Unpolished Thai rice prevents ACF formation and dysplastic progression in AOM-induced rats and induces apoptosis through redox alteration in CaCo-2 cells.

Oxidative stress is associated with colon carcinogenesis including aberrant crypt foci (ACF) formation and it plays an important role in pathophysiological changes in cancer cells. The aims of this study were to investigate the effects of dietary unpolished Thai rice (UTR) on ACF formation and dysplastic progression in azoxymethane (AOM)-treated rats. Anti-cancer efficacy of UTR regarding apoptotic induction and oxidative redox status in human colon cancer (CaCo-2) cells was also investigated. Rats given 20% and 70% of UTR in the diet showed significantly and dose-dependently decreased total number of ACF. UTR treatment also was strongly associated with the low percentage of dysplastic progression and mucin depletion. In addition, we found that UTR significantly induced cancer cell apoptosis, increased cellular oxidants, and decreased the level of GSH/GSSG ratio in CaCo-2 cells. Our study suggests that UTR supplementation may be a useful strategy for CRC prevention with the inhibition of precancerous progression, with induction of cancer cell apoptosis through redox alteration.

High collagen I gene expression as an independent predictor of adverse renal outcomes in lupus nephritis patients with preserved renal function.

CONTEXT: The deposition of extracellular matrix is a major pathogenic mechanism leading to fibrosis and progressive decline in renal function in patients with lupus nephritis (LN). Currently, available clinicopathologic features cannot predict renal outcome consistently. OBJECTIVE: To test that the expression of renal fibrogenic genes correlates with renal fibrosis at the time of biopsy and is predictive of renal outcomes. DESIGN: Renal gene expression levels of transforming growth factor ß-1 (TGFB1), and collagen I (COL1) were studied by real-time multiplex quantitative polymerase chain reaction in a prospective cohort of patients with LN (n = 39). Extracellular matrix index (ECMI) and collagen I/III matrix index were measured from Picro-Sirius Red-stained slides under normal and polarized light, respectively. RESULTS: After follow-up (median, 43.9 months), renal failure (50% reduction in glomerular filtration rate [GFR] or dialysis) had developed in 13 subjects. The expression levels of renal fibrogenic genes were increased as compared to controls without LN. COL1 correlated with collagen I/III matrix index at baseline. Both high expression of TGFB1 or COL1 tended to predict renal failure by univariate analysis. By multivariate analysis, high ECMI and low GFR were predictive of renal failure. In patients with baseline GFR of 60 mL/min/1.73 m(2) or greater, high renal COL1 expression was an independent (hazard ratio = 4.4, P = .04) predictor of renal failure. CONCLUSIONS: High renal COL1 expression is a strong predictor of adverse renal outcome in patients with LN and preserved baseline GFR. These findings support larger prospective studies to confirm the benefits of COL1 in identifying patients at high risk of progression to renal disease.

Monoclonal antibody against recombinant Fasciola gigantica cathepsin L1H could detect juvenile and adult cathepsin Ls of Fasciola gigantica.

Cathepsin Ls (CatLs), the major cysteine protease secreted by Fasciola spp., are important for parasite digestion and tissue invasion. Fasciola gigantica cathepsin L1H (FgCatL1H) is the isotype expressed in the early stages for migration and invasion. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1H (rFgCatL1H) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with recombinant proFgCatL1H (rproFgCatL1H). This MoAb is an immunoglobulin (Ig)G1 with κ light chain isotype. The MoAb reacted specifically with rproFgCatL1H, the native FgCatL1H at a molecular weight (MW) 38 to 48 kDa in the extract of whole body (WB) of metacercariae and newly excysted juvenile (NEJ) and cross-reacted with rFgCatL1 and native FgCatLs at MW 25 to 28 kDa in WB of 2- and 4-week-old juveniles, adult, and adult excretory-secretory (ES) fractions by immunoblotting and indirect ELISA. It did not cross-react with antigens in WB fractions from other parasites, including Gigantocotyle explanatum, Paramphistomum cervi, Gastrothylax crumenifer, Eurytrema pancreaticum, Setaria labiato-papillosa, and Fischoederius cobboldi. By immunolocalization, MoAb against rFgCatL1H reacted with the native protein in the gut of metacercariae and NEJ and also cross-reacted with CatL1 in 2- and 4-week-old juveniles and adult F. gigantica. Therefore, FgCatL1H and its MoAb may be used for immunodiagnosis of both early and late fasciolosis in ruminants and humans.

Identification and expression of Fasciola gigantica thioredoxin.

In the present study, a cDNA encoding Trx from F. gigantica (FgTrx) was cloned by polymerase chain reaction (PCR). The sequence of FgTrx, analyzed by BLAST, SignalP, and ClustralW programs, showed 315 bp of an open reading frame (ORF), 12 bp 5'UTR, 78 bp 3'UTR, and the putative FgTrx peptide comprising of 104 amino acids, with a molecular weight of 11.68 kDa, with the active site containing five amino acids (tryptophan, cysteine, glycine, proline, cysteine) with a conserved dithiol motif from the two cysteines, and pI 5.86. The peptide had no signal sequence; hence, it was not a secreted protein. The recombinant FgTrx was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTrx). The FgTrx protein expression, estimated by indirect ELISA using the rabbit anti-rFgTrx as probe, showed high levels in eggs, 2- and 4-week-old juveniles, and adult parasite. In a functional test, the rFgTrx exhibited specific activity that could be suppressed by an inhibitor (PX12). When tested by immunoblotting and immunohistochemistry, rabbit anti-rFgTrx reacted with natural FgTrx at a molecular weight of 11.68 kDa from eggs, metacercariae, NEJ, 2- and 4-week-old juveniles, and adult F. gigantica. The FgTrx protein was distributed at high levels in the tegument of 2- and 4-week-old juveniles, and the tegument, parenchyma, eggs, and reproductive organs of adult parasites. FgTrx may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or drug target.

Production and characterization of a monoclonal antibody against recombinant saposin-like protein 2 of Fasciola gigantica.

A monoclonal antibody (MoAb) against recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with rFgSAP-2. This MoAb is an IgG1, κ light chain isotype. By immunoblotting and indirect ELISA, the MoAb reacted specifically with rFgSAP-2, the natural FgSAP-2 at 10kDa in whole body (WB) and excretory-secretory (ES) fractions of F. gigantica. It did not cross react with antigens in WB fractions from other parasites, including Opisthorchis viverrini, Schistosoma mansoni which are human parasites, Haemonchus placei, Setaria labiato-papillosa, Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gigantocotyle explanatum, Gastrothylax crumenifer, and Paramphistomum cervi which are ruminant parasites. By immunohistochemistry, the FgSAP-2 protein was localized only in the cytoplasm of caecal epithelial cells of 4-week-old juvenile and adult stages, but not in metacercariae, newly excysted juvenile (NEJ), 2- and 3-week-old juveniles. This finding indicated that FgSAP-2 is an abundantly expressed parasite protein that is released into the ES, hence SAP-2 and its MoAb may be used for immunodiagnosis of ruminant and human fasciolosis.

Characterization and localization of saposin-like protein-2 (SAP-2) in Fasciola gigantica.

Fasciola gigantica saposin-like protein-2 (FgSAP-2) belongs to a family of lipid-interacting proteins that are involved in the cytolysis of target cells. In this study, we have cloned and expressed FgSAP-2 and produced the antibody against this recombinant protein. Rabbit antiserum against rFgSAP-2 reacted with a similar native protein in the whole body extracts of the 4-week-old juvenile and adult stage, as well as a protein in their excretion-secretion, but not in the tegument. In situ hybridization and immunofluorescence detection revealed the presence of SAP-2 mRNA transcripts and proteins in the cecal epithelial cells of 4-week-old juvenile and adult parasites, but not in the metacercariae and newly excysted juveniles. Moreover, SAP-2 is present only in the cecal epithelial cells lining the distal part of the digestive tract, but not in the tegumental-type epithelium lining the proximal part of the digestive tract. The rFgSAP-2 reacted with antisera from rabbits infected with F. gigantica metacercariae collected at 5 weeks, but not at 2 weeks after infection. Anti-rFgSAP-2 did not exhibit any cross-reactivity with the other parasites' antigens, including Opisthorchis viverrini, Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gigantocotyle explanatum, Paramphistomum cervi, Setaria labiato-papillosa, and Haemonchus placei. This finding indicated that SAP-2 is a unique protein that is expressed only in late juvenile and adult F. gigantica, and it could be considered for immunodiagnostic and as a vaccine candidate for fasciolosis.

Gene expression profile of duodenal epithelial cells in response to chronic metabolic acidosis.

Chronic metabolic acidosis (CMA) affects ion transport, permeability, and metabolism of the intestinal absorptive cells. Most effects of CMA on the intestine are long-term adaptations at genomic level. To identify the CMA-regulated genes, the Illumina's microarray featuring high-performance BeadArray technology was performed on RNA samples from the rat duodenal epithelial cells exposed to long-standing acidemia. After 21 days of CMA, we found 423 transcripts upregulated and 261 transcripts downregulated. Gene ontology analysis suggested effects of CMA on cellular processes, such as cell adhesion, proliferation, fuel metabolism, and biotransformation. Interestingly, 27 upregulated transcripts (e.g., Aqp1, Cacnb1, Atp1a2, Kcnab2, and Slc2a1) and 13 downregulated transcripts (e.g., Slc17a7, Slc9a4, and Slc30a3) are involved in the absorption of water, ions, and nutrients. Some upregulated genes, such as Slc38a5 and Slc1a7 encoding glutamine transporters, may be parts of the total body adaptation to alleviate negative nitrogen balance. Therefore, the present results provided a novel genome-wide information for further investigations of the mechanism of CMA effect on the intestine.

Genetic instability in cervical cancer detected by arbitrarily primed polymerase chain reaction.

The genetic instability in 54 Thai cervical cancer tissues were analyzed by Arbitrarily Primed Polymerase Chain Reaction (AP-PCR). The band alterations produced from 54 arbitrary primers were compared between the DNA finger printing from the patients and their corresponding normal cervical tissues. Results revealed 7 arbitrary primers provided DNA alteration patterns. Of these, an allelic loss in tumor DNA was found in DNA fingerprinting obtained from primers F-2 (64.8%), F-11 (68.5%), U-8 (51.9%), AE-3 (75.9%), AE-11 (53.7%), respectively. Moreover, DNA amplification was exhibited in patterns with primers B-12 (42.6%), J-16 (24.1%) and U-8 (70.4%). When genetic instability was investigated for associations with clinicopathological features, only the DNA amplified fragment with primer U-8 was significantly associated with stage II (P=0.030). Likewise, allelic loss amplified from arbitrary primer AE-3 showed significantly associate with age lower than 50 years old (P=0.003). Our findings suggest that the DNA alteration fragments produced from arbitrary primers of U-8 and AE-11 might be relevant to the pathogenesis of cervical cancer in Thai patients.

Schistosoma mekongi: the in vitro effect of praziquantel and artesunate on the adult fluke.

The efficacy and tolerance of 80 microg/ml praziquantel (PZQ) and 40 microg/ml artesunate (ATS) against adult stage Schistosoma mekongi in vitro were investigated after 3, 6, 12, and 24h incubation by monitoring worm motility and compared tegumental changes using scanning electron microscopy (SEM). Thirty mice were infected with S. mekongi cercaria for 49 days. Adult worms were collected by perfusion method and prepared for in vitro study. Contraction and decreased motor activity were observed after as little as 3h incubation with PZQ and ATS. Some of the worms were immobile 12h after exposure, and died within 24h. The tegument of S. mekongi showed severe swelling, vacuolization and disruption, fusion of the tegumental ridges, collapse and peeling. After 12-24h incubation, PZQ induced similar but they less severe, tegumental changes to those observed after exposure to ATS. The direct observation of the fluke motility and SEM study suggest that ATS is more effective than PZQ in causing tegumental damage in adult S. mekongi, and provides a basis for subsequent clinical trials.

Effects of praziquantel and artesunate on the tegument of adult Schistosoma mekongi harboured in mice.

The effects of praziquantel and artesunate on the tegument of adult Schistosoma mekongi harboured in mice were compared using scanning electron microscopy (SEM). Forty-two mice infected with S. mekongi for 49 days were treated intragastrically with either 300 mg/kg praziquantel or 300 mg/kg artesunate. Mice were sacrificed 1 or 3 days post-treatment. Worms were collected by perfusion and examined by SEM. One to 3 days after administration of artesunate, the tegument of S. mekongi showed severe swelling, vacuolization, fusion of the tegumental ridges and loss or shortening of the spines on the trabeculae, collapse and peeling. Praziquantel induced similar tegumental alterations as those observed after administration of artesunate, but they were less severe. Three days post-treatment, there was evidence of recovery only in the case of praziquantel. The results of our study suggest that artesunate is more effective than praziquantel in causing tegumental damage in adult S. mekongi, and provides a basis for subsequent clinical trials.