Expressed sequence tag analysis of adult Clonorchis sinensis, the Chinese liver fluke

Expressed sequence tag (EST) pools represent partial profiles of the gene expressions of organisms. In an effort to construct a Clonorchis sinensis EST pool, 2,387 ESTs were collected from an adult C. sinensis cDNA library and assembled into 1,573 clusters. Of these clusters, 1,225 ESTs (51%) were singletons and 348 clusters consisted of more than two ESTs. There were 848 clusters (54%) that shared significant identity with previously reported proteins, and of these, 401 clusters were categorized into 11 major functional protein classes. Three cDNA clones of fructose-1,6-bisphosphate (FBP) aldolase were selected from the C. sinensis EST pool and analyzed for phylogenic clustering. FBP clones encoded a complete polypeptide, which shared significant identity to those of vertebrate and invertebrate animals and clustered with those of trematodes. We believe that the EST pool described can be confidently used as a platform in multigene researches on C. sinensis gene expression.     

An EF-handed Ca2+-binding protein of Chinese liver fluke Clonorchis sinensis

A cDNA clone encoding 8 kDa protein was retrieved from an EST pool of Chinese liver fluke Clonorchis sinensis. A deduced polypeptide of the cDNA clone was similar to 8 kDa Ca²⁺-binding proteins from other parasitic trematodes, and, thus, named as CsCa8, containing two EF-hand Ca²⁺-binding sites. Homology models predicted CsCa8 to be a single globular structure having four helices and molecular folds similar to Ca²⁺-binding state of other small Ca²⁺-binding proteins. Recombinant CsCa8 protein showed specific Ca²⁺-binding affinity and shifting in native gel mobility assay. Mouse immune sera raised against recombinant CsCa8 protein recognized native CsCa8 from adult C. sinensis worm extract. CsCa8 was localized in oral and ventral suckers, vitelline follicles and subtegumental tissues. These findings suggest that CsCa8 might be involved in cellular Ca²⁺ signal transduction for muscle contraction and egg production.     

Pathology and immunology of Clonorchis sinensis infection of the liver

The existence of clonorchiasis cases among the Asian immigrants and the major clinical and pathologic features encountered in those patients is emphasized. Owing to the longevity of the parasite, clinical symptoms may occur long after endemic exposure. The common manifestations among the immigrants are recurrent pyogenic cholangitis and pancreatitis. The relationship between clonorchiasis and cholangiocarcinoma and the possible pathogenesis of this tumor are discussed. The specific immunologic phenomenon in clonorchiasis is described.     

Characterization of the secreted cathepsin B cysteine proteases family of the carcinogenic liver fluke Clonorchis sinensis

Clonorchis sinensis excretory/secretory products (ESP) have gained high attentions because of their potential to be vaccine candidates and drug targets in C. sinensis prevention. In this study, we extensively profiled the characteristics of four C. sinensis cathepsin B cysteine proteases (CsCB1, CsCB2, CsCB3, and CsCB4). Bioinformatics analysis showed all CsCBs contained signal peptides at the N-terminal. Functional domains and residues were found in CsCB sequences. We expressed four CsCBs and profiled immune responses followed by vaccine trials. Recombinant CsCBs could induce high IgG titers, indicating high immunogenicity of CsCB family. Additionally, ELISA results showed that both IgG1 and IgG2a levels apparently increased post-immunization with all four CsCBs, showing that combined Th1/Th2 immune responses were triggered by CsCB family. Both Real-time polymerase chain reaction (RT-PCR) and Western blotting confirmed that four CsCBs have distinct expression patterns in C. sinensis life stages. More importantly, we validated our hypothesis that CsCBs were C. sinensis excretory/secretory products. CsCBs could be recognized by C. sinensis-infected sera throughout the infection period, indicating that secreted CsCBs are immune triggers during C. sinensis infection. The protective effect was assessed by comparing the worm burden and egg per gram (EPG) between CsCB group and control group, showing that worm burden (P?P?CsCB2 and CsCB3 groups were significantly lower than in control group. In conclusion, we profiled secreted cathepsin B cysteine proteases family for the first time and demonstrated that all CsCB family were C. sinensis excretory/secretory products that may regulate host immune responses.     

我院就诊人群华支睾吸虫感染的临床分析

目的掌握华支睾吸虫感染和患病情况的临床特点,探讨临床简便的确诊方法,指导相应的防治对策。方法对我院2007-2009年随机就诊的广东人群进行大便常规检查,并对发现华支睾吸虫卵的患者的临床资料进行统计分析。结果 56140例受检患者发现,华支睾吸虫感染1029例（1.83%）。主要临床症状为肝区疼痛486例（47.2%）、上腹疼痛297例（28.9%）、腹泻173例（16.8%）、乏力147例（14.3%）、食欲不振107例（10.4%）,主要疾病有胆囊炎462例（44.9%）、胆囊息肉213例（20.7%）、胆管壁增厚182例（17.7%）、胆石症132例（12.8%）。实验室辅助检查中,华支睾吸虫感染组的嗜酸性粒细胞比例（EOS%）及血清γ谷氨酰转肽酶活性（GGT）显著高于非感染患者组（Z=-10.837,P〈0.01;Z=-6.622,P〈0.01）和健康对照组（Z=-7.320,P〈0.01;Z=-6.920,P〈0.01）。丙氨酸氨基转移酶活性（ALT）在华支睾吸虫感染有症状组中显著高于非感染患者组（Z=-3.528,P〈0.01）和健康对照组（Z=-4.948,P〈0.01）,而在华支睾吸虫感染无症状组中与非感染患者组和健康对照组无显著性差异（Z=-1.561,P=0.119;Z=-1.834,P=0.067）。结论肝区疼痛和上腹不适是华支睾吸虫病的主要临床表现,在EOS%和GGT增高的患者中华支睾吸虫卵的检出率明显提高。     

Clonorchis sinensis infection is positively associated with atopy in endemic area

Background Epidemiologic studies have suggested that helminth infections play a protective role against allergy; this inverse association, however, has not been consistent. Clonorchis sinensis, the liver fluke of human, is prevalent in the Far East. The association between C. sinensis infection and allergy has not yet been reported. Objective We evaluated the association between clonorchiasis and atopy or allergic diseases in adults in endemic areas of clonorchiasis. Methods A total of 1116 subjects (males 419, females 697; age range, 30–86; mean age=61 years) were recruited from two endemic areas of C. sinensis in Korea. Clonorchiasis was confirmed by stool examination. Allergic symptoms were evaluated with a modified ISAAC questionnaire, and atopy was defined by skin prick test for common inhalant allergens. Total serum IgE and C. sinensis‐specific IgE level was measured by ELISA and methacholine bronchial provocation test was performed to evaluate airway hyperresponsiveness (AHR). Results Clonorchiasis was positively associated with atopy [odds ratio (OR), 1.856; 95% confidence interval (CI), 1.199–2.873] and high levels of total serum IgE (OR, 1.455; 95% CI, 1.050–2.016). Higher association with clonorchiasis was shown in subjects who showed both atopy and high total serum IgE levels (OR, 2.540; 95% CI, 1.448–4.455). Clonorchiasis had no association with wheezing, AHR, asthma or allergic rhinitis. Conclusion and Clinical Relevance Clonorchiasis was positively associated with atopy in adults in endemic area. Cite this as: M‐H Choi, Y‐S Chang, M. K. Lim, Y. M. Bae, S‐T Hong, J‐K Oh, E. H. Yun, M‐J Bae, H‐S Kwon, S‐M Lee, H‐W Park, K‐U Min, Y‐Y Kim and S‐H Cho, Clinical & Experimental Allergy, 2011 (41) 697–705.     

Gene expression profiling in mouse liver infected with Clonorchis sinensis metacercariae

Clonorchis sinensis, the parasite that causes clonorchiasis, is endemic in many Asian countries, and infection with the organism drives changes in the liver tissues of the host. However, information regarding the molecular events in clonorchiasis remains limited, and little is currently known about host–pathogen interactions in clonorchiasis. In this study, we assessed the gene expression profiles in mice livers via DNA microarray analysis 1, 2, 4, and 6 weeks after induced metacercariae infection. Functional clustering of the gene expression profile showed that the immunity-involved genes were induced in the livers of the mice at the early stage of metacercariae infection, whereas immune responses were reduced in the 6-week liver tissues after infection in which the metacercariae became adult flukes. Many genes involved in fatty acid metabolism, including Peci, Cyp4a10, Acat1, Ehhadh, Gcdh, and Cyp2 family were downregulated in the infected livers. On the other hand, the liver tissues infected with the parasite expressed Wnt signaling molecules such as Wnt7b, Fzd6, and Pdgfrb and cell cycle-regulating genes including cyclin-D1, Cdca3, and Bcl3. These investigations constitute an excellent starting point for increased understanding of the molecular mechanisms underlying host–pathogen interaction during the development of C. sinensis in the host liver.     

肝吸虫感染大鼠红细胞免疫功能的动态变化

目的:探讨大鼠感染肝吸虫前后红细胞免疫功能的动态变化.方法:取健康成年Wistar大鼠48只,随机分为正常对照组8只和感染组40只,感染组大鼠口服肝吸虫囊蚴建立肝吸虫病动物模型,分别于灌胃后0、2、4、6、8和14周处死.对肝吸虫病大鼠的红细胞C3b受体花环率(RBC-C3bRR),红细胞免疫复合物花环率(RBC-ICR),红细胞粘附促进因子(RFER)及红细胞粘附抑制因子(RFIR)的活性分别进行动态检测.结果:肝吸虫病大鼠RBC-C3bRR和RFER水平降低,而ICRR和RFIR水平升高,与正常对照组比较差异显著,有统计学意义.结论:肝吸虫病大鼠的红细胞免疫功能发生改变,表现为继发性红细胞免疫功能异常和红细胞免疫调节功能紊乱,说明肝吸虫感染可损伤宿主的红细胞免疫功能.检查肝吸虫病大鼠红细胞免疫功能对了解其免疫状态具有一定的意义.     

Intraductal papillary neoplasm of the bile duct associated with Clonorchis sinensis infection

Columnar cell lesions (CCLs) are one of the most common abnormalities in the adult female human breast, characterized by the presence of columnar-shaped epithelial cells lining enlarged terminal-duct lobular units. CCLs are being seen increasingly in core biopsies taken for the non-palpable calcifications. The increased incidence may reflect improved delineation and recognition of CCLs by pathologists or a true increase in incidence related to biological and/or environmental factors. Columnar cell-like lesions have been described under a variety of names such as blunt duct adenosis, flat epithelial atypia, and ductal intraepithelial neoplasia type DIN1a. The current histologic classification used by some pathologists divides them into simple columnar cell change and columnar cell hyperplasia, both of which can occur with or without atypia. Columnar cells lack mature luminal or basal/myoepithelial phenotype markers, but they are usually positive for estrogen receptor-alpha. The cellular origin of CCLs and their possible relationship to either expansion or metaplasia of a preexisting normal cell phenotype remains unclear. CCLs are frequently associated with lobular and ductal in situ tumors and invasive lobular and tubular carcinomas. The relationship and natural history of CCLs to invasive ductal carcinoma is enigmatic, but they may prove of clinical relevance when detected by screening mammography.     

Epidemiological investigation of Clonorchis sinensis infection in freshwater fishes in the Pearl River Delta

Pearl River Delta region is a high clonorchiasis-endemic area in China. However, no complete epidemiological data exist regarding its infection in freshwater fishes, an important epidemic factor for Clonorchis sinensis. The present study collected freshwater fishes and shrimps from 32 sites of nine cities in the Pearl River Delta, and the encysted metacercariae of C. sinensis were detected by digesting these specimens with artificial gastric juice. The mean infection rate of freshwater fishes was 37.09% (2,160/5,824) with a mean number of 14.269 encysted metacercariae in every infected fish and 0.460 encysted metacercariae in every gram of fish meat. Of these freshwater fishes, 5,219 were domesticated, and the infection rate was 36.69% with a mean number of 10.743 encysted metacercariae in every infected fish and 0.312 encysted metacercariae in every gram of fish meat; the other 605 were wild, and the infection rate was 40.50% with a mean number of 41.829 encysted metacercariae in every infected fish and 8.812 encysted metacercariae in every gram of fish meat. A total of 228 shrimps were examined, and 3.07% of them were infected with a mean number of 1.00 encysted metacercariae in every infected shrimp. Pseudorasbora parva and Ctenopharyngodon idellus had the highest infection rate and degree of infection in the fishes studied. The results demonstrated a high incidence of C. sinensis infection in freshwater fishes and shrimps within Pearl River Delta region and a great difference in the infection rate among different collection sites and different fish species.     

Protection against translocating Salmonella typhimurium infection in mice by feeding the immuno-enhancing probiotic Lactobacillus rhamnosus strain HN001

The probiotic lactic acid bacterium Lactobacillus rhamnosus (strain HN001) is known to stimulate enhanced innate and acquired immune responses in mice. following oral delivery. Here, the ability of HN001 to confer immune enhancement and protection against an oral challenge of Salmonella tYphimurium was investigated. HN001-fed and non-probiotic-fed control BALB/c mice were challenged with either a single dose of S. typhimurium (ATCC strain 1772), or with five repeated daily doses of the pathogen; post-challenge clinical, behavioural, bacteriological and immunological parameters were assessed. Mice began to show ostensible signs of infection 3-4 days following the initiation of Salmonella challenge, and the first mortalities were observed after 6 days. Following single-dose Salmonella challenge, HN001-fed mice maintained a higher mean pre-mortality general health score than control mice; retained significantly greater food and water intake and weight gain, produced higher titres of serum and intestinal tract anti-Salmonella antibodies, and showed greater overall survival of infection (27/30 mice surviving at 21 days post-challenge, compared to 2/29 in the control group). Following repeated-dose Salmonella challenge, HN001-fed mice had significantly lower mean pathogen burdens in visceral organs (spleen, liver) compared to controls, and additionally, blood and peritoneal leucocytes obtained from HN001-fed mice exhibited significantly higher ex vivo phagocytic capacity compared to control-mice. This study affirms that Lb. rhamnosus strain HN001 displays immuno-enhancing properties in S. typhimurium-infected mice, and demonstrates that oral delivery of this probiotic can promote increased protection against a highly virulent enteric bacterial pathogen.     

Dynamic gut microbiome changes following regional intestinal lymphatic obstruction in primates

The pathogenesis of inflammatory bowel disease (IBD) has been linked with lymphostasis, but whether and how lymphatic obstruction might disturb the intestinal microbiome in the setting of Crohn’s Disease (CD) is currently unknown. We employed a new model of CD in African Green monkeys, termed ‘ATLAS’ (African green monkey truncation of lymphatics with obstruction and sclerosis), to evaluate how gut lymphatic obstruction alters the intestinal microbiome at 7, 21 and 61 days. Remarkable changes in several microbial sub- groupings within the gut microbiome were observed at 7 days post-ATLAS compared to controls including increased abundance of Prevotellaceae and Bacteroidetes-Prevotella-Porphyromonas (BPP), which may contribute to disease activity in this model of gut injury. To the best of our knowledge, these findings represent the first report linking lymphatic structural/gut functional changes with alterations in the gut microbiome as they may relate to the pathophysiology of CD.     

The role of the gut microbiome and diet in the pathogenesis of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, with a prevalence that is increasing in parallel with the global rise in obesity and type 2 diabetes mellitus. The pathogenesis of NAFLD is complex and multifactorial, involving environmental, genetic and metabolic factors. The role of the diet and the gut microbiome is gaining interest as a significant factor in NAFLD pathogenesis. Dietary factors induce alterations in the composition of the gut microbiome (dysbiosis), commonly reflected by a reduction of the beneficial species and an increase in pathogenic microbiota. Due to the close relationship between the gut and liver, altering the gut microbiome can affect liver functions; promoting hepatic steatosis and inflammation. This review summarises the current evidence supporting an association between NAFLD and the gut microbiome and dietary factors. The review also explores potential underlying mechanisms underpinning these associations and whether manipulation of the gut microbiome is a potential therapeutic strategy to prevent or treat NAFLD.     

The probiotic bacterium Lactobacillus casei induces activation of the gut mucosal immune system through innate immunity

The mechanisms by which probiotic bacteria affect the immune system are unknown yet, but many of them are attributed to an increase in the innate or in the acquired immune response. To study the influence of the probiotic bacterium Lactobacillus casei in the expression of receptors involved in the innate immune response, this bacterium was orally administered to BALB/c mice. After, they were sacrificed; the small intestine and intestinal fluids were collected to measure secretory immunoglobulin A (IgA) specific for L. casei. Mononuclear cells from Peyer's patches were isolated to determine the CD-206 and TLR-2 receptors. In histological slices we determined the number of IgA+, CD4+, CD8+, and CD3+ cells and two cytokines (interleulin-5 [IL-5] and IL-6). CD-206 and TLR-2 increased with respect to the untreated control. We did not observe an increase in the T population or in the IL-5-positive cells. IgA+ cells and IL-6-producing cells increased after 7 days of L. casei administration. We did not find specific antibodies against L. casei. The main immune cells activated after oral L. casei administration were those of the innate immune response, with an increase in the specific markers of these cells (CD-206 and TLR-2), with no modification in the number of T cells.     

阳山县华支睾吸虫病综合防治示范区中期评估结果

目的了解阳山县华支睾吸虫病综合防治示范区人群华支睾吸虫感染状况,评估示范区一年来综合防治效果。方法按国家中期考核评估方案要求分东西南北中5个片随机抽取调查点,采用改良加藤厚涂片法对人群进行虫卵粪检。结果东片青莲、西片黎埠、南片七拱、北片小江、中片阳城5个片调查点华支睾吸虫感染率分别为7.26%、6.09%、14.80%、7.14%和10.80%,平均为9.28%。感染率由2006年的14.01%下降到9.28%,下降率为33.76%。结论阳山县居民华支睾吸虫感染率较2006年基线调查结果明显下降,综合防治效果明显。