Combination of Praziquantel and Aspirin Minimizes Liver Pathology of Hamster Opisthorchis viverrini Infection Associated Cholangiocarcinoma.

Opisthorchiasis is one of the major risk factors for cholangiocarcinoma (CCA) in northeastern Thailand. An effective drug for killing this parasite is praziquantel. Recently, several reports have shown that with frequent use, praziquantel may itself be a CCA risk and can cause liver cell damage from an immunopathological response after parasite death. Aspirin has many properties including anti-inflammation and anti-cancer. Therefore, we use of aspirin (As) and praziquantel (Pz) to improve hepatobiliary system function in hamsters infected with Opisthorchis viverrini (OV) and or administered N-nitrosodimethylamine (ND). Livers of OVNDAsPz, appeared healthy macroscopically, suggesting slow progression of cholangiocarcinoma evident by extent of fibrosis and bile duct cell proliferation was less than OVND although aggregations of inflammatory cells remained. Proliferating cell nuclear antigen (PCNA), cytokeratin 19 (CK19), and cancer antigen (CA19-9) staining were strongly positive in OVND, but were only slight in OVNDAs. Moreover, OVNDAsPz, appeared a few inflammatory infiltrations, bile duct proliferation, fibrosis and CCA area than the OVNDAs group. Thirty seven point five percent of hamster in this group could not develop CCA. These findings suggest that using aspirin combination with praziquantel treatment can improve the hepatobiliary system after O. viverrini infection and reduce the risk of CCA.

DNA barcode identification of freshwater snails in the family Bithyniidae from Thailand.

Freshwater snails in the family Bithyniidae are the first intermediate host for Southeast Asian liver fluke (Opisthorchis viverrini), the causative agent of opisthorchiasis. Unfortunately, the subtle morphological characters that differentiate species in this group are not easily discerned by non-specialists. This is a serious matter because the identification of bithyniid species is a fundamental prerequisite for better understanding of the epidemiology of this disease. Because DNA barcoding, the analysis of sequence diversity in the 5' region of the mitochondrial COI gene, has shown strong performance in other taxonomic groups, we decided to test its capacity to resolve 10 species/ subspecies of bithyniids from Thailand. Our analysis of 217 specimens indicated that COI sequences delivered species-level identification for 9 of 10 currently recognized species. The mean intraspecific divergence of COI was 2.3% (range 0-9.2 %), whereas sequence divergences between congeneric species averaged 8.7% (range 0-22.2 %). Although our results indicate that DNA barcoding can differentiate species of these medically-important snails, we also detected evidence for the presence of one overlooked species and one possible case of synonymy.

Species-specific primers designed from RAPD products for Bithynia funiculata, the first intermediate host of liver fluke, Opisthorchis viverrini, in North Thailand.

Bithynia funiculata is the first intermediate host of the human liver fluke Opisthorchis viverrini in northern Thailand but its identification through morphological analysis is often problematic due to the shortage of gastropod taxonomists. As a consequence, we focused on the development of species-specific primers for use as an identification tool. Our work involved recovery of a 502-base pair (bp) amplicon of unknown function through species-specific primers whose effectiveness was tested by analyzing specimens of B. funiculata from 3 locations in northern Thailand. This primer set did not amplify other species in the Bithyniidae or in other gastropod families. By providing a tool to confirm morphological identifications of B. funiculata, and by enabling the identification of juvenile specimens and those with damaged shells, these primers will improve estimates of the prevalence of parasitic infections in this snail.

A simple PCR condition for detection of a single cyst of Acanthamoeba species.

Acanthamoeba is a free-living protozoan with a worldwide distribution in a variety of natural and artificial habitats. It has even been found in contact lens solution. Acanthamoeba spp. can cause infections such as granulomatous amoebic encephalitis and amoebic keratitis. Specific and sensitive diagnosis of Acanthamoeba infections can prevent clinical symptoms from worsening. Recently, PCR technique has been used for Acanthamoeba diagnosis. Unfortunately the dormant cyst of Acanthamoeba is resistant to chemical reagents; thus, most extraction of DNA uses a commercial DNA extraction kit for obtaining DNA for further use in polymerase chain reaction (PCR). Therefore in the present study, we improved the ability to diagnose Acanthamoeba using a simplified PCR technique. Interestingly, heating at 94°C for 10 min could release DNA which is amplified with specific primers designed from 16S rRNA. The PCR product is about 180 bp. This technique is a simple and efficient method for detection of Acanthamoeba-even a single cyst-and does not require high-cost reagents or complicated procedures to extract DNA.

Curcumin induces a nuclear factor-erythroid 2-related factor 2-driven response against oxidative and nitrative stress after praziquantel treatment in liver fluke-infected hamsters.

Praziquantel has been used for the treatment of liver fluke infection, but an oxidative/nitrative stress may occur after a short-term treatment and participate in side effects. In an attempt to reduce the adverse effects, we administered curcumin, an anti-inflammatory agent, to Opisthorchis viverrini-infected hamsters treated with praziquantel. At 12h after treatment, curcumin decreased eosinophil infiltration and increased mononuclear cell infiltration in parallel with nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 expression at the transcriptional and protein levels. Curcumin also enhanced the expression of genes involved in the Nrf2-regulated stress pathway (Kelch-like ECH-associated protein 1, NAD(P)H:quinine oxidoreductase 1, glutamate cysteine ligase, and activating transcription factor 3, peroxiredoxin 3, peroxiredoxin 6, manganese superoxide dismutase, and catalase), leading to increased ferric antioxidant capacity in the plasma. In contrast, curcumin decreased the level of oxidative and nitrative stress markers such as urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine, plasma levels of malondialdehyde and nitrate/nitrite, and activity of plasma alanine transaminase, a liver injury marker. This correlated with the suppression of nuclear factor-kappaB (NF-κB) and related molecules (cyclooxygenase-2 and inducible nitric oxide synthase) and pro-inflammatory cytokines (IL-1ß and TNF-α). In conclusion, curcumin may be an effective chemopreventive agent against oxidative and nitrative stress derived from praziquantel treatment during O. viverrini infection via induction of Nrf2 and suppression of NF-κB-mediated pathways. Nrf2 may also be a novel therapeutic target for not only parasitic diseases but other types of inflammation-mediated diseases.

Purification and characterization of Heteroscorpine-1 (HS-1) toxin from Heterometrus laoticus scorpion venom.

Crude venom from Thai giant scorpion, Heterometrus laoticus, most commonly found in the northeastern area of Thailand, was evaluated for PD50 (paralytic dose 50) activities from abdominal injection to cricket (Gryllus sp.) and activities against various kinds of microorganisms. It exhibited good results in disc diffusion assay for Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. After purification, toxin showed acceptable results for PD50 determination in entrapped cricket as well as inhibitory activity against B. subtilis, K. pneumoniae, and P. aeruginosa with activities over 300 times higher than that of the crude venom. The purified fraction was showed to contain a single band in SDS-PAGE. MALDI-TOF-MS/MS analysis showed one peak of major protein with 8293Da. Partial amino acid sequence show high homology to Scorpine-a polypeptide toxin family with potassium channel blocking and defensin activity. The novel toxin was named "Heteroscorpine-1" (HS-1) as the first Scorpine from genus Heterometrus. After full length determination by PCR, HS-1 gene was found to be composed of two exons, separated by an intron. Deduction revealed 95 amino acid residues with 19 residues as the leading sequence. It showed about 80% similarity to Panscorpine and Opiscorpine.

Population genetic data on D1S80, D17S5, ApoB, COL2A1 and Ig-JH in Northeastern Thais.

Allele frequency distributions at five VNTR loci namely; D1S80, D17S5, ApoB, COL2A1 and Ig-JH were examined in Northeastern Thais. The number of alleles at each locus were 19, 13, 14, 6 and 8 with the heterozygous frequencies of 0.814, 0.818, 0.676, 0.579 and 0.288, respectively. No significant deviations were found by statistical tests for Hardy-Weinberg equilibrium. The combined power of discrimination for all five loci was 0.99998.