the medial nucleus were revealed to project to the PVN. These results suggest that NO-producing neurons in the medial amygdala directly innervate PVN neurons and regulate neuroendocrine systems such as vasopressin and corticotropin releasing factor release.     

Age-related changes in the morphology of preganglionic neurons projecting to the paracervical ganglion of nulliparous and multiparous rats

The aim of this study was to investigate age-related changes in preganglionic neurons in the lumbar and sacral spinal cord of the female rat that may underlie impaired control of the urogenital system in old age. Preganglionic sympathetic and parasympathetic neurons of young adult, aged nulliparous and aged multiparous rats were identified by retrograde tracing with cholera toxin subunit-B and subsequent immunocytochemistry. Labeled preganglionic neurons were scanned, processed and analyzed using the confocal microscope. Measurements were made of soma area, number of primary dendrites, number of dendritic branch points and total dendritic length. There were significant decreases in the number of primary dendrites, number of dendritic branch points and total dendritic length of sympathetic preganglionic neurons in both nulliparous and multiparous aged rats compared to the young adult group. No significant differences were found in the dendritic morphology of aged parasympathetic preganglionic neurons. Soma area was not significantly different between age groups for sympathetic or parasympathetic preganglionic neurons. These changes in dendritic morphology may result in altered control of the lower urogenital tract in aged nulliparous and multiparous female rats.     

The effect of nicotinic and muscarinic receptor blockade on cholera toxin induced intestinal secretion in rats and cats

The effects of hexamethonium (cholinergic nicotinic receptor antagonist) and atropine (cholinergic muscarinic receptor antagonist) on cholera toxin induced secretion were investigated in denervated segments of the small intestine of rats and cats. While there was no effect of atropine, hexamethonium markedly inhibited choleraic secretion and turned it into a net fluid absorption in many animals. This observation further strengthens our hypothesis that the enteric nervous system is involved in cholera secretion.     

2002-2011年霍乱疫情分析及防控对策

目的:掌握霍乱流行规律,为政府预防控制提供科学决策依据。方法:搜集2002-2011年霍乱疫情发生信息资料,分析霍乱发生的相关因素及所采取的措施,评估效果。结果:2002-2011年间安县发生的三起霍乱疫情均为农村群体聚餐,菌群为霍乱弧菌0139,菜品中均有甲鱼,在三起霍乱疫情发生中有两起怀疑感染来源于甲鱼,其中一起从同一批次的甲鱼中检出霍乱弧菌0139与病例同源,为厨师未按生熟食品分开进行加工制作,导致食品交叉污染所致。结论:加强农村群体聚餐、农村厨师的管理,规范厨师加工制作食品,避免食物交叉污染,加强居民消化道传染病预防控制知识的健康教育,加强监测,在防控策略上关口前移,重心下沉是预防控制霍乱等消化道传染病的金钥匙。     

Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold

V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boosting with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response.     

Stimulation of cAMP signalling allows isolation of clonal pancreatic precursor cells from adult mouse pancreas

Aims/hypothesis Duct cells of the pancreas are thought to include latent progenitors of islet endocrine cells that can be induced to differentiate by appropriate morphogens. Here we developed a method for isolating pancreatic ductal epithelial cells from adult mice that overcomes the shortcomings of previous methods. Materials and methods Pancreatic ductal cells were grown in serum-free DMEM/F12 medium in the presence of cholera toxin or 8-bromo-cyclic adenosine monophosphate, which is known to be an intracellular cAMP generator. Single cell cloning was performed by limiting dilution in serum-free medium. Results The isolated clonal cells expressed high levels of cytokeratin and Ipf1 (formerly known as Pdx-1). Adenovirus-mediated expression of ngn3 (also known as Neurog3) and Ptf1a in these cells induced expression of insulin and somatostatin, and of carboxypeptidase A, respectively. Furthermore, albumin production was induced by dexamethasone or by long-term culture in serum-containing medium. Conclusions/interpretation Stimulation of the cAMP-dependent signalling allowed us to isolate clonal pancreatic ductal cells from adult mice. These cells are able to partially differentiate into endocrine cells, exocrine cells and hepatocyte-like cells and are therefore considered to have the characteristics of endodermal progenitor cells.     

Positive and negative regulation of water channel aquaporins in human small intestine by cholera toxin

Analysis of osmotic water permeability of aquaporin (AQP) 1, AQP3 and AQP4, which are expressed in human small intestine, in the presence or absence of cholera toxin (CT) was performed using a Xenopus oocyte expression system. When treated with CT, water permeability of AQP4 was facilitated while that of AQP3 was suppressed. AQP1 did not show any significant change of water permeability when treated with CT. An adenylyl cyclase accelerator forskolin showed similar effects as CT did, suggesting that changes of the water permeability of AQP4 and AQP3 were due to an increase of intracellular cAMP concentration. A possibility that these AQPs are responsible molecules for causing acute secretory diarrhoea as in cholera is considered.     

幽门螺杆菌BIB原核表达工程菌的构建及微生物学特性分析

目的构建融合了幽门螺杆菌尿素膜通道蛋白（Ure I）、尿素酶B亚单位（UreB）的表位基因及霍乱毒素B亚单位（CTB）的原核表达质粒pET28a（＋）／ct B／ure I-B（简称BIB）及其原核表达工程菌,并研究其生物学特性。方法参考基因库中ure l、ure B、ct B的基因序列,合成不舍信号肽的ct B基因及ure I、ure B优势表位基因,串联融合,形成多靶点重组ct B／ure I-B基因（简称BIB基因）,并构建pET28a（＋）／BIB原核表达质粒,经限制性内切酶Nco I、Xho I酶切以及DNA测序鉴定正确后,将重组质粒导入大肠杆菌BL21（DE3）中,构建含BIB基因的原核表达工程菌。经乳糖诱导后,SDS-PAOE电泳检测重组蛋白（rBIB）,Western blot及肌注BALB／c小鼠实验检测rBIB的免疫反应性和免疫原型。结果构建的原核表达质粒pET28a（＋）／BIB,经双酶切和测序分析显示,构建的BIB基因与设计序列100％一致。乳糖诱导后,SDS-PAGE电泳显示在33kD左右出现一条明显蛋白条带,Western blot检测在33kD左右出现特异反应条带,肌注免疫BALB／c小鼠产生了较高的抗体水平。结论成功构建了pET28a（＋）／BIB原核表达质粒及BIB原核表达工程菌,该工程菌可表达重组蛋白rBIB,且该重组蛋白具有良好的免疫反应性及免疫原性。