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Angiostrongylus cantonensis constitutes a major etiologic agent of eosinophilic meningoencephalitis. The detection methods for angiostrongyliasis mainly depend on morphology or immunology. A firmer diagnosis could be reached by directly detecting the parasite in the cerebrospinal fluid or through laboratory assays that are specific for Angiostrongylus-induced antibodies or the parasite's DNA. A. cantonensis detection could be carried out by larva release from the tissue upon pepsin digestion. However, the procedure requires live mollusks, which might complicate the analysis of large amounts of samples. Since morphological assays are limited, multiple molecular techniques have been put forward for detecting A. cantonensis, including PCR amplification of targets followed by fragment length or DNA sequence analysis. This allows rapid and accurate identification of A. cantonensis for efficient infection management and epidemiological purposes. In this study, we reviewed the current methods, concepts, and applications of molecular approaches to better understand the genetic characterization, molecular detection methods, and practical application of molecular detection in A. cantonensis.

Different miRNAs are involved in the life cycles of Schistosoma japonicum. The aim of this study was to examine the expression profile of miRNAs in individual S. japonicum of different sex before and after pairing (18 and 24 dpi). The majority of differential expressed miRNAs were highly abundant at 14 dpi, except for sja-miR-125b and sja-miR-3505, in both male and female. Moreover, it was estimated that sja-miR-125b and sja-miR-3505 might be related to laying eggs. sja-miR-2a-5p and sja-miR-3484-5p were expressed at 14 dpi in males and were significantly clustered in DNA topoisomerase III, Rap guanine nucleotide exchange factor 1 and L-serine/L-threonine ammonia-lyase. Target genes of sja-miR-2d-5p, sja-miR-31- 5p and sja-miR-125a, which were expressed at 14 dpi in males but particularly females, were clustered in kelch-like protein 12, fructose-bisphosphate aldolase, class I, and heat shock protein 90 kDa beta. Predicted target genes of sja-miR-3483-3p (expressed at 28 dpi in females but not in males) were clustered in 26S proteasome regulatory subunit N1, ATPdependent RNA helicase DDX17. Predicted target genes of sja-miR-219-5p, which were differentially expressed at 28 dpi in females but particularly males, were clustered in DNA excision repair protein ERCC-6, protein phosphatase 1D, and ATPase family AAA domaincontaining protein 3A/B. Moreover, at 28 dpi, eight miRNAs were significantly up-regulated in females compared to males. The predicted target genes of these miRNAs were significantly clustered in heat shock protein 90 kDa beta, 26S proteasome regulatory subunit N1, and protein arginine N-methyltransferase 1. To sum up, differentially expressed miRNAs may have an essential role and provide necessary information on clarifying this trematode's growth, development, maturation, and infection ability to mammalian hosts in its complex life cycle, and may be helpful for developing new drug targets and vaccine candidates for schistosomiasis.

The objective of this study was to investigate the mechanism of Toll-like receptor (TLR4)- mediated dendritic cell (DC) immune against Cryptosporidium parvum infection. C. parvum sporozoites were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester. Murine bone marrow-derived DCs were isolated, and divided into TLR4 antibody blocking (TAB; infected with 2 × 105 labeled sporozoites and 0.5 μg TLR4 blocking antibody), TLR4 antibody unblocking (TAU; infected with 2 × 105 labeled sporozoites), and blank control (BC; with 1.5 mL Roswell Park Memorial Institute 1640 medium) groups. The adhesion of Cryptosporidium sporozoites to DCs and CD11c+ levels were examined by fluorescence microscopy and flow cytometry. Male KM mice were orally injected with C. parvum. The proliferation of T lymphocytes in spleen, expression of cytokines in peripheral blood, and TLR4 distribution features in different organs were further determined by immunohistochemistry. A significantly higher expression of CD11c+ and higher C. parvum sporozoite adhesion were found in the TAU group compared with other groups. The expression of CD4+CD8- /CD8+CD4- in the spleen were obviously differences between the TAB and TAU groups. The expression of TLR4, interleukin IL-4, IL-12, IL-18 and IFN-γ improved in the TAU group compared with TAB group. Higher expression of TLR4 was detected in the lymph nodes of mice in the TAU group, with pathological changes in the small intestine. Hence, TLR4 could mediate DCs to recognize C. parvum, inducing Th1 immune reaction to control C. parvum infection.

Babesia microtiis a protozoan that infects red blood cells. Babesiosis is becoming a new global threat impacting human health. Rhoptry neck proteins (RONs) are proteins located at the neck of the rhoptry and studies indicate that these proteins play an important role in the process of red blood cell invasion. In the present study, we report on the bioinformatic analysis, cloning, and recombinant gene expression of two truncated rhoptry neck proteins 2 (BmRON2), as well as their potential for incorporation in a candidate vaccine for babesiosis. Western blot and immunofluorescence antibody (IFA) assays were performed to detect the presence of specific antibodies against BmRON2 in infected mice and the localization of N-BmRON2 inB. microtiparasites.In vitroexperiments were carried out to investigate the role of BmRON2 proteins during theB. microtiinvasion process andin vivoexperiments to investigate immunoprotection. Homologous sequence alignment and molecular phylogenetic analysis indicated that BmRON2 showed similarities with RON2 proteins of otherBabesiaspecies. We expressed the truncated N-terminal (33–336 aa, designated rN-BmRON2) and C-terminal (915–1171 aa, designated rC-BmRON2) fragments of the BmRON2 protein, with molecular weights of 70 and 29 kDa, respectively. Western blot assays showed that the native BmRON2 protein is approximately 170 kDa, and that rN-BmRON2 was recognized by serum of mice experimentally infected withB. microti.Immunofluorescence analysis indicated that the BmRON2 protein was located at the apical end of merozoites, at the opposite end of the nucleus.In vitrored blood cell invasion inhibition studies withB. microtirBmRON2 proteins showed that relative invasion rate of rN-BmRON2 and rC-BmRON2 group is 45 and 56%, respectively. Analysis of the host immune response after immunization andB. microtiinfection showed that both rN-BmRON2 and rC-BmRON2 enhanced the immune response, but that rN-BmRON2 conferred better protection than rC–BmRON2. In conclusion, our results indicate that truncated rhoptry neck protein 2, especially its N-terminal fragment (rN-BmRON2), plays an important role in the invasion of host red blood cells, confers immune protection, and shows good potential as a candidate vaccine against babesiosis.

Background Human African trypanosomiasis (HAT) is one of the most complex parasitic diseases known to humankind. It usually occurs in endemic areas in Africa, but is occasionally detected in returning travelers and migrants in non-endemic countries. Case presentation In August 2017, a case of HAT was diagnosed in China in a traveler returning from the Masai Mara area in Kenya and the Serengeti area in Tanzania. The traveler visited Africa from 23 July to 5 August, 2017. Upon return to China, she developed a fever (on 8 August), and Trypanosoma brucei rhodesiense infection was confirmed by laboratory tests (on 14 August) including observation of parasites in blood films and by polymerase chain reaction. She was treated with pentamidine followed by suramin, and recovered 1 month later. Conclusions This is the first imported rhodesiense HAT case reported in China. This case alerts clinical and public health workers to be aware of HAT in travelers, and expatriates and migrants who have visited at-risk areas in Africa. Electronic supplementary material The online version of this article (10.1186/s40249-018-0432-5) contains supplementary material, which is available to authorized users.

// Pei-Hua Lu 1, 2, * , Min-Bin Chen 3, * , Yuan-Yuan Liu 3, * , Mian-Hua Wu 2 , Wen-Ting Li 2 , Mu-Xin Wei 4 , Chao-Ying Liu 1 , Shu-Kui Qin 5 1 Department of Medical Oncology, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi, China 2 Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine (TCM) Prevention and Treatment of Tumor of Nanjing University of Chinese Medicine, Nanjing, China 3 Department of Oncology, Kunshan First People’s Hospital Affiliated to Jiangsu University, Kunshan, China 4 Department of Traditional Chinese Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing, China 5 People’s Liberation Army Cancer Center, 81st Hospital of People’s Liberation Army, Nanjing, China * Co-first authors Correspondence to: Shu-Kui Qin, email: drqinshukuinj@126.com Min-Bin Chen, email: cmb1981@163.com Keywords: hepatocellular carcinoma (HCC), icaritin, sphingosine kinase 1 (SphK1), ceramide Received: September 20, 2016 Accepted: November 08, 2016 Published: February 09, 2017 ABSTRACT Hepatocellular carcinoma (HCC) is a highly aggressive neoplasm. We aim to explore the anti-HCC activity by a natural prenylflavonoid icaritin. Icaritin was cytotoxic and pro-apoptotic when added to established (HepG2, KYN-2 and Huh-7 lines) and primary human HCC cells. At the signaling level, icaritin inhibited sphingosine kinase 1 (SphK1) activity in HCC cells, which led to pro-apoptotic ceramide production and JNK1 activation. SphK1 inhibition or silence (by shRNA/microRNA) mimicked icaritin-mediated cytotoxicity, and almost nullified icaritin’s activity in HepG2 cells. Reversely, exogenous over-expression of SphK1 sensitized icaritin-induced HepG2 cell apoptosis. In vivo , oral administration of icaritin dramatically inhibited HepG2 xenograft growth in SCID mice. Further, SphK1 activity in icaritin-treated tumors was largely inhibited. In summary, icaritin exerts potent anti-HCC activity in vitro and in vivo . SphK1 inhibition could be the primary mechanism of its actions in HCC cells.

Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.

Here we evaluated the anti-cancer activity of aqueous Oldenlandia diffusa (OD) extracts (ODE) in colorectal cancer (CRC) cells. We showed that ODE exerted potent anti-proliferative, cytotoxic and pro-apoptotic activities against a panel of established CRC lines (HCT-116, DLD-1, HT-29 and Lovo) and primary (patient-derived) human CRC cells. ODE activated AMP-activated protein kinase (AMPK) signaling, which led to subsequent mTORC1 inhibition and Bcl-2/HIF-1α downregulation in CRC cells. In ODE-treated CRC cells, AMPKα1 formed a complex with p53. This might be important for p53 activation and subsequent cancer cell apoptosis. Inhibition of AMPK signaling, though dominant negative (dn) mutation or shRNA/siRNA knockdown of AMPKα1 attenuated ODE-exerted CRC cytotoxicity. In vivo, i.p. administration of ODE inhibited HCT-116 xenograft tumor growth in SCID mice. In addition, AMPK activation, mTORC1 inhibition and p53 activation were observed in ODE-treated HCT-116 xenograft tumors. These results suggest that ODE inhibits CRC cells in vitro and in vivo, possibly via activation of AMPK-dependent signalings.

The development of a method to rapidly diagnose Neospora caninum infection is highly desirable. Recombinase polymerase amplification (RPA), combined with lateral flow (LF) strips, is a novel approach to rapidly amplify and visualize DNA. We have developed a prototype LF-RPA assay, using primers and a probe that targeted a specific sequence in the N. caninum NC-5 gene. The N. caninum-specific LF-RPA assay was first tested on purified DNA from oocysts and amplified N. caninum DNA to detectable levels in 10?min, at a constant temperature and without the need for an expensive thermocycler. The designed RPA primers and probe displayed 100% specificity for detecting N. caninum without any cross-reaction with DNA from nine related protozoan spp. (eg Toxoplasma gondii, Sarcocystis gigantean, Sarcocystis zuoi, Hammondia hammondi, Hammondia heydorni, Eimeria cylindrica, Plasmodium falciparum, Theileria annulata and Babesia bigemina). Although, LF-RPA assay detected amounts as low as 50?fg of N. caninum DNA, it was nearly 5-fold less sensitive than previously published qPCR and nested PCR assays. We tested the diagnostic performance of the LF-RPA assay for the detection of N. caninum DNA in aborted bovine fetal tissue samples, and compared the results with those obtained from nested PCR. Out of the 75?samples examined, 18 (24%) and 17 (22.6%) tested positive using LF-RPA and nested PCR, respectively. Our results indicate that LF-RPA is a suitable assay for the rapid and reliable detection of N. caninum.

c-Myc, a key activator of cell proliferation and angiogenesis, promotes the development and progression of breast cancer. Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) is a multifunctional scaffold protein that suppresses the proliferation of breast cancer cells. In this study we investigated whether the cancer-suppressing effects of EBP50 resulted from its regulation of c-Myc signaling in human breast cancer MCF-7 cells in vitro and in vivo. We first found a significant correlation between EBP50 and c-Myc expression levels in breast cancer tissue, and demonstrated that EBP50 suppressed cell proliferation through decreasing the expression of c-Myc and its downstream proteins cyclin A, E and Cdc25A in MCF-7 cells. We further showed that EBP50 did not regulate c-Myc mRNA expression, but it promoted the degradation of c-Myc through the autophagic lysosomal pathway. Moreover, EBP50 promoted integration between c-Myc and p62, an autophagic cargo protein, triggering the autophagic lysosomal degradation of c-Myc. In EBP50-silenced MCF-7 cells, activation of autophagy by Beclin-1 promoted the degradation of c-Myc and inhibited cell proliferation. These results demonstrate that the EBP50/Beclin-1/p62/c-Myc signaling pathway plays a role in the proliferation in MCF-7 breast cancer cells: EBP50 stimulates the autophagic lysosomal degradation of c-Myc, thereby inhibits the proliferation of MCF-7 cells. Based on our results, promoting the lysosomal degradation of c-Myc might be a promising new strategy for treating breast cancer.

Use of the conventional cancer chemotherapy (i.e. vincristine) is limited in tumor cells exhibiting pre-existing or acquired resistance. Here, we found that C6 ceramide (C6) dramatically sensitized vincristine's activity. In vitro, C6 and vincristine coadministration induced substantial necrosis and apoptosis in multiple human cancer cell lines, which were accompanied by a profound AMP-activated protein kinase (AMPK) activation, subsequent p53 activation, mTORC1 inactivation and Bcl-2/HIF-1α downregulation. Such synergistic effects were attenuated by AMPK inactivation through genetic mutation or short hairpin RNA silencing. Coadministration-activated p53 translocated to mitochondria, and formed a complex with cyclophilin-D, leading to mitochondrial permeability transition pore opening and cell necrosis. Disrupting p53-Cyp-D complexation through pharmacological or genetic means reduced costimulation-induced cytotoxicity. In vivo, a liposomal C6 was synthesized, which dramatically enhanced the antiproliferative activity of vincristine on HCT-116 or A2780 xenografts. Together, C6 sensitizes vincristine-induced anticancer activity in vivo and in vitro, involving activating AMPK-p53 signaling.

Background Fasciolopsis buski is a zoonotic intestinal fluke infecting humans and pigs, but it has been seriously neglected. It is yet to know whether there is any genetic diversity among F. buski from different geographical locations, particularly in sequences of nuclear ribosomal DNA (rDNA) and mitochondrial (mt) DNA. Therefore, we determined the sequences of partial 18S, the complete internal transcribed spacer (ITS) rDNA and the complete mt genome of F. buski from China, compared the rDNA and mtDNA sequences with those of isolates from India and Vietnam, and assessed the phylogenetic relationships of this fluke and related fasciolid trematodes based on the mtDNA dataset. Results The complete mt genome sequence of F. buski from China is 14,833 bp, with 36 genes, including 12 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes (rrnL and rrnS). The AT content of F. buski from China is 65.12%. The gene content and arrangement of the F. buski mt genome is similar to that of Fascioloides magna. Genetic distances between isolates of F. buski from China and India were high (28.2% in mtDNA, 13.2% in ITS-1 and 9.8% in ITS-2) and distinctly higher than the interspecific differences between Fasciola hepatica and Fasciola gigantica. The rDNA and mtDNA datasets for F. buski from China (isolate from pigs) and Vietnam (isolates from humans) were identical. The intergeneric differences in amino acid and nucleotide sequences among the genera Fasciolopsis, Fascioloides and Fasciola ranged between 24.64–25.56% and 26.35–28.46%, respectively. Conclusions Our results indicate that F. buski from China and India may represent distinct taxa, while F. buski in Vietnam and China represent the same species. These findings might have implications for the implementation of appropriate control strategies in different regions. Further studies are needed to decode mtDNA and rDNA sequences of F. buski from various geographical isolates for the better understanding of the species complex of F. buski. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2039-2) contains supplementary material, which is available to authorized users.

The aim of this study was to study the relation between histopathological classification of gastric carcinoma and Helicobacter pylori (H. pylori) infection. 200 patients with confirmed gastric carcinoma between January 2010 and January 2012 from our hospital were included. All these patients went through endoscopy and histological examinations for gastric carcinoma and immunological test for H. pylori infection. Patients were grouped according to the histological classification, and the infection rates of H. pylori in different groups were compared. Types of gastric cancer that was closely related to H. pylori infection were identified. Infection rate in patients with the intestinal type of gastric cancer was significantly higher compared to those with the diffuse type. For tubular type of carcinoma, the well to medium-differentiated group had a significantly higher infection rate than the poorly differentiated group. Helicobacter pylori infection and histological types were relevant. The effect of H. pylori infection on the intestinal type was more significant than that on the diffuse type. The infection rate of well-differentiated group was higher than that of the poorly differentiated group, which suggested an association between H. pylori infection and the degree of differentiation of tubular cancerous tissue.

Taenia solium and Taenia asiatica are zoonotic parasites which are transmitted between pigs and humans, with pigs acting as the intermediate host and humans as the definitive host. The aim of the present study was to compare the microRNA (miRNA) profiles between T. solium and T. asiatica by Solexa deep sequencing and bioinformatic analyses. A total of 18.26 and 15.24 million reads with high quality and three new miRNAs were obtained in each species, respectively. The two cestodes shared nearly the same level of total sRNA, but the known miRNAs were in a very different manner. Nucleotide guanine (G) and uracil (U) were the most frequently used first nucleotide in both T. solium and T. asiatica, respectively. Furthermore, it was also found that there were great differences in the kinds and copy numbers of miRNAs among the two Taenia species, which might indicate the different evolutions in the two parasites. To our knowledge, this is the first report on the characterization of the miRNA profiles for the two Taenia cestodes, and it would lay a foundation for further functional studies of miRNAs of T. solium and T. asiatica. Key words: Taenia solium, Taenia asiatica, microRNA profiles, human, pig.

Helicobacter pylori infection is a risk factor for gastric cancer. In addition, toll-like receptor 4 (TLR4) plays a fundamental role in pathogen recognition and activation of innate immunity. This study investigated the association of TLR4 polymorphisms with a risk of intestinal metaplasia (IM) and intraepithelial neoplasia (IN) in a Chinese Han population. This study analyzed TLR4 gene polymorphisms in 333 patients (IM, 193 cases; IN, 140 cases) and 312 atypia-free controls in a Chinese Han population using a Taqman allelic discrimination assay. The TLR4 single nucleotide polymorphisms +896A/G and +1196C/T were not associated with the risk of IM or IN. However, the single-locus analysis showed that the C allele of TLR4+2856T/C had significantly reduced risk of IM and IN [adjusted odds ratio (OR)=0.42; 95%CI=0.29-0.62 and OR=0.62; 95%CI=0.41-0.93, respectively] compared with the wild-type homozygote (TT). The frequencies of TLR4+2856T/C TC and T carrier were significantly lower in patients with Sydney's slight IM and low grade IN (P

Background Neglected tropical diseases (NTDs) are a heterogeneous group of mainly chronic, debilitating and often stigmatizing diseases that largely affects low-income and politically marginalized populations, causing a large burden of public health, social and economies in the NTDs endemic countries. NTDs are caused by infections with a range of pathogen, including bacteria, parasites, protozoa and viruses. The accurate diagnosis of NTDs is important for reducing morbidity, preventing mortality and for monitoring of control programs. External Quality Assessment (EQA), a component of laboratory quality assurance, aims to assess the performance of participating laboratories in detecting parasitic infections. The aim of this paper is to report the findings and put forward the recommendations on capacity build from the EQA results of participating NTDs laboratories in selected countries in the WHO Western Pacific Region from 2012 to 2015. Methods Reference or public health laboratories at national level working on NTDs in 6 countries participated in EQAs organized by the National Institute of Parasitic Diseases (NIPD) of Chinese Center for Disease Control and Prevention (CDC) based in Shanghai, China. Two representatives of each participating laboratory were invited to NIPD to detect NTDs’ parasitic infections using the same prepared samples for serological tests (IHA and ELISA) and helminth eggs’ morphological tests (Direct smear and Kato-Katz). All of the results were scored and analyzed by using SPSS statistics 19.0 software. Results The percentage of participants who had EQA score ≥ 60 during 2012–2015 for direct smear test were 80.00% (2012), 71.43% (2013), 100% (2014) and 75.00% (2015), whereas for Kato-Katz test were 80.00% (2012), 57.14% (2013), 100% (2014) and 37.50% (2015), respectively. The detection rate of helminth eggs varied in different species, with Ascaris lumbricoides being the highest at 94.07% in average. All laboratories did very well with ELISA tests as shown by the high scores in all four years except Lab A in the first and last EQA. For the positive or negative judgments of serum samples, the total coincidence rates of ELISA between 2012 and 2015 were 90.00%, 99.29%, 94.29% and 98.75%, respectively. While the total coincidence rates of IHA were respectively 100%, 95.00%, 90.00% and 97.50%. However, detecting low levels of serum antibody remained problematic for IHA when the titres of samples were taken into consideration. Conclusion This study demonstrate that EQA scheme have been beneficial to the participating laboratories. The EQA programme identifies certain deficiencies which were needed to overcome and improved the laboratories’ performance in helminthiasis diagnosis. However, further optimization of accuracy and uniformity in NTDs diagnosis remains a big challenge. Electronic supplementary material The online version of this article (doi:10.1186/s40249-017-0319-x) contains supplementary material, which is available to authorized users.

Fasciolopsis buski is a food-borne zoonotic parasite which is transmitted by aquatic plants, with pigs and humans as the definitive hosts. The objective of the present study was to characterize the microRNA (miRNA) profiles of this parasite by Solexa deep sequencing and bioinformatic analysis. Approximately 12 million high-quality reads were obtained from adult F. buski. A total of 286 miRNA candidates were found and 24 miRNA candidates were conserved miRNAs in the miRBase database. Three novel miRNAs were identified and confirmed by stem-loop reverse transcriptase polymerase chain reaction (RT-PCR). The miRNAs found in the present study belong to 13 families whose members showed high bias. The guanine (G) was the dominant nucleotide at the beginning and middle of the conserved miRNAs, particularly at the positions of 2nd, 6th, and 13th. To our knowledge, this is the first report of the miRNA profiles of F. buski, which would lay a foundation for further functional studies of miRNAs of F. buski.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been implicated in the onset of cystic fibrosis and other clinical respiratory disorders. In the present study, we investigated the role of CFTR variations, poly-T, TG-repeats, and M470V in susceptibility to bronchial asthma and chronic bronchitis in a Chinese population in Jiangsu province, China. A total of 72 bronchial asthma patients, 68 chronic bronchitis patients, and 117 healthy subjects were included in this study. The Tn-TGm haplotype was sequenced and the CFTR variant M470V was detected using restriction fragment length polymorphism (RFLP). We found that the frequency of T5-TG12-V470 in chronic bronchitis patients was 0.07%, which was notably higher than that in healthy subjects (0.01%) and bronchial asthma patients (0.04%). Thus, the presence of the T5-TG12 haplotype of the CFTR gene is likely to play a role in the development and progression of respiratory conditions, such as chronic bronchitis.

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1pg of purified genomic DNA, 5EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent.

Angiostrongylus cantonensis causes eosinophilic meningitis and eosinophilic pleocytosis in humans and is of significant socio-economic importance globally. microRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in gene expression regulation, cellular function and defense, homeostasis and pathogenesis. They have been identified in a diverse range of organisms. The objective of this study was to determine and characterize miRNAs of female and male adults of A. cantonensis by Solexa deep sequencing. A total of 8,861,260 and 10,957,957 high quality reads with 20 and 23 conserved miRNAs were obtained in females and males, respectively. No new miRNA sequence was found. Nucleotide bias analysis showed that uracil was the prominent nucleotide, particularly at positions of 1, 10, 14, 17 and 22, approximately at the beginning, middle and the end of the conserved miRNAs. To our knowledge, this is the first report of miRNA profiles in A. cantonensis, which may represent a new platform for studying regulation of genes and their networks in A. cantonensis.

We have read with great interest the article ‘‘Three common TP53 polymorphisms in susceptibility to breast cancer: evidence from meta-analysis’’ published in April 2010 issue of ‘‘Breast Cancer Research and Treatment’’ [1]. The results suggest that IVS3 16 bp Del/Ins is likely an important genetic marker contributing to the susceptibility of breast cancer, and codon 72 has a potential role in the association with breast cancer risk within certain populations or regions. Thus, some methodological issues need to be addressed concerning the data provided in the meta-analysis by Hu et al. [1]. We would like to comment and raise some concerns. The minor variants of IVS3 16 bp (rs17878362) polymorphism in the cases and controls were three and one, respectively [2]. However, a small number of subjects do not always indicate that they are minor variants in the meta-analysis. The data reported by Hu et al. [1] for the study did not seem in accordance with the data provided by Weston et al. [2] in their original publication. The numbers reported by Hu et al. [1] for RR, RP, and PP of codon 72 (rs1042522) polymorphism in the cases and controls were 32-27-6 and 72-42-3. Interestingly enough, after carefully studying the data presented by Weston et al. [2], the frequencies we retrieved were 6-27-32 and 3-42-72, which corresponded to the genotypes of 1-1, 1-2, and 2-2 [2]. In Weston’s research, the minor variants data were 32 and 72, indicating the large number of subjects. Similarly, the numbers reported by Hu et al. [1] for GG, GA, and AA of IVS6 ? 62A[G (rs1625895) polymorphism in the cases and controls were 43-20-2 and 95-22-0. The frequencies corresponding to the genotypes of 1-1, 1-2, and 2-2 [2] were 2-20-43 and 0-22-95. We can conclude that a small number of subjects do not always indicate that they are minor variants data for inclusion in a pooled analysis. Hu et al. [1] confused the original data and made the results not entirely credible. Thus, the odds ratio and the 95% confidence interval for codon 72 (rs1042522) and IVS6 ? 62A[G (rs1625895) polymorphisms in the metaanalysis significantly differed from those calculated by the original data of Weston’s research [2]. It would be valuable if the authors could provide a new and more accurate estimation of the pooled odds ratio after considering our comment. More importantly, the research by Weston et al. [2] was a nested case–control study involving 182 Caucasians, 56 Hispanics, and 46 African-Americans. This implies that the genotypes of 1-1, 1-2, and 2-2 by Hu et al. [1] were only subjects for Caucasians. The genotypes of 56 Hispanics and 46 African-Americans were not included even if they Pei-Hua Lu, Min-Bin Chen and Mu-Xin Wei contributed equally to this work and should be considered as co-first authors.

Trinucleotide repeat containing 9 (TNRC9) is a gene located at chromosome 16q12. Although of an uncertain function, it is a newly described risk factor for breast cancer. It contains a putative high-mobility group box motif, suggesting its possible role as transcription factor; it has been implicated in breast cancer metastasis. Published studies on the association between TNRC9 polymorphisms and breast cancer risk remain inconclusive, and a meta-analysis is required to verify the association. This pioneering research performed a meta-analysis of eight studies comprising a total of 25,828 cases and 36,177 controls. Significantly elevated breast cancer risk was associated with TNRC9 rs3803662 polymorphism when all studies were pooled in the meta-analysis (T vs. C allele contrast model: OR 1.18, 95% CI 1.09-1.28; TT vs. CC homozygote codominant model: OR 1.26, 95% CI 1.02-1.55; TT vs. CC+CT recessive model: OR 1.23, 95% CI 1.06-1.42). For TNRC9 rs12443621 polymorphism, no significant association was detected in all genetic models. For TNRC9 rs12443621 polymorphism, meanwhile, no significant association was observed in all comparison models. Conclusively, this meta-analysis suggests that TNRC9 rs3803662 polymorphism was significantly correlated with breast cancer risk and the variant T allele of TNRC9 rs3803662 polymorphism is a low-penetrant risk factor for developing breast cancer. There is no significant association between TNRC9 rs12443621 and rs8051542 polymorphisms and risk of breast cancer in current literature.

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs in two bioactive forms, PACAP-38 and PACAP-27 that have identical N-terminal sequences but differ by the presence of a C-terminal 11 residue elongation in the former. Although VIP and PACAP have several similar biological actions due to their amino acid sequence similarity, we have found that they evoke opposite responses in the guinea pig gallbladder smooth muscle, where PACAP induces contraction while VIP causes relaxation. In addition the response to PACAP-38 is four times lower than that of PACAP-27. In a previous study we have reported the role of the N-terminal α-helical regions of PACAP-27 which play a key role in gallbladder contraction. In the present study the biological action on the guinea pig gallbladder was investigated using a synthetic mini-library of C-terminally deleted peptides related to PACAP-38. The effects caused by residues within the C-terminus are not a result of a response via the M-receptor or Na+ channel, but most likely arise from a delicate balance between the differential effects of PACAP-38 on specific PAC1 and VPACs receptors.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction, whereas VIP induces relaxation. Here, we have attempted to identify key residues responsible for their interactions with PACAP (PAC1) and VIP (VPAC) receptors in the guinea pig gallbladder. We synthesized PACAP-27/VIP hybrid peptides and compared their actions on isolated guinea pig gallbladder smooth muscle strips using isotonic transducers. [Ala4]- and [Val5]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [Ala4, Val5]- and [Ala4, Val5, Asn9]PACAP-27 induced relaxation similarly to VIP. [Asn9]-, [Thr11]-, or [Leu13]PACAP-27 had 20–70% contractile activity of PACAP-27, whereas [Asn24,Ser25,Ile26]PACAP-27 showed no change in the activity. All VIP analogs, including [Gly4,Ile5,Ser9]VIP, induced relaxation. In the presence of a PAC1 receptor antagonist, PACAP(6–38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of PAC1 receptor, “hop” splice variant, and VPAC1 and VPAC2 receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via PAC1/hop receptors. Gly4 and Ile5 are the key NH2-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors. However, both the NH2-terminal and α-helical regions of PACAP-27 are required for initiating gallbladder contraction.

Objective: To study the effects of L. F04, the active fraction ofLycopus lucidus, on erythrocytes rheological property so as to investigate its mechanism in promoting blood circulation and removing blood stasis.Method: The effects of L. F04 (used for treatment for 10 days in different dosages) on deformability, aggregation and membrane liquidity of erythrocytes (MLE) as well as whole blood apparent viscosity (ηb) were examined on the basis of rat model of blood-stasis syndrome induced by venous injection of high molecular weight dextran.Result: As compared with the normal control group, the model group’s RBC deformability and MLE were lower, and the aggregation of erythrocytes and ηb were higher. Compared with the model group, both L. F04 0. 612g/kg and 0.306g/kg showed significant effect in improving deformability and inhibiting aggregation of red blood cells (RBC) and reducing blood viscosity. The trend of improving MLE was also shown.Conclusion: L. F04 could significantly improve the abnormal rheological property of erythrocytes.

Background Schistosomiasis remains a serious public health problem in affected countries, and routine, highly sensitive and cost-effective diagnostic methods are lacking. We evaluated two immunodiagnostic techniques for the detection of Schistosoma japonicum infections: circulating antibody and circulating antigen assays. Methods A total of 1864 individuals (between 6 and 72 years old) residing in five administrative villages in Hubei province were screened by serum examination with an indirect hemagglutination assay (IHA). The positive individuals (titer ≥20 in IHA) were reconfirmed by stool examination with the Kato-Katz method (three slides from a single stool specimen). Samples of good serum quality and a volume above 0.5 ml were selected for further testing with two immunodiagnostic antibody (DDIA and ELISA) and two antigen (ELISA) assays. Results The average antibody positive rate in the five villages was 12.7%, while the average parasitological prevalence was 1.50%; 25 of the 28 egg-positive samples were also circulating antigen-positive. Significant differences were observed between the prevalence according to the Kato-Katz method and all three immunodiagnostic antibody assays (P-value

Background Accurate detection of blood protozoa from clinical samples is important for diagnosis, treatment and control of related diseases. In this preliminary study, a novel DNA microarray system was assessed for the detection of Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii and Babesia in humans, animals, and vectors, in comparison with microscopy and PCR data. Developing a rapid, simple, and convenient detection method for protozoan detection is an urgent need. Methodology/Principal Findings The microarray assay simultaneously identified 18 species of common blood protozoa based on the differences in respective target genes. A total of 20 specific primer pairs and 107 microarray probes were selected according to conserved regions which were designed to identify 18 species in 5 blood protozoan genera. The positive detection rate of the microarray assay was 91.78% (402/438). Sensitivity and specificity for blood protozoan detection ranged from 82.4% (95%CI: 65.9% ~ 98.8%) to 100.0% and 95.1% (95%CI: 93.2% ~ 97.0%) to 100.0%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) ranged from 20.0% (95%CI: 2.5% ~ 37.5%) to 100.0% and 96.8% (95%CI: 95.0% ~ 98.6%) to 100.0%, respectively. Youden index varied from 0.82 to 0.98. The detection limit of the DNA microarrays ranged from 200 to 500 copies/reaction, similar to PCR findings. The concordance rate between microarray data and DNA sequencing results was 100%. Conclusions/Significance Overall, the newly developed microarray platform provides a convenient, highly accurate, and reliable clinical assay for the determination of blood protozoan species., Author Summary More than 1 billion people are infected with blood protozoan diseases worldwide. The most common blood protozoa in humans, animals, and vectors include Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii and Babesia. Due to similar morphology among different blood protozoan species, misdiagnosis always occurs. Most molecular techniques are only carried out in laboratories, with a small number of samples detected simultaneously. Meanwhile, common detection methods may not be convenient for field investigation of large amounts of samples. In order to better manage blood protozoan infection, proper tools are required for the monitoring of these pathogens. Here, a comprehensive and sensitive DNA microarray was developed and tested, which allowed the parallel detection of 18 blood protozoan species.

AIM: To systematically review pathological changes of gastric mucosa in gastric atrophy (GA) and intestinal metaplasia (IM) after Helicobacter pylori (H. pylori) eradication. METHODS: A systematic search was made of PubMed, Web of Science, EMBASE, ClinicalTrials.gov, OVID and the Cochran Library databases for articles published before March 2013 pertaining to H. pylori and gastric premalignant lesions. Relevant outcomes from articles included in the meta-analysis were combined using Review Manager 5.2 software. A Begg’s test was applied to test for publication bias using STATA 11 software. χ2 and I2 analyses were used to assess heterogeneity. Analysis of data with no heterogeneity (P > 0.1, I2 < 25%) was carried out with a fixed effects model, otherwise the causes of heterogeneity were first analyzed and then a random effects model was applied. RESULTS: The results of the meta-analysis showed that the pooled weighted mean difference (WMD) with 95%CI was 0.23 (0.18-0.29) between eradication and non-eradication of H. pylori infection in antral IM with a significant overall effect (Z = 8.19; P

Purpose: To investigate the association between various risk factors and precancerous lesion of gastric cancer (PLGC) in patients from eastern China. Materials and Methods: 501 cases of PLGC and 523 cases of superficial gastritis were included. A comparative study of the relation between different risk factors and PGLC was performed. Results: Statistical differences were noted in a series of indexes including Helicobacter pylori (HP) infection, family history of esophageal cancer (EC), gastric cancer (GC) and chronic atrophic gastritis (CAG), a history of CAG, gastric polyps (GP) and gastric ulcer (GU), usage of non-steroids (e.g., aspirin), gastroesophageal reflux disease (GERD), consuming alcohol, eating food rich in nitroso compounds, irregular eating habits with no breakfast, ingestion of smoked meat, fried food and spicy food, anxiety and depression. The risk factors associated with PLGC ranked in an order of a history of CAG, GP, family history of GC, usage of non-steroids (e.g., aspirin), ingestion of spicy food frequently, HP infection, family history of EC, consuming alcohol, anxiety, a history of GU, GERD and family history of CAG. Conclusions: A history of CAG was most associated with PLGC in patients from eastern China, followed by a history of GP and family history of GC.

Here we report that activation of AMP-activated protein kinase (AMPK) mediates plumbagin-induced apoptosis and growth inhibition in both primary cultured human colon cancer cells and cell lines. Knocking-down of AMPKα by the target shRNA significantly inhibits plumbagin-induced cytotoxicity in cultured colon cancer cells, while forced activation of AMPK by introducing a constitutively active AMPK (CA-AMPK), or by the AMPK activator, inhibits HT-29 colon cancer cell growth. Our Western-blots and immunoprecipitation (IP) results demonstrate that plumbagin induces AMPK/Apoptosis signal regulating kinase 1 (ASK1)/TNF receptor-associated factor 2 (TRAF2) association to activate pro-apoptotic c-Jun N-terminal kinases (JNK)-p53 signal axis. Further, after plumbagin treatment, activated AMPK directly phosphorylates Raptor to inhibit mTOR complex 1 (mTORC1) activation and Bcl-2 expression in colon cancer cells. Finally, we found that exogenously-added short-chain ceramide (C6) enhances plumbagin-induced AMPK activation and facilitates cell apoptosis and growth inhibition. Our results suggest that AMPK might be the key mediator of plumbagin's anti-tumor activity.

Fascioliasis is a common parasitic disease in livestock in China. However, human fascioliasis is rarely reported in the country. Here we describe an outbreak of human fascioliasis in Yunnan province. We reviewed the complete clinical records of 29 patients and performed an epidemiological investigation on the general human population and animals in the outbreak locality. Our findings support an outbreak due to Fasciola gigantica with a peak in late November, 2011. The most common symptoms were remittent fever, epigastric tenderness, and hepatalgia. Eosinophilia and tunnel-like lesions in ultrasound imaging in the liver were also commonly seen. Significant improvement of patients' condition was achieved by administration of triclabendazole®. Fasciola spp. were discovered in local cattle (28.6%) and goats (26.0%). Molecular evidence showed a coexistence of F. gigantica and F. hepatica. However, all eggs seen in humans were confirmed to be F. gigantica. Herb (Houttuynia cordata) was most likely the source of infections. Our findings indicate that human fascioliasis is a neglected disease in China. The distribution of triclabendazole®, the only efficacious drug against human fascioliasis, should be promoted.

MicroRNAs (miRNAs) are key regulators of gene expression at the post-transcription level. The present study specifically explored and compared the miRNA expression profiles of F. gigantica and F. hepatica using an integrated sequencing and bioinformatics platform and quantitative real-time PCR. Nineteen and 16 miRNA candidates were identified from F. gigantica and F. hepatica, respectively. The two parasites shared 11 miRNAs, with 8 also showing similarity to miRNAs of Schistosoma japonicum. Another 8 miRNAs were identified as F. gigantica-specific and 5 as F. hepatica-specific, most of which were novel. Predicted target analysis with 11465 mRNA and EST sequences of F. hepatica and F. gigantica revealed that all of the miRNAs had more than one target, ranging from 2 to 398 with an average of 51 targets. Some functions of the predicted targets were only found in F. gigantica, such as "transcription regulator", while some others were only found in F. hepatica, such as "reproduction" and "response to stimulus", indicating the different metabolism and gene regulation patterns of the two parasites. The present study represents the first global comparative characterization of miRNA expression profiles of F. gigantica and F. hepatica, which has provided novel valuable resources for a better understanding of the two zoonotic trematodes.

OBJECTIVES Mutation in the Pkhd1 gene that encodes a ciliary protein, fibrocystin, causes multiple cysts in the kidneys and liver in the polycystic kidney (PCK) rat, a model for human autosomal recessive PCK disease. To clarify the role of primary cilia in the pancreatic duct, we examined the structure and function of the exocrine pancreas of PCK rats. METHODS Pancreatic juice and bile were collected from anesthetized rats. Pancreatic ductal structure was analyzed by microdissection and immunohist0chemistry. RESULTS Histologically pancreatic acini were apparently normal, and no cysts were detected in the pancreas. Larger pancreatic ducts were irregularly dilated with enhanced expression of AQP1 in epithelial cells. The pancreatic duct of PCK rats exhibited significantly (P < 0.05) higher distensibility than that of wild-type (WT) rat at a physiological luminal pressure (3 cm H2O). Pancreatic fluid secretion stimulated with a physiological dose of secretin (0.03 nmol/kg per hour) in PCK rats was significantly smaller than that in WT, but the differences were not significant at higher doses. The amylase responses to carbamylcholine were not different between PCK and WT rats. CONCLUSIONS These findings suggest that fibrocystin/primary cilia-dependent mechanisms may play a role in the regulation of pancreatic ductal structure and fluid secretion.

Background Food-borne helminthiases (FBHs) have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. Methodology/Principal Findings In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI): 95.3–98.7%) to 100.0% (95% CI: 100.0%) in the protein microarray and from 97.7% (95% CI: 96.2–99.2%) to 100.0% (95% CI: 100.0%) in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1–96.3%) to 92.1% (95% CI: 83.5–100.0%) in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4–92.6%) to 92.1% (95% CI: 83.5–100.0%). Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. Conclusions/Significance The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening., Author Summary Food-borne helminthiases (FBHs) have caused significant problems in public health and also posed socio-economic concerns. Common FBHs, such as cysticercosis, trichinellosis, paragonimiasis, sparganosis and angiostrongyliasis, have a worldwide distribution with high morbidity and even death. The objective of the present study was to develop and test a rapid assay suitable for large-scale screening for FBHs that would also allow differential diagnosis between the various parasite species. We tested archived, well-characterized serum specimens and prioritized tests for future evaluation in rapid and simultaneous screening of five different FBHs, i.e. cysticercosis, trichinellosis, paragonimiasis, sparganosis and angiostrongyliasis. This was done with a multiplex protein microarray assay equipped with semi-purified antigens capable of detecting disease-specific antibodies. Results showed that the protein microarray developed displayed a good specificity, ranging from 97.0% to 100.0%, and a sensitivity, ranging from 85.7% to 92.1%, with a Youden index variation from 0.83 to 0.92. It was concluded that the protein microarray provides a sensitive, high-throughput technique for the simultaneous detection of multiple FBHs overcoming the limitations of conventional diagnostics.

Background Clonorchiasis, caused by Clonorchis sinensis, is one of the major parasitic zoonoses in China, particularly in China's southern Guangdong province where the prevalence of C. sinensis infection in humans is high. However, little is known of the prevalence of C. sinensis infection in its reservoir hosts dogs and cats. Hence, the prevalence of C. sinensis infection in dogs and cats was investigated in Guangdong province, China between October 2006 and March 2008. Results A total of 503 dogs and 194 cats from 13 administrative regions in Guangdong province were examined by post-mortem examination. The worms were examined, counted, and identified to species according to existing keys and descriptions. The average prevalences of C. sinensis infection in dogs and cats were 20.5% and 41.8%, respectively. The infection intensities in dogs were usually light, but in cats the infection intensities were more serious. The prevalences were higher in some of the cities located in the Pearl River Delta region which is the most important endemic area in Guangdong province, but the prevalences were relatively lower in seaside cities. Conclusions The present investigation revealed a high prevalence of C. sinensis infection in its reservoir hosts dogs and cats in China's subtropical Guangdong province, which provides relevant "base-line" data for conducting control strategies and measures against clonorchiasis in this region.

Background IgY isolated from egg yolk has been widely used in immunodiagnostic tests, including tests to detect circulating antigen (soluble egg antigen or SEA) of Schistosoma japonicum . Results A sandwich ELISA was established using a combination of anti- S. japonicum SEA-IgY polyclonal antibodies and IgM monoclonal antibodies. To explore sensitivity and specificity of the sandwich ELISA, serum samples from 43 patients infected with S. japonicum were tested. All acute cases and 91.3% of the chronic cases showed a positive reaction. Only 5% of the control sera from healthy persons gave a positive response. Cross-reactions with antibodies to nine other parasites were rare. Conclusion The developed immunoassay is reasonably sensitive and specific. It could be used for field research and treatment efficacy assessments.

Liver flukes belonging to the genus Fasciola are among the causes of foodborne diseases of parasitic etiology. These parasites cause significant public health problems and substantial economic losses to the livestock industry. Therefore, it is important to definitively characterize the Fasciola species. Current phenotypic techniques fail to reflect the full extent of the diversity of Fasciola spp. In this respect, the use of molecular techniques to identify and differentiate Fasciola spp. offer considerable advantages. The advent of a variety of molecular genetic techniques also provides a powerful method to elucidate many aspects of Fasciola biology, epidemiology, and genetics. However, the discriminatory power of these molecular methods varies, as does the speed and ease of performance and cost. There is a need for the development of new methods to identify the mechanisms underpinning the origin and maintenance of genetic variation within and among Fasciola populations. The increasing application of the current and new methods will yield a much improved understanding of Fasciola epidemiology and evolution as well as more effective means of parasite control. Herein, we provide an overview of the molecular techniques that are being used for the genetic characterization, detection and genotyping of Fasciola spp.., This work was supported, in part, by the National S & T Major Program (Grant No. 2008ZX10004-011, 2009ZX10004-302, 2009ZX10004-201), the National Key Technology R & D Program (Grant No. 2008BAI56B03), the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, the Yunnan Provincial Program for Introducing High-level Scientists (Grant No. 2009CI125) and the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT0723).

Each of BALB/c mice was infected with 50 Angiostrongylus cantonensis larvae. One group of mice received an intraperitoneal injection of 50 μg 12D5 monoclonal antibody (mAb) against a 98 kDa antigen of adult worms at 10 days post-infection (dpi), with a booster injection of 25 μg at 12 dpi. Five mice from each group were sacrificed at 14 dpi for pathological examination and RNA extraction. The infiltration of eosinophils and severity of eosinophilic meningitis were reduced in 12D5 mAb-treated mice compared with the infected mice without 12D5 treatment. The levels of eotaxin mRNA expression in spleen significantly increased and the expression of the Th2-type cytokine IL-5 significantly decreased. However, the expression of IL-4 was not changed. 12D5 mAb can observably enhance the survival rate of infected mice and reduce symptoms of angiostrongyliasis. A. cantonensis infection is a major cause of eosinophilic meningoencephalitis. The results of this study could be helpful for the development of treatment of human angiostrongylosis.

With the use of 2 monoclonal antibodies (MAbs) against excretory/secretory (ES) antigens of adult Angiostrongylus cantonensis, a new method was developed for double antibody sandwich ELISA for the detection of circulating antigens (CAg). To evaluate the sensitivity of the new procedure, the CAg in sera of rats (80) and mice (15) infected with A. cantonensis, as well as CAg in sera of clinically confirmed angiostrongyliasis patients (70), were evaluated. Cross-reaction testing was used to determine the specificity of serum from patients infected with Ascaris lumbricoides, Trichinella spiralis , Toxoplasma gondii , Schistosoma japonicum, Paragonimus westermani, Clonorchis sinensis, Echinococcus granulosus, Spirometra, and Taenia solium, as well as normal healthy people. The results proved that the sensitivity and the specificity of the new method were totally effective for the detection of A. cantonensis CAg. The assay is highly sensitive, specific, and reproducible, with easy handling and excellent cost effectiveness, and thereby provides a new method for the accurate diagnosis of angiostrongyliasis.

Background In recent years, surveys of Toxoplasma gondii infection in dogs have been reported worldwide, including China. However, little is known about the prevalence of T. gondii in pet dogs in Northwest China. In the present study, the prevalence of T. gondii in pet dogs in Lanzhou, China was investigated using the modified agglutination test (MAT). Results In this survey, antibodies to T. gondii were found in 28 of 259 (10.81%) pet dogs, with MAT titers of 1:20 in 14 dogs, 1:40 in nine, 1:80 in four, and 1:160 or higher in one dog. The prevalence ranged from 6.67% to 16.67% among dogs of different ages, with low rates in young pet dogs, and high rates in older pet dogs. The seroprevalence in dogs >3 years old was higher than that in dogs ≤1 years old, but the difference was not statistically significant (P > 0.05). The seroprevalence in male dogs was 12.50% (17 of 136), and in female dogs it was 8.94% (11 of 123), but the difference was not statistically significant (P > 0.05). Conclusions A high prevalence of T. gondii infection was found in pet dogs in Lanzhou, Northwest China, which has implications for public health in this region. In order to reduce the risk of exposure to T. gondii, further measures and essential control strategies should be carried out rationally in this region.

Angiostrongylus cantonensis, a rat lungworm, can cause eosinophilic meningitis and angiostrongyliasis in humans following ingestion of contaminated foods or intermediate/paratenic hosts with infective larvae. The snail Achatina fulica is one of the important intermediate hosts of A. cantonensis and is commonly eaten by humans in some countries. In the present study, we developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of A. cantonensis in Ac. fulica. Primers for LAMP were designed based on the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) of A. cantonensis. Specificity tests showed that only the products of A. cantonensis were detected when DNA samples of A. cantonensis and the heterologous control samples Anisakis simplex s.s, Trichuris trichiura, Toxocara canis, Trichinella spiralis and Ascaris lumbricoides were amplified by LAMP. Sensitivity evaluation indicated that the LAMP assay is 10 times more sensitive than the conventional polymerase chain reaction (PCR) assay. The established LAMP assay is rapid, inexpensive and easy to be performed. It can be used in clinical applications for rapid and sensitive detection of A. cantonensis in snails, which has implications for the effective control of angiostrongyliasis.

Published data on the association between mitogen-activated protein kinase kinase kinase 1 (MAP3K1) gene rs889312 polymorphism and breast cancer risk are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. Crude ORs with 95% CIs were used to assess the strength of association between them. A total of seven eligible articles including 26,015 cases and 33,962 controls based on the search criteria were involved in this meta-analysis. We observed that the MAP3K1 rs889312 polymorphism was significantly correlated with breast cancer risk from the fixed effects model when all studies were pooled into the meta-analysis (the allele contrast model: OR 1.09, 95% CI 1.07–1.12; the homozygote codominant: OR 1.22, 95% CI 1.15–1.29; the heterozygote codominant: OR 1.07, 95% CI 1.04–1.11; the dominant model: OR 1.10, 95% CI 1.06–1.13; the recessive model: OR 1.18, 95% CI 1.12–1.25). No significant association was found in the BRCA1 mutation carriers in all genetic models. When stratified by BRCA2 mutation carriers status, statistically significantly elevated risk was found in this meta-analysis (C vs. A: OR 1.12, 95% CI 1.01–1.23; CC vs. AA: OR 1.35, 95% CI 1.06–1.71; the recessive model: OR 1.31, 95% CI 1.05–1.65). There was no evidence for significant association between MAP3K1 rs889312 polymorphism and breast cancer risk in BRCA1 and BRCA2 positive cohort for all comparison models. In conclusion, this meta-analysis suggests that the MAP3K1 rs889312 C allele is a low-penetrant risk factor for developing breast cancer, and there is limited evidence to indicate that MAP3K1 rs889312 polymorphism is associated with increased risk of breast cancer in BRCA1 mutation carriers.

Aims Innate and adaptive immune responses are associated with the development of hypertension-induced myocardial hypertrophy and fibrosis. As a result, we investigated whether heat shock protein (HSP) 70, which is a molecule of damage-associated molecular patterns, could induce inflammation in the myocardium and promote the development of hypertension-induced cardiac hypertrophy and fibrosis. Methods and results We found that HSP70 serum levels, as well as the amount of HSP70 translocation to the cardiomyocyte membranes and the interstitial space, were elevated in the hypertensive mice caused by abdominal aortic constriction (AAC). Transcriptional inhibition of HSP70 expression by a specific heat shock transcript factor inhibitor, KNK437, reduced the serum level, and the re-distribution of HSP70. It promoted myocardial hypertrophy and cardiac dysfunctions although it protected animals from AAC-induced cardiac fibrosis. On the other hand, the functional antagonism of HSP70 by an anti-HSP70 antibody attenuated AAC-induced cardiac hypertrophy and fibrosis without adverse haemodynamic effects. The cardioprotective effect of the anti-HSP70 antibody was largely attributed to its ability to block AAC-activated immune response in the heart, as was indicated by suppressing the hypertension-enhanced conjugation of HSP70 with toll-like receptor 4, reducing heart-infiltrating macrophages, decreasing the expression of pro-inflammatory factor monocyte chemoattractant protein-1 and profibrotic factor transforming growth factor beta 1, and attenuating pro-hypertrophy signal MAPK P38 and ERK. Conclusion These results indicate that intracellular and extracellular HSP70 have different roles in the regulation of cardiac remodelling and function in response to hypertension. Extracellular HSP70 is a potential therapeutic target against cardiac hypertrophy and fibrosis.

To the Editor: Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans after they ingest infective larvae in freshwater and terrestrial snails and slugs, paratenic hosts (such as freshwater fish, shrimps, frogs, and crabs), or contaminated vegetables. With the increase of income and living standards, and the pursuit of exotic and delicate foods, populations around the world have seen angiostrongyliasis become an important foodborne parasitic zoonosis (1–9). Shenzhen municipality is situated in the most southern part of mainland People’s Republic of China between the northern latitudes of 22°27′ to 22°52′ and eastern longitudes of 113°46′ to 114°37′; it shares a border with the Hong Kong Special Administrative Region, China, in the south. The climate is subtropical, with an average annual temperature of 23.7°C. The city is 1,952.84 km2 and has a population of 10 million. Since 2006, thirty-two sporadic cases of human eosinophilic meningitis caused by consumption of undercooked aquacultured snails have been documented in Shenzhen (Shenzhen Center for Disease Control and Prevention, unpub. data). To identify the source of these infections and assess the risk for an outbreak of eosinophilic meningitis, we conducted a survey to investigate whether A. cantonensis occurs in wild rats and snails in Shenzhen. To examine A. cantonensis infection in intermediate host snails, 302 terrestrial snails (Achatina fulica) were collected from 10 investigation sites across Shenzhen, and 314 freshwater snails (Pomacea canaliculata)were sampled from 6 investigation sites. We examined the snails for A. cantonensis larvae by using pepsin digestion standardized procedures (3). To survey the prevalence of adult A. cantonensis in definitive host rats, we collected 187 Rattus norvegicus rats and 121 R. flavipectus rats collected from 4 sites where positive snails positive for A. cantonensis were found. These rats were examined for the presence of adult A. cantonensis in their cardiopulmonary systems. A. cantonensis larvae were found in 96 (15.6%) of 616 examined snails. Of these, P. canaliculata had an average infection rate of 20.7% (65/314), significantly higher (p

A seroepidemiological survey was carried out in China during 2009-2010 to determine the extent of circulating antigens (CAg) for Angiostrongylus cantonensis in the Chinese population using the gold immunochromatographic assay, with the objective of elucidating the nationwide prevalence of angiostrongyliasis in China. A total of 1,730 blood samples was collected and assayed from the general adult population (the "general group"), and those involved in aquaculture or processing of snails Achatina fulica and Pomacea canaliculat (the "occupational group") from 5 provinces (Fujian, Hunan, Guangdong, Guangxi, and Zhejiang) and 1 municipal city (Beijing). The overall seroprevalence for the "occupational group" was 7.4% (40/540), which was significantly higher (P0.001) than that of the "general group" (0.8%, 9/1,190). The seroprevalence in males (9.5%) was significantly higher than in females (4.2%) (P0.05). These results demonstrate that angiostrongyliasis represents a significant zoonotic disease in China, requiring the strengthening of food safety for control of this food-borne disease.