Novel Potential Biomarkers for Opisthorchis viverrini Infection and Associated Cholangiocarcinoma.

BACKGROUND/AIM: Early detection of disease is a pivotal factor for determining prognosis and clinical outcome of patients with cancer. As cholangiocarcinoma (CCA) is currently difficult to detect and most cases of such cancer present with late-stage disease at the time of initial diagnosis, we employed proteomic analysis of the bile to identify potential candidate biomarkers for Opisthorchis viverrini (OV)-associated CCA. MATERIALS AND METHODS: Proteins in pooled bile samples from patients with CCA and OV infection, with CCA without OV infection, with OV infection but no CCA, and with neither OV infection nor CCA were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel trypsin digestion and analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: According to our analysis, three proteins, namely aristaless-like homeobox1 isoform X1 (ALX1), major histocompatibility complex polypeptide-related sequence A (MICA), and uncharacterized protein C14orf105 isoform X12 were found to be potential markers for OV infection, as they were predominantly found in all OV-infected groups. Although these proteins were detected in both OV-infected patients with and without CCA, their abundance was 2.90-, 7.06-and 3.65-fold higher, respectively, in those with CCA. In patients with CCA, potential novel biomarkers wre immunoglobulin heavy chain, translocated in liposarcoma (TLS), visual system homeobox 2 (VSX2) and an unnamed protein product. CONCLUSION: We provided novel information regarding potential biomarkers for OV infection and CCA. These two protein profiles could benefit diagnosis as well as monitoring of CCA.

Chromosome Abnormalities and Absolute Telomere Lengths of Leukocytes from Silk Weavers with Emphasis on Potential Genotoxicity and Mutagenicity of Silk Dyes

Objectives: This study is aimed to assess the possible genotoxicity and mutagenicity of silk dyes on silk weavers. Methods: Peripheral blood leukocytes were obtained from 24 silk weavers and 24 age- and sex-matched controls in northeastern Thailand. After mitogen stimulation in culture, chromosome abnormalities were examined using Giemsa banding and the absolute telomere length (aTL) was measured with SYBR green qRT-PCR. To confirm genotoxic and mutagenic effects of silk dyes, leukocytes from one each of healthy male and female volunteers were cultured with various concentrations of 3 dark red silk dyes under the presence of mitogen. Chromosome abnormalities and the telomere length were determined as above. Results: The proportion of normal metaphase in the silk weaving workers was significantly lower than that in controls. The frequency of chromosome aberrations was higher in the silk weavers than in control group. Polyploidy was detected only in the silk weavers. The aTL was significantly shorter in the silk weavers than in control group (p < 0.05). When leukocytes from normal volunteers were stimulated with mitogen under the presence of various concentrations of 3 silk dyes, suppressed the mitotic index (MI) and normal metaphase, whereas the proportion of prophase and the incomplete chromosome forming increased significantly. All dyes induced polyploidy. Dye #CA5 induced structural changes in male leukocytes, whereas #30 induced the changes in female leukocytes. The #CA5 increased aTL of normal leukocytes in a dose-dependent manner. Conclusions: All dyes, especially #CA5, have high genotoxicity and mutagenicity to induce chromosome aberrations and telomeric instability. Taken all those results together, regular health checking of silk weavers who have been exposed to those dyes is critically necessary to prevent various chemical-induced carcinogenesis.

Viability and morphological changes of Acanthamoeba spp. cysts after treatment with Effective microorganisms (EM).

Acanthamoeba is a free-living opportunistic protozoan parasite that is found in diverse environments. It can cause keratitis, mostly related to inappropriate use of contact lenses, as well as life threatening diseases including encephalitis, disseminated sinusitis, and skin ulcers. This study investigated morphological changes and fine structures of the cyst form of Acanthamoeba spp. after treatment with effective microorganisms (EM™) using light and scanning electron microscopies. Acanthamoeba cysts treated with 1:2, 1:4, 1:6, and undiluted EM™ showed higher percentages of non-viable cysts than those treated with 1:8, 1:10, 1:100, 1:200, and 1:400 EM™ and at 5 days post-treatment developed from cystic stage to trophozoite stage. Acanthamoeba cysts treated at concentrations of 1:2, 1:4, 1:6, and undiluted EM™ exhibited cytoplasmic clumping and shrinkage of amoeba cells away from cyst walls. The effective EM™ concentration lethal to Acanthamoeba spp. cyst could provide information to monitor the environmental control system.

Morphological features of Acanthamoeba causing keratitis contaminated from contact lens cases.

OBJECTIVE: To study the morphological characteristics of genus Acanthamoeba which is an opportunistic organism associated with wearing contact lenses that the biofilm phenomenon in contact lens cases contained Acanthamoeba causing keratitis by conventional culture technique. MATERIAL AND METHOD: A total of 150 contact lens cases were biofilm scraped in March till September 2007, at an institution in Nakhornpathom Province, Thailand. The 'gold standard' culture technique was used for the excystation growth development observation. Cysts of Acanthamoeba spp. contained 50 microlitres of Escherichia coli and contact lens solution were incubated and observed for the presence of cysts and/or trophozoites for 12 days. An infected slide was stained with giemsa solution and other non-stained and non-fixed slides were carried out for morphological characteristics study by different microscopes. RESULTS: The prevalence of Acanthamoeba spp. in scraping of contact lens cases was 6.7% (10/150). These Acanthamoeba isolates at temperature around 37 degrees C were consisted of all three groups, which in summary; the average diameter of cysts in Astronyxids (group I) was relatively large. They were > or = 18 micrometers, while those of Polyphagids (group II) and Culbertsonids (group III) were < or = 18 micron. The typical morphology of Acanthamoeba trophozoites moving freely in water were recognized by the presence of lobopodium and acanthopodia within 12 observed days. The average size of Acanthamoeba trophozoites was in the range of 12-45 micron. Three different images of cyst were feature studied. CONCLUSION: Three Acanthamoeba groups by biofilm scraping from contact lens cases should be differentiated. Morphological characteristics cysts and trophozoites should be confirmed. In addition, to improve contact lens wearer education, compliance with contact lens cases, hygiene recommendations and regular disposal of contact lens cases might help to solve contact lens cases.

TaqMan real-time PCR assay for specific detection of Opisthorchis viverrini DNA in Thai patients with hepatocellular carcinoma and cholangiocarcinoma.

The aim of this study was to develop TaqMan real-time PCR assay that detected Opisthorchis viverrini DNA from 18 normal and 18 tumor tissue specimens from Thai patients with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), who underwent liver resection from October 2005 to May 2006. Control liver specimens were seven non-primary liver cancers. A conserved probe representing 100% sequence homology was used as a reference for O. viverrini-specific probe. Five of six tumors (83%) and all six normal tissues from CCA group; and seven of twelve tumors (58%) and ten of twelve normal tissues (83%) from HCC group were found to have O. viverrini DNA. The O. viverrini DNA detection among HCC and CCA patients were not associated (p=0.193; 90%CI). This RT-PCR will be a useful tool for investigating the relationship between cancer type and presence of the parasite and also for conducting epidemiological surveys.

Detection of VacA gene specific for Helicobactor pylori in hepatocellular carcinoma and cholangiocarcinoma specimens of Thai patients.

In order to investigate and compare the presence of Helicobacter pylori VacA in primary liver cancer specimens (12 hepatocellular carcinoma and 6 cholangiocarcinoma) and control liver specimens (7 non-primary liver cancer) from Thai patients who underwent liver resection, H. pylori VacA gene was assayed in extracted DNA by real-time polymerase chain reaction. The selected amplicons revealed high homology compared with H. pylori VacA sequence. H. pylori VacA gene was detected in all primary liver cancer specimens and in 71% (5/7) of control liver specimens.