Sirintip Boonjaraspinyo

A Cross-Sectional Study on Intestinal Parasitic Infections in Rural Communities, Northeast Thailand

Despite the existence of effective anthelmintics, parasitic infections remain a major public health problem in Southeast Asia, including Thailand. In rural communities, continuing infection is often reinforced by dietary habits that have a strong cultural basis and by poor personal hygiene and sanitation. This study presents a survey of the prevalence of intestinal parasitic infections among the people in rural Thailand. The community-based cross-sectional study was conducted in villages in Khon Kaen Province, northeastern Thailand, from March to August 2013. A total of 253 stool samples from 102 males and 140 females, aged 2-80 years, were prepared using formalin-ethyl acetate concentration methods and examined using light microscopy. Ninety-four individuals (37.2%) were infected with 1 or more parasite species. Presence of parasitic infection was significantly correlated with gender (P=0.001); nearly half of males in this survey (49.0%) were infected. Older people had a higher prevalence than younger members of the population. The most common parasite found was Opisthorchis viverrini (26.9%), followed by Strongyloides stercoralis (9.5%), Taenia spp. (1.6%), echinostomes (0.4%), and hookworms (0.4%). The prevalence of intestinal protozoa was Blastocystis hominis 1.6%, Entamoeba histolytica 0.8%, Entamoeba coli 0.8%, Balantidium coli 0.4%, Iodamoeba bütschlii 0.4%, and Sarcocystis hominis 0.4%. Co-infections of various helminths and protozoa were present in 15.9% of the people. The present results show that the prevalence of parasitic infections in this region is still high. Proactive education about dietary habits, personal hygiene, and sanitation should be provided to the people in this community to reduce the prevalence of intestinal parasite infections. Moreover, development of policies and programs to control parasites is needed.

Turmeric reduces inflammatory cells in hamster opisthorchiasis

The curcumin compound from turmeric is effective in the treatment of many inflammatory diseases. The aim of our present study was to evaluate the efficacy of turmeric on reducing the histopathological changes of hamster opisthorchiasis. Hamsters were infected with Opisthorchis viverrini and then administered turmeric. Using light microscopic observation, liver function tests for alanine transaminase (ALT), alkaline phosphatase, and direct bilirubin were investigated. The resulting histopathological changes show that turmeric has anti-inflammatory properties—during both N-nitrosodimethylamine administration and O. viverrini infection—by reducing the aggregation of inflammatory cells surrounding the hepatic bile ducts, which correlates with a decreased serum ALT level. The decrease in direct bilirubin levels in the hamsters treated with turmeric suggests that turmeric may enhance biliary contraction. The present study found that turmeric clearly reduces the inflammatory cells in hamster opisthorchiasis at an early stage. This finding may be connected with a reduction in the risk factors of cholangiocarcinoma development.

Animal models for Opisthorchis viverrini infection

We investigated the utility of various animal models for the study of opisthorchiasis in humans and its common sequel of cholangiocarcinoma (CCA). Rats, mice, gerbils, and hamsters were infected with Opisthorchis viverrini metacercariae. Worms from the infected animal hosts were recovered from livers and counts made of eggs per gram of feces. Worms were observed in and recovered from hamsters and gerbils but not rats and mice. The recovered worms from the infected gerbils were larger and more physiologically developed than those from the infected hamsters. The results suggest that gerbils are more susceptible to infection by Opisthorchis viverrini and thus more suitable for modeling opisthorchiasis and its connection to CCA.

Overexpression of PDGFA and its receptor during carcinogenesis of Opisthorchis viverrini-associated cholangiocarcinoma

Cholangiocarcinoma (CCA) is a crucial health problem in northeastern part of Thailand, which is caused by a combination of Opisthorchis viverrini infection and nitrosamine. A better understanding of its molecular mechanism is an important step to discover and develop the new diagnostics and therapies for CCA. To reveal the involvement of potential genes in the development of CCA, the present study investigated the expression kinetics of platelet-derived growth factor alpha (Pdgfa) and its receptor (Pdgfra) during the tumorigenesis of CCA induced by O. viverrini infection with quantitative RT-PCR, and confirmed the expression with immunohistological staining. The results showed that in the hamster model of opisthorchiasis-associated CCA, the expression of Pdgfa was increased after infection plus N-nitrosodimethylamine (NDMA) administration, reached its peak at 2 months post infection, and remained at the high level until 6 months. Similarly, the expression of Pdgfra was increased time-dependently. The positive immunostaining for PDGFA proteins was observed in the cytoplasm of epithelial tumor cells of hamster CCA. Moreover, the analysis of the expression of these genes in 10 cases of human opisthorchiasis-associated CCA showed that Pdgfa was overexpressed in 80%, and Pdgfra was overexpressed in 40% cases (>3.0 folds, compared with the expressions of adjacent normal tissues). This result suggests that PDGFA is likely involved in the tumorigenesis of opisthorchiasis-associated CCA, and may be a promising candidate biomarker for diagnosis and treatment strategies of CCA.

Candidate genes involving in tumorigenesis of cholangiocarcinoma induced by Opisthorchis viverrini infection

Opisthorchiasis-associated cholangiocarcinoma (CCA) is one of main public health problems in Opisthorchis viverrini endemic areas. Although the definite relationship between prevalence of CCA and the parasite infection has been demonstrated, the molecular mechanism of tumorigenesis is still unknown. In the present study, by using animal model of opisthorchiasis-associated CCA, a kinetic analysis of cDNA microarray was performed to screen the candidate genes that involve in the development of opisthorchiasis-associated CCA. Microarray analysis revealed that the expressions of 131 genes were up-regulated during the development of CCA, including the genes relative to cell proliferation, differentiation and transformation, cell growth and cycle regulation, apoptosis, DNA repair, and cytoskeletal structure. The expressions of 145 genes were down-regulated, including the genes relative to metabolic enzymes, tumor suppressor, apoptosis, and oxidative response and oxidation reduction. The present study listed up the candidate genes involving tumorigenesis, provided molecular information on the development of opisthorchiasis-associated CCA and the potential biomarkers for diagnosis and therapy, and suggested that the increased expression of cell differentiation, proliferation, transformation-related genes, and decreased expression of metabolic enzymes may play important roles in the tumorigenesis of CCA.

Significance of S100P as a biomarker in diagnosis, prognosis and therapy of opisthorchiasis-associated cholangiocarcinoma

Cholangiocarcinoma (CCA) is a malignancy of bile duct with the difficulty in early diagnosis, poor prognosis and less alternation in therapy. S100P is a member of S100 family proteins and plays important roles in cancers. We investigated the S100P expression and its correlation with clinicopathology in 78 cases of opisthorchiasis‐associated CCA, and the effects of S100P knockdown with shRNA interference on the proliferation, cell cycle, migration, apoptosis and sensitivity to anti‐cancer drug. Extremely high expression of S100P mRNA was detected in the CCA tumor tissues. The increased S100P protein expression was immunohistochemically confirmed and localized in the CCA cytoplasm and/or nuclei as well as in the hyperneoplasia and dysplasia bile ducts, but not in normal bile ducts. The intensity of immunostaining was correlated with survival, tumor stage and metastasis, and the high expression could be an independent prognostic factor. High levels of S100P were detected in the serum and bile fluid of CCA patients. The shRNA‐mediated knockdown of S100P expression inhibited the proliferation in vitro and in vivo, and migration of CCA cells, arrested cell cycle with the up‐regulated expression of cell cycle arrest related factors, p21, p27, GADD45A, and 14‐3‐3 zeta. S100P knockdown also promoted CCA cell apoptosis by up‐regulating expression of apoptosis related factors, DR5, TRADD, caspase 3 and BAX, and increased the sensitivity of CCA cells to the chemotherapeutic agents sunitinib and apigenin. Taken together, this study indicates that S100P might be a promising biomarker for the diagnosis, prognosis and therapy of CCA.

Down-Regulated Expression of HSP70 in Correlation with Clinicopathology of Cholangiocarcinoma

Cholangiocarcinoma is a crucial health problem in northeast Thailand. Although rare, it is a highly fatal disease and the prognosis of CCA patients is very poor. To determine if expression of specific genes is useful for diagnosis and prognosis for CCA. We examined the expression of HSP70, HSP90, RB1, cyclin D1, and HDAC6 in 50 resections of human CCA tissues by quantitative real-time PCR. The expression of HSP70, RB1, and HDAC6 was “dominant down-regulation,” while the expression of cyclin D1 and HSP90 was “dominant up-regulation.” There were no correlations between RB1, cyclin D1, HSP90, and clinicopathological parameters such as status, histology type, histological grading, stage of CCA, and metastasis. A significant association was found between HDAC6 and CCA staging (p = 0.000), CCA gross type and HSP70 (p = 0.046) as well as RB1 expression (p = 0.046). Patients with down-regulation of HSP70 had significantly poorer prognosis than those in the up-regulation group (p = 0.002). Expression of HSP70 may be useful as a new prognostic marker for CCA.

Indirect effect of a turmeric diet: enhanced bile duct proliferation in Syrian hamsters with a combination of partial obstruction by Opisthorchis viverrini infection and inflammation by N-nitrosodimethylamine administration

The present study revealed the indirect effect of a turmeric (TUR) diet on the histopathological changes and proliferating cell nuclear antigen staining in Syrian hamsters with partial obstruction by liver fluke (Opisthorchis viverrini) infection and inflammation by N-nitrosodimethylamine (NDMA) administration. The result of the analysis of histopathological changes shows that a TUR diet has an anti-inflammatory property in the case of a single condition of NDMA administration or O. viverrini infection, as has been reported previously. Unfortunately, an adverse indirect effect of TUR was observed in the combination of infection with O. viverrini and administration of NDMA, with a 30–50% increase in new bile duct formation, correlated with an increase in proliferating cell nuclear antigen. Our present result suggests that the properties of curcumin are anti-inflammation and antioxidant including enhancing biliary contraction and bile flow. Thus, a combination of factors (treated with O. viverrini, NDMA, and TUR diet) result in an increasing bile duct proliferation which may cause from biliary homeostasis.

Opisthorchis viverrini infection causes liver and biliary cirrhosis in gerbils

Opisthorchis viverrini infection causes many hepatobiliary diseases, including cholangiocarcinoma. Hence, the study of O. viverrini infection in humans is subject to ethical limitations, so an animal model, the Syrian hamster, is often used. O. viverrini can develop into the adult stage not only in Syrian hamsters but also in other animals, including gerbils, but until now, there has been no report on pathology and susceptibility in gerbils. The present study revealed the pathology of O. viverrini infection in gerbils through gross appearance, histopathology, and worm recovery at various time points. Gerbils were infected with 50 O. viverrini metacercariae and then sacrificed at the time of observation. The gross appearance of the liver showed micronodules at the liver surface, suggesting liver and biliary cirrhosis. Light microscopic observation was correlated to the gross appearance with cholecystitis, fatty liver changes, fibrous septa, and generalized cirrhosis. The range of worm burden fluctuated from 1 to 25 worms with large body size, which was correlated with pathology. These novel findings indicate that O. viverrini infection can cause liver and biliary cirrhosis in gerbils, depending on the worm burden, worm size, and habitat.

Alteration of galectin-1 during tumorigenesis of Opisthorchis viverrini infection-induced cholangiocarcinoma and its correlation with clinicopathology

Galectin-1 is a beta-galactoside-binding lectin to function in cell adhesion, proliferation, differentiation, and might be involved in tumor progression and metastasis. In the present study, the expression kinetics of galectin-1 during the tumorigenesis of a parasite Opisthorchis viverrini infection-induced cholangiocarcinoma (CCA) was investigated in model animal hamsters, and the expression was confirmed in human CCA cases. It was found that galectin-1 was overexpressed at mRNA and protein levels with the tumor progression. The mRNA expression was elevated in very early stage during tumorigenesis and the increase was time dependent. Galectin-1 protein expression profiles indicated that the increased expression was mainly located in the epithelium of extensively proliferated and hyperplasia small bile ducts at early stage of CCA development in model animal and mainly in the extensive tumor stroma tissues in both model animals and human CCA cases at later stage. The analysis of correlation of the overexpression with clinicopathology in human cases suggested that high expression of galectin-1 was associated with advanced stage and metastasis and with shorter cumulative overall survival of the patients. Multivariate Cox regression analysis revealed that galectin-1 expression was of independent prognostic significance for CCA. Our results suggest that galectin-1 is likely involved in the tumorigenesis and expected to serve as a tumor stroma marker in diagnosis and prediction of metastasis and poor prognosis of the opisthorchiasis-associated CCA.