Plasma IgG autoantibody against actin-related protein 3 in liver fluke Opisthorchis viverrini infection.

Opisthorchiasis secondary to Opisthorchis viverrini infection leads to cholangiocellular carcinoma through chronic inflammation of the bile ducts and possibly inducing autoimmunity. It was hypothesized that plasma autoantibodies directed against self-proteins are biomarkers for opisthorchiasis. Plasma from patients with opisthorchiasis was tested using proteins derived from immortalized cholangiocyte cell lines, and spots reacting with plasma were excised and subjected to LC-MS/MS. Seven protein spots were recognized by IgG autoantibodies, and the highest matching scored protein was actin-related protein 3 (ARP3). The antibody against ARP3 was tested in plasma from 55 O. viverrini-infected patients, 24 patients with others endemic parasitic infections and 17 healthy controls using Western blot and ELISA. Immunoreactivity against recombinant ARP3 was significantly more prevalent in opisthorchiasis compared to healthy controls at Western blotting and ELISA (P < 0.05). Plasma ARP3 autoantibody titres were also higher in opisthorchiasis compared to healthy individuals (P < 0.01) and other parasitic infections including Strongyloides stercoralis (P < 0.001), echinostome (P < 0.05), hookworms (P < 0.001) and Taenia spp. (P < 0.05). It was further characterized in that the ARP3 autoantibody titre had a sensitivity of 78.18% and specificity of 100% for opisthorchiasis. In conclusion, it may be suggested that plasma anti-ARP3 might represent a new diagnostic antibody for opisthorchiasis.

Circulating CD14(+) CD16(+) monocyte levels predict tissue invasive character of cholangiocarcinoma.

Chronic inflammation as a risk factor for cancer development is driven in part by monocyte/macrophages, which in many cancers exhibit pro-tumorigenic activity. In this study we identified elevation in CD14(+) CD16(+) , a minor blood monocyte subpopulation in cholangiocarcinoma (CCA) patients, compared to normal and biliary disease patient specimens. Tumour association was suggested by the observation that this elevated level decreased to normal after tumour resection. Moreover, the elevated level of CD14(+) CD16(+) monocytes in CCA patient blood correlated with degree of MAC387-positive (recent blood-derived macrophage migrant-specific marker) tumour-associated macrophage infiltration as determined by immunohistochemistry. These CD14(+) CD16(+) monocytes were suggested to enhance tumour progression as this subpopulation possesses (i) high expression of adhesion molecules (CD11c, CD49d, and CD54) and scavenger receptor (CD163), which enable them to adhere strongly to endothelial cells, and (ii) that peripheral blood monocytes from CCA patients express high levels of growth and angiogenic factor-related genes (epiregulin, VEGF-A and CXCL3). Elevation of peripheral CD14(+) CD16(+) monocyte levels was associated with features associated with poor prognosis CCA parameters (non-papillary type and high number of tissue macrophages). These data indicate that the CD14(+) CD16(+) monocytes from CCA patients with pro-tumorigenic characteristics may associate with rapid tumour progression and poor patient outcome. If confirmed in subsequent studies, the level of CD14(+) CD16(+) monocytes may serve as a marker for disease activity in CCA patients and serve as a target for pathogenic macrophage specific drug development.

Proteomic identification of peroxiredoxin 6 for host defence against Opisthorchis viverrini infection.

Opisthorchis viverrini infection causes opisthorchiasis and is a risk factor for cholangiocarcinoma via chronic inflammation. To investigate the mechanism of O. viverrini -induced liver disease, we applied a proteomic approach to examine alterations in hepatic protein levels in O. viverrini -infected hamsters. Two-dimensional gel electrophoresis (2DE) revealed that O. viverrini infection induced upregulation (1.5- to 4.3-fold) of 25 proteins and downregulation (1.5 to 2.5-fold) of 24 proteins compared with uninfected animals. Expression of proteins related to stress response, DNA replication and repair, and cell structure was significantly increased, whereas that of proteins associated with normal liver function, such as metabolism, blood volume maintenance and fatty acid cycle was decreased. Among the upregulated proteins, a 2.7-fold increase in peroxiredoxin 6 (Prdx6), an antioxidant protein, was confirmed by 2DE and immunoblot analysis, Western blot and quantitative PCR. Immunohistochemical analysis showed that Prdx6 expression was observed mainly in the cytoplasm of inflammatory cells. These results suggest that Prdx6 is important for host defence against O. viverrini infection. This study provides basic information for Prdx6 as a potential biomarker and therapeutic target for opisthorchiasis.

Apoptosis-related gene expression in hamster opisthorchiasis post praziquantel treatment.

The aim of this study was to investigate the apoptosis-related gene expression in hamster opisthorchiasis after praziquantel treatment. Hamsters were infected with Opisthorchis viverrini metacercariae then treated with praziquantel. The expression of apoptosis-related genes [i.e., apoptosis gene Bcl-2-associated protein X (BAX), caspase 9, p53, and protein kinase B (PKB)] was detected by real-time reverse transcription polymerase chain reaction. Histopathological analyses of liver tissues were studies by staining the sections with hematoxylin and eosin using light microscopy. Apoptotic assay was used to localize the apoptotic cell death. The results show that BAX, Akt/PKB, p53, and caspase 9 expression level were significantly increased on day 30 post infection and at 6 h post treatment and gradually decreased nearly to the uninfected control and 24 h post treatment, perhaps due to a decrease in inflammatory cells. Apoptotic staining was positive reaction at inflammatory cells and nuclei of epithelial bile ducts. Although using praziquantel has an advantage in killing parasites, our results show the effect of praziquantel treatment from host immune response that induces increased apoptosis-related genes in the short term due to an increase in inflammatory cells surrounding the bile ducts.

Apoptosis-related gene expressions in hamsters re-infected with Opisthorchis viverrini and re-treated with praziquantel.

Our objective was to reveal whether host immune response in hamster opisthorchiasis post-praziquantel treatment could induce apoptotic cell death in inflammatory cells. We, therefore, investigated apoptosis-related gene expression in hamsters re-infected with Opisthorchis viverrini (OV) and re-treated with praziquantel. Hamsters were re-infected with OV metacercariae then re-treated with praziquantel. The expression of apoptosis-related genes (i.e. apoptosis gene Bcl-2 associated protein X [BAX], caspase 9, p53 and protein kinase B [PKB]) was detected by real-time reverse transcription-polymerase chain reaction. Histopathological analyses of liver tissues were performed by staining the sections with haematoxylin and eosin using light microscopy. The results show that BAX, Akt/PKB, p53 and caspase 9 expression levels were significantly increased on day 30 post-infection and at 6 h post-treatment and gradually decreased to a level near the uninfected control and at 24 h post-treatment, perhaps because of a decrease in inflammatory cells. Apoptotic cell death was observed at the nuclei of epithelial cells of the bile ducts and of T cells. Our results suggest that repeated infection with OV and re-treatment with praziquantel induces a host immune response that increases inflammatory cells, which in turn leads to increase, apoptosis-related gene expression in the short term post-treatment.

Seroprevalence of specific total immunoglobulin (Ig), IgG and IgM antibodies to Toxoplasma gondii in blood donors from Loei Province, Northeast Thailand.

A total samples from 345 healthy blood donors from Loei Province, Northeast Thailand were examined for anti-Toxoplasma gondii antibodies by ELISA. The seroprevalence of the anti-Toxoplasma gondii total Ig, IgG and IgM antibodies was 4.9%, 4.1% and 4.3%, respectively. Overall seropositive rate was 33 out of 345 individuals (9.6%). Among the seropositve cases, 5 (15.2%), 2 (6.1%) and 13 (39.4%) of the samples were determined by using each type of anti-T. gondii total Ig, IgG and IgM antibodies, respectively. The seropositive sera was also determined by combining of anti-T. gondii antibodies (anti-T. gondii total Ig with IgG and anti-T. gondii total Ig with IgM antibodies). These results were 10 (30.3%) and 2 (6.1%) cases, respectively. Only one (3%) sample had all types of anti-T. gondii antibodies. In addition, the frequency distribution curves of ELISA optical densities of anti-T. gondii total Ig, IgG and IgM antibodies in blood donor presented "unimodal" curves. The negative results were found in the age group that less than 20 years old and more than 51. The highest seropositive results were found in two age groups (21-30 and 31-40 years old), and males were significantly higher than female (p < 0.05). These results demonstrated that when using anti-T. gondii total Ig, IgG and IgM antibodies for determining the seroprevalence, the sensitivity was twice that with the anti-T. gondii, total Ig antibody alone.

K-ras oncogene and p53 gene mutations in cholangiocarcinoma from Thai patients.

Paraffin embedded tissues from twenty Thai patients with intrahepatic cholangiocarcinomas were studied for K-ras gene mutations at codon 12, 13 and 61 and for p53 gene mutations in exon 5 to 8 using polymerase chain reaction and thermal cycle sequencing. Results showed that point mutations at these regions in K-ras oncogene were not present in all the samples. One case harbored a p53 gene mutation in codon 282 in exon 8, CGG (arginine) to TGG (tryptophan), but the mutation was not found in other patient's tissues with similar histological features.

Centrocestus formosanus: surface morphology of metacercaria, adult and egg

The surface morphology of metacercariae isolated from Puntius spp., adult worms from infected hamsters and eggs of Centrocestus formosanus (Digenea: Heterophyidae) were studied using scanning electron microscopy. It was found that the surfaces of the metacercariae and adult worms were closely similar in appearance. The oral sucker was surrounded by a circumoral expansion with two rows of 32 spines (16 spines each). The ventral sucker, with six large nonciliated papillae on the lip, and the genital opening were located mid-ventrally. The excretory pore was terminal. The body surface was covered with pectinate scale-like spines of varying sizes. The scales in the middle area were larger than those in the anterior and posterior parts of the body. Sensory papillae, mostly uniciliated, were present in greater abundance at the anterior region of both stages. However, morphological variations were observed among the adults depending on the age of the worms. At 4 weeks, the oral sucker and circumoral expansion were cobblestone-like in structure and the grooves of spines split compared with their smooth surfaces at 10 days. In addition, the ventral sucker became depressed and its large papillae on the lip disappeared at 10 weeks post infection. Multiciliated papillae were also present in 10-week old worms. Each egg of this parasite possessed a prominent operculum and a latticed design on the egg shell.