Suda Riengrojpitak

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 Freunds 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.

Histopathological effects of triphenyltin hydroxide on liver, kidney and gill of Nile tilapia (Oreochromis nilotica)

The histopathological effects of triphenyltin hydroxide (TPTH) on the liver kidney and gill of Nile tilapia (Oreochromis nilotica) one month old was studied by light microscopy. Two concentrations of TPTH were used: 1 mg l -1 and 3 mg 1 -1 . The fish were sacrificed at the end of one, two, three and four months. The results showed that the hepatocytes underwent a variety of changes from congestion and dilatation of sinusoidal space, pallor of cytoplasm, vacuolation and accumulation of hyaline droplets. Subcapsular and scattered focal necrosis was also observed. In the kidney, hydropic degeneration and accumulation of hyaline droplets in the tubular epithelial cells were noted. In addition, a congestion of peritubular capillaries and detachment of tubular epithelial cells were observed. In more severe case there was a collapse of glomerular capillary tuft with a widening of the Bowmans capsule. There were some changes in the gill filaments and lamellae, namely hyperplasia of the covering gill epithelium, congestion of gill capillaries and vessels, and aneurysmal formation of gill lamellar capillaries. These alterations were time-and dose-dependent.

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.

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.

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.

V3 sequence diversity of HIV-1 subtype E in infected mothers and their infants

To elucidate genetic characteristics of HIV-1 subtype E involved in vertical transmission, V3 regions of HIV-1 subtype E isolated from 17 infected mothers (M1-M17) and their infants (I1-I17) at 1 month after birth were sequenced after cloned into pCRII vectors. At least three clones of each sample were collected. All mothers were asymptomatic and had been infected through a heterosexual route. Nine infants (I9-I17) showed mild symptomatic and immunosuppression within the first year of life. The interpatient nucleotide distance of mothers and infants in this group (0.065+/-0.008) were of greater diversity than those of a nonimmunosuppression group (0.039+/-0.006) by a significant amount (Fischers exact test, p = .003). The substitution with asparagine (N) at threonine (T) at position 13 and aspartic acid (D) at position 29 of the V3 sequence were significantly associated with nonimmunosuppression in the first year of life (F-test, p = 0.003). Either a single or multiple viral variants could transmit from mothers to their infants.

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

BackgroundDespite 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.MethodsSince 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.ResultsThe 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.ConclusionsIt 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.

Simple and rapid screening of the thiocyanate level in saliva for the identification of smokers and non-smokers by capillary electrophoresis with contactless conductivity detection

A simple and rapid capillary electrophoresis method with contactless conductivity detection for direct determination of thiocyanate in saliva to differentiate between smokers and non-smokers is proposed. Simple saliva preparation was performed by centrifugation and the dilute and shoot method (1:1, v/v) with a running buffer prior to hydrodynamic injection. The running buffer was 20.0 mM His/MES at pH 6.00 containing 0.1 mM CTAB as the electroosmotic flow modifier. Maleate anions were employed as the internal standard for the precision of the response factor of electrophoresis separation which was carried out at −8.0 kV (266 V cm−1) with a total analysis time of 3.5 min per sample. A linear calibration curve for thiocyanate in the concentration range 0.2–4.0 mM was obtained (r2 > 0.999). The limit of quantification was 0.12 mM which is sufficient for the quantitative determination of thiocyanate in saliva for the purpose of identification of smokers and non-smokers. Intra-day and inter-day precisions were 0.7–1.7% RSD and 2.4–6.4% RSD, respectively. Accuracy based on the percentage recovery of spiked saliva samples was in the range of 72–141% (n = 18). The measured thiocyanate levels were significantly different to classify non-smokers from smokers; a cut-off value of 1.0 mM is proposed to separate the two groups. This method is convenient and rapid, suitable for screening of a large number of subjects, such as in clinical work or health-care surveys.

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.