Cytosolic superoxide dismutase can provide protection against Fasciola gigantica.

Superoxide dismutases (SOD), antioxidant metallo-enzymes, are a part of the first line of defense in the trematode parasites which act as the chief scavengers for reactive oxygen species (ROS). A recombinant Fasciola gigantica cytosolic SOD (FgSOD) was expressed in Escherichia coli BL21 (DE3) and used for immunizing rabbits to obtain polyclonal antibodies (anti-rFgSOD). This rabbit anti-rFgSOD reacted with the native FgSOD at a molecular weight of 17.5kDa. The FgSOD protein was expressed at high level in parenchyma, caecal epithelium and egg of the parasite. The rFgSOD reacted with antisera from rabbits infected with F. gigantica metacercariae collected at 2, 5, and 7 weeks after infection, and reacted with sera of infected mice. Anti-rFgSOD exhibited cross reactivity with the other parasites' antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. A vaccination was performed in imprinting control region (ICR) mice by subcutaneous injection with 50µg of rFgSOD combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 15 metacercariae by oral route. IgG1 and IgG2a in the immune sera were determined to indicate Th2 and Th1 immune responses. It was found that the parasite burden was reduced by 45%, and both IgG1 and IgG2a levels showed correlation with the numbers of worm recoveries.

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.

Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase.

The Fasciola gigantica thioredoxin-glutathione reductase (FgTGR) gene is a fusion between thioredoxin reductase (TR) and a glutaredoxin (Grx) gene. FgTGR was cloned by polymerase chain reaction (PCR) from adult complementary DNA (cDNA), and its sequences showed two isoforms, i.e., the cytosolic and mitochondrial FgTGR. Cytosolic FgTGR (cytFgTGR) was composed of 2370 bp, and its peptide had no signal sequence and hence was not a secreted protein. Mitochondrial FgTGR (mitFgTGR) was composed of 2506 bp with a signal peptide of 43 amino acids; therefore, it was a secreted protein. The putative cytFgTGR and mitFgTGR peptides comprised of 598 and 641 amino acids, respectively, with a molecular weight of 65.8 kDa for cytFgTGR and mitFgTGR, with a conserved sequence (CPYC) of TR, and ACUG and CVNVGC of Grx domains. The recombinant FgTGR (rFgTGR) was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTGR). The FgTGR protein expression, estimated by indirect ELISA using the rabbit anti-rFgTGR as probe, showed high levels of expression in eggs, and 2- and 4-week-old juveniles and adults. The rFgTGR exhibited specific activities in the 5,5'-dithiobis (2-nitro-benzoic acid) (DTNB) reductase assay for TR activity and in ß-hydroxyethul disulfide (HED) for Grx activity. When analyzed by immunoblotting and immunohistochemistry, rabbit anti-rFgTGR reacted with natural FgTGR at a molecular weight of 66 kDa from eggs, whole body fraction (WB) of metacercariae, NEJ, 2- and 4-week-old juveniles and adults, and the tegumental antigen (TA) of adult. The FgTGR protein was expressed at high levels in the tegument of 2- and 4-week-old juveniles. The FgTGR 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 a drug target.

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.

Saposin-like protein 2 has an immunodiagnostic potential for detecting Fasciolosis gigantica.

Saposin-like protein 2 (SAP-2) plays an important role in the digestive process of Fasciola gigantica (Fg). It is one of the major proteins synthesized by the caecal epithelial cells and released into fluke's excretion-secretion. Therefore, FgSAP-2 is a plausible target for detecting fasciolosis. A polyclonal antibody (PoAb) against recombinant FgSAP-2 was produced by immunizing rabbits with the recombinant protein (rFgSAP-2), and used in sandwich ELISA assay to detect the circulating FgSAP-2 in sera of mice experimentally infected with F. gigantica metacercariae. The assay could detect rFgSAP-2 and the native FgSAP-2 in the excretory-secretory (ES) and whole body (WB) fractions of adult F. gigantica at the concentrations as low as 38 pg/ml, 24 ng/ml, and 102 ng/ml, respectively. As well, the sera from mice experimentally infected with F. gigantica were tested positive by this sandwich ELISA, which exhibited sensitivity, specificity, false positive rate, false negative rate and accuracy at 99.99, 98.67, 1.33, 0.01 and 99.32%, respectively. Therefore, this assay could be used for diagnosis of fasciolosis by F. gigantica.

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.

Fatal firearm injuries in autopsy cases at central Bangkok, Thailand: a 10-year retrospective study.

Even though there have been previously published reports on firearm injuries in various countries, the incidence and pattern of death from firearm injuries in Thailand have not been studied before. In present study, 149 fatal firearm injuries from 2002 to 2011 were reviewed. At total of 7126 autopsies, fatal firearm injuries comprised of 2.09% (n = 149) of total autopsies cases. Among those victims, 136 were male (91.3%), 13 (8.7%) were female. The youngest age of victim was 10 years and the oldest was 79 years. Mean age of the victims was 33.79 years and median age was 30 years. Outdoor incident was the most common scene of crime. Night time incident (18:00 PM-05:59 AM) was higher than day time one. Most of the cases occurred in week ends (n = 52). Homicide (77.2%) was the most frequent manner of death. Head/face and chest were the most common sites of entrance. The autopsy report also study on entrance wound, range and types of projectiles. Blood alcohol concentration was examined in 122 cases and 38 victims showed positive results, 11 cases revealed using of illegal substances in blood and urine analysis. This study also included the association between manner of death and other factors. Age group, time of incidence, place of incidence, number of entrance wound and range showed statistically significant association with manner of death.

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.

Vaccine potential of recombinant saposin-like protein 2 against Fasciolosis gigantica in mice.

Saposin-like protein 2 (SAP-2) is a protein that adult of Fasciola spp. use to lyse plasma membrane of red blood cells, so that their contents can be digested by proteases for the parasites' nutrients. Thus SAP-2 is a plausible target for vaccination against these parasites. Recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was expressed in Escherichia coli BL21 (DE3). A vaccination was performed in ICR mice (n=10) by subcutaneous injection with 50µg of rFgSAP-2 combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 30 F. gigantica metacercariae by oral route. The percentages of protection of rFgSAP-2 vaccine against F. gigantica were estimated to be 76.4-78.5% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. The antibodies in immune sera of vaccinated mice were shown by immuno-blotting to react with native FgSAP-2 in the extract of 2- and 4-week-old juvenile parasites. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune responses, it was found that both Th1 and Th2 humoral immune response were significantly increased in rFgSAP-2 immunized group compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rFgSAP-2-immunized group showed no significant difference from those of the non-immunized and infected group, indicating that early juvenile parasites induced liver parenchyma damage, even though the numbers of worm recoveries were significantly different. This study indicates that rFgSAP-2 has a high potential as a vaccine candidate against F. gigantica in mice, and this potential will be tested in larger economic animals.

Immunization with recombinant leucine aminopeptidase showed protection against Fasciola gigantica in mice.

Leucine aminopeptidase (LAP) is expressed in all stages of Fasciola gigantica and, hence, is considered as a potential vaccine candidate. In this study, we have tested a vaccine potential of LAP and the types of immune responses it elicited in vaccinated mice. Recombinant F. gigantica leucine aminopeptidase (rFgLAP) was expressed in Escherichia coli, BL21 (DE3). The imprinting control region mice subcutaneously immunized with 50 µg of rFgLAP combined with Freund's adjuvant (n = 10) exhibited a significant reduction in worm recoveries when compared with non-immunized and Freund's adjuvant controls at 60.8 and 64.3%, respectively, and both T helper (Th)1 and Th2 humoral immune responses were elicited in the hosts as reflected by the levels of IgG1 and IgG2a, with Th2 predominating. The levels of IgG1- and IgG2a-specific antibodies to rFgLAP were inversely and significantly correlated with the numbers of worm recoveries. The rFgLAP-vaccinated mice showed significantly reduced levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase and liver damage. These indicated that rFgLAP has a potential as a vaccine candidate against F. gigantica, whose efficacy will be studied further in economic animals including cattle, sheep, and goat.

A member of the CPW-WPC protein family is expressed in and localized to the surface of developing ookinetes.

BACKGROUND: Despite the development of malaria control programs, billions of people are still at risk for this infectious disease. Recently, the idea of the transmission-blocking vaccine, which works by interrupting the infection of mosquitoes by parasites, has gained attention as a promising strategy for malaria control and eradication. To date, a limited number of surface proteins have been identified in mosquito-stage parasites and investigated as potential targets for transmission-blocking vaccines. Therefore, for the development of effective transmission-blocking strategies in epidemic areas, it is necessary to identify novel zygote/ookinete surface proteins as candidate antigens. METHODS: Since the expression of many zygote/ookinete proteins is regulated post-transcriptionally, proteins that are regulated by well-known translational mediators were focused. Through in silico screening, CPW-WPC family proteins were selected as potential zygote/ookinete surface proteins. All experiments were performed in the rodent malaria parasite, Plasmodium yoelii XNL. mRNA and protein expression profiles were examined by RT-PCR and western blotting, respectively, over the course of the life cycle of the malaria parasite. Protein function was also investigated by the generation of gene-disrupted transgenic parasites. RESULTS: The CPW-WPC protein family, named after the unique WxC repeat domains, is highly conserved among Plasmodium species. It is revealed that CPW-WPC mRNA transcripts are transcribed in gametocytes, while CPW-WPC proteins are expressed in zygote/ookinete-stage parasites. Localization analysis reveals that one of the CPW-WPC family members, designated as PyCPW-WPC-1, is a novel zygote/ookinete stage-specific surface protein. Targeted disruption of the pycpw-wpc-1 gene caused no obvious defects during ookinete and oocyst formation, suggesting that PyCPW-WPC-1 is not essential for mosquito-stage parasite development. CONCLUSIONS: It is demonstrated that PyCPW-WPC-1 can be classified as a novel, post-transcriptionally regulated zygote/ookinete surface protein. Additional studies are required to determine whether all CPW-WPC family members are also present on the ookinete surface and share similar biological roles during mosquito-stage parasite development. Further investigations of CPW-WPC family proteins may facilitate understanding of parasite biology in the mosquito stage and development of transmission-blocking vaccines.

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.

Molecular cloning and characterization of leucine aminopeptidase from Fasciola gigantica.

M17 leucine aminopeptidase (LAP) is one of a family of metalloexopeptidases, of which short peptide fragments are cleaved from the N-terminals. In this study, the full length of cDNA encoding Fasciola gigantica LAP (FgLAP) was cloned from adult parasites. The amino acid sequences of FgLAP showed a high degree of identity (98%) with that from Fasciola hepatica and a low degree of identities (11% and 9%) with those from cattle and human. Phylogenetic analysis revealed that the FgLAP was closely related and grouped with F. hepatica LAP (FhLAP). Northern analysis showed that FgLAP transcriptional products have 1800 base pairs. Analysis by RNA in situ hybridization indicated that LAP gene was expressed in the cecal epithelial cells of adult parasites. A polyclonal antibody to a recombinant FgLAP (rFgLAP) detected the native LAP protein in various developmental stages of the parasite. In a functional test, this rFgLAP displayed aminolytic activity using a fluorogenic Leu-MCA substrate, and was significantly inhibited by bestatin. Its maximum activity was at pH 8.0 and enhanced by Mn(2+) ions. Localization of LAP proteins by immunohistochemistry and immunofluorescence techniques indicated that the enzyme was distributed in the apical cytoplasm of cecal epithelial cells. Because of its important metabolic role and fairly exposed position, FgLAP is a potential drug target and a possible vaccine candidate against fasciolosis.

Fasciola gigantica: the in vitro effects of artesunate as compared to triclabendazole on the 3-weeks-old juvenile.

The in vitro effect of artesunate (ATS) on the 3-week-old juveniles of Fasciola gigantica was compared with triclabendazole (TCZ) by incubating the parasites in M-199 medium containing the drugs at concentrations of 20, 40, and 80 µg/ml for 1, 3, 6, 12, and 24h. The anthelmintic activities of these drugs were evaluated based on the relative motility value (RM) and the alterations of the tegument as observed by scanning (SEM) and transmission (TEM) electron microscopy. The RM values of TCZ-treated flukes decreased significantly from 6 to 24h for all dosages. For ATS-treated flukes, RM value decreased markedly from 12 to 24h, but the rates of decline were less than TCZ at the same doses. When observed by SEM, the tegument showed similar sequence of morphological changes after treatments with both drugs, comprising of swelling of tegumental ridges, followed by blebbing and later rupturing of the blebs, leading to erosion and lesion, and disruption of the tegument. When examined by TEM, ultrastructural changes in the tegument and associated structures after treatments with TCZ and ATS were similar which comprised of swelling, blebbing of the tegument, dilation of basal infoldings, and depolymerization of the microtrabecular network. After a longer incubation time, the tegument was completely sloughed off and the tegument cell bodies became necrotic. Additionally, in ATS-treated flukes, mitochondria showed severe swelling, rupturing of outer membrane, and their interior filled with flocculent materials.

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.

Claudin expression in the bone-lining cells of female rats exposed to long-standing acidemia.

Besides enhancing osteoclast-mediated bone resorption, chronic metabolic acidosis (CMA) induces mineral efflux across the epithelial-like bone membrane formed by bone-lining cells (inactive osteoblasts), possibly via the paracellular pathway. However, there was a compensatory mechanism that restricted bone loss in the late phase of CMA, and changes in the expression of claudins, which are tight junction proteins known to regulate epithelial barrier function, were therefore anticipated in bone-lining cells. Herein, primary rat osteoblasts were found to express several transcripts of claudins, i.e., claudin-5, -11, -14, -15 and -16. Their protein expressions in bone-lining cells were demonstrated by immunohistochemistry in decalcified tibial sections. After exposure to CMA induced by oral administration of 1.5% NH(4)Cl for 21 days, expression of claudin-14, which normally seals the paracellular space and restricts ion movement, was increased, whereas that of claudin-15 and -16 which form pores for ion transport were decreased. Expressions of claudin-5 and -11 were not changed by CMA. In conclusion, the bone-lining cells of rats exposed to CMA for 21 days upregulated an ion-restrictive claudin (i.e., claudin-14), while downregulating ion-permeable claudins (i.e., claudin-15 and -16). These cellular responses might be parts of a compensatory mechanism accounting for deceleration of bone loss in late CMA.

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.

Fasciola gigantica and Schistosoma mansoni: vaccine potential of recombinant glutathione S-transferase (rFgGST26) against infections in mice.

Recombinant Fasciola gigantica glutathione S-transferase (rFgGST26) was expressed in Escherichia coli. This protein had 86% and 56% sequence identity with 26 kDa GST from Fasciola hepatica and Schistosoma mansoni, respectively. Polyclonal antibody raised in ICR mice against rFgGST26 recognized immunoblotted 26 kDa native GSTs from F. gigantica and S. mansoni. rFgGST26 was used as a vaccine in combination with Freund's adjuvant to evaluate the induction of immune responses and protection against F. gigantica and S. mansoni infection in mice. Mice were immunized via subcutaneous (s.c.), intramuscular (i.m.) or intradermal (i.d.) routes. Strong protection (77-84%) against F. gigantica was observed in all routes. Immunization via s.c. route induced immune response with IgG1 isotype predominating, while i.m. and i.d. routes resulted in mixed IgG1/IgG2a immune responses. Passive intraperitoneal transfer of IgG1 predominating antisera from s.c. rFgGST26-immunized donors to naive recipient mice resulted in 47% protection against F. gigantica infection. This suggests that the mechanism of resistance depends on the presence of specific antibody against rFgGST26. Immunization with rFgGST26 via i.m. and i.d. routes resulted in significant cross protection (55%) against S. mansoni infection in the i.d. route with mixed IgG1/IgG2a response with IgG1 isotype predominating. This indicated that rFgGST26 is a good vaccine candidate against F. gigantica in mice and could also provide cross protection against S. mansoni.

Genotypic resistance mutations in treatment-naïve and treatment-experienced patients under widespread use of antiretroviral drugs in Thailand: implications for further epidemiologic surveillance.

The aims of this study were to illustrate the prevalence and determinants of mutations associated with antiretroviral drug resistance in a group of antiretroviral-naive and treatment-experienced patients in Thailand, where antiretroviral drugs are widely used. One hundred and thirteen treatment-naive (92 CRF01_AE and 21 subtype B patients) and 1,709 treatment-experienced patients were recruited. Genotypic resistance to antiretroviral drugs was studied by sequencing the isolated viruses. Mutation frequencies in treatment-naive patients were reported along with those for treatment-experienced patients. The results showed that all of the patients with treatment-experienced patients showed the same pattern of genotypic resistance. The results also showed that only 14 drug-naive patients (12.4%) carried HIV-1, with at least one drug-resistant mutation. Moreover, four drug-naive patients were found to carry the marker mutations for transmission of drug resistance. The most commonly found marker in drug-naive patients was M36I/V/L (n=90, 81.1%), which is a common natural polymorphism among HIV-1 subtype CRF01_AE individuals. In order to prevent the rapid emergence of resistant virus strains, a national program to monitor antiretroviral drug resistance should be established. We also recommend routine genotypic testing in treatment-naive patients before starting antiretroviral therapy to prevent subtherapeutic response and viral failure.

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.