Proteomic identification of peroxiredoxin 6 for host defence against Opisthorchis viverrini infection.

Opisthorchis viverrini infection causes opisthorchiasis and is a risk factor for cholangiocarcinoma via chronic inflammation. To investigate the mechanism of O. viverrini -induced liver disease, we applied a proteomic approach to examine alterations in hepatic protein levels in O. viverrini -infected hamsters. Two-dimensional gel electrophoresis (2DE) revealed that O. viverrini infection induced upregulation (1.5- to 4.3-fold) of 25 proteins and downregulation (1.5 to 2.5-fold) of 24 proteins compared with uninfected animals. Expression of proteins related to stress response, DNA replication and repair, and cell structure was significantly increased, whereas that of proteins associated with normal liver function, such as metabolism, blood volume maintenance and fatty acid cycle was decreased. Among the upregulated proteins, a 2.7-fold increase in peroxiredoxin 6 (Prdx6), an antioxidant protein, was confirmed by 2DE and immunoblot analysis, Western blot and quantitative PCR. Immunohistochemical analysis showed that Prdx6 expression was observed mainly in the cytoplasm of inflammatory cells. These results suggest that Prdx6 is important for host defence against O. viverrini infection. This study provides basic information for Prdx6 as a potential biomarker and therapeutic target for opisthorchiasis.

Evaluation of immunoglobulin G4 subclass antibody in a peptide-based enzyme-linked immunosorbent assay for the serodiagnosis of human fascioliasis.

SUMMARYTo improve the diagnosis of human fascioliasis caused by Fasciola gigantica, we developed a peptide-based enzyme-linked immunosorbent assay (peptide-based ELISA) based on the detection of specific IgG4 subclass antibody. Two identified B-cell epitopes of F. gigantica cathepsin L1 were synthesized as single synthetic peptides, acetyl-DKIDWRESGYVTELKDQGNC-carboxamide (peptide L) and acetyl-DKIDWRESGYVTEVKDQGNC-carboxamide (peptide V), and their diagnostic potential was evaluated. The sera of 25 patients infected with F. gigantica, 212 patients with other parasitic infections, 32 cholangiocarcinoma patients and 57 healthy controls were analysed. The sensitivity, specificity, accuracy, and positive and negative predictive values of this assay were the same with both peptides at 100%, 99.7%, 99.7%, 96.2% and 100%, respectively. These highly sensitive and specific peptide-based ELISAs for the detection of specific IgG4 antibody could be useful for laboratory diagnosis of human fascioliasis in future large-scale surveys throughout Southeast Asia where this disease is prevalent.

Genomic characterization of lung flukes, Paragonimus heterotremus, P. siamensis, P. harinasutai, P. westermani and P. bangkokensis by RAPD markers.

Random amplified polymorphic DNA (RAPD) markers were assayed in an attempt to discriminate among five species of Paragonimus. Genomic DNAs of two strains of Paragonimus heterotremus from two provinces in Thailand, Saraburi and Phitsanulok, as well as of P. siamensis, P. harinasutai, P. westermani and P. bangkokensis were extracted and amplified by an arbitrary primer, namely P2 (5-GTTTCGCTCC-3). RAPD patterns showed that those five species were genetically distinct, although they shared genomic DNA to some extent. This primer could also distinguish between two strains of P. heterotremus. The polymorphism observed allowed to construct a relationship dendrogram. The phylogenetic dendrogram showed that the P. heterotremus strains were closest to P. harinasutai, followed by P. siamensis, P. bangkokensis and P. westermani.

Characterisation of Fasciola gigantica adult 27-kDa excretory-secretory antigen in human fascioliasis.

The 27-kDa excretory-secretory antigen partially purified from adult Fasciola gigantica adult worms (FG27) has been used as the sensitive and specific antigen for immunodiagnosis of human fascioliasis. Lectin-blot analyses have shown that the FG27 possesses both N - and O -glycans. In two-dimensional electrophoresis, silver staining, lectin blotting and immunoblotting, FG27 displayed at least four antigenic-glycosylated protein spots at the pI values of 4.8, 4.9, 5.2 and 5.4, respectively. After removing glycan moieties from the antigen by alkaline treatment, the molecular mass of the FG27 was reduced to 26.5 kDa as a prominent deglycosylated band. Immunoblotting analysis has revealed that the 26.5-kDa band reacts with the pooled human fascioliasis sera without any loss of antigenicity. These results suggest that the major antigenicity of the FG27 is due to its protein epitopes.

Detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes by a PCR-based method.

A PCR procedure for the detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes was developed. This procedure was based on primers designed from a pOV-A6 specific probe sequence giving a 330 base-pair product. The detection was accomplished in host tissue homogenates to which a single cercaria or metacercaria was introduced. PCR can detect as little as a single cercaria artificially inoculated in a snail or a single metacercaria artificially inoculated in a fish sample. The method gave a 100% positivity rate for all infected snails or fishes. The method did not yield a 330 base-pair amplified product with other digenean fluke DNAs such as Haplorchis taichui, Centrocestus spp., Echinostoma malayanum, Fasciola gigantica, animal schistosomes, Paragonimus heterotremus or Haplorchoides spp. The assay has great potential for application in epidemiological surveys of both snail and fish intermediate hosts as well as for investigation of foodborne parasites in freshwater fishes.

Serum total sialic acid in cholangiocarcinoma patients: an ROC curve analysis.

OBJECTIVES: This study was undertaken to establish the diagnostic utility of serum total sialic acid (TSA) for patients with cholangiocarcinoma (CCA). DESIGN AND METHODS: Serum TSA was determined in 89 histologically confirmed CCA patients, 38 with benign hepatobiliary diseases (BHD) and 43 healthy persons. To check whether the test could adequately discriminate between these groups, complete statistical Receiver Operating Characteristic (ROC) curves were analyzed. RESULTS: The mean value of serum TSA in CCA patients (2.75 +/- 0.67 mmol/L) was significantly higher than that in the BHD (2.33 +/- 0.69 mmol/L p < 0.002) and healthy persons (1.89 +/- 0.46 mmol/L p < 0.001) groups. The areas under the ROC curves were 0.6699 and 0.8558, respectively. A cut-off value of 2.33 mmol/L discriminated between the CCA, BHD and healthy groups with a sensitivity of 71.9% and a positive predictive value range of 80 to 89%. CONCLUSION: Determination of TSA yielded high diagnostic values for differentiating between CCA, BHD and healthy persons. The determination of serum TSA would be most useful as an adjunct diagnosis rather than an early detection and screening tool because of the apparent nonspecificity of SA to a given disease.

Antimicrobial activity of Streblus asper leaf extract.

Bactericidal activity was found in the 50% ethanol (v/v) extract of Streblus asper leaves. The extract possessed a selective bactericidal activity towards Streptococcus, especially to Streptococcus mutans which has been shown to be strongly associated with dental caries. The extract had no effect on cultures of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, Staphylococcus coagulase positive, Staphylococcus coagulase negative, Serratia marcescens, Klebsiella pneumoniae, Enterobacter, Pseudomonas aeruginosa, Burkholderia pseudomeallei and Candida albicans. The minimum growth inhibitory concentration and the minimum bactericidal concentration of S. asper extract against 10(8) CFU/mL of S. mutans was 2 mg/mL. The active compound is partially polar, partially heat labile, precipitated by 80% ammonium sulphate, and possesses a molecular weight larger than 10 000 Da. The potential for using S. asper extract as a natural product for controlling dental caries is discussed.

Correlation between Na, K-ATPase activity and potassium and magnesium contents in skeletal muscle of renal stone patients.

Samples of external oblique muscles were surgically removed from 45 renal stone patients and analyzed for their K, Na and Mg content. The muscle samples were also measured for membrane Na, K-ATPase activity from the assay of its K+-dependent 3-0-methyl fluorescein phosphatase (K+-dependent 3-0-MFPase) activity. The results showed that the mean muscle contents +/- SEM of K, Na and Mg were 65.2 +/- 1.7 (range, 41.1 to 86.1), 45.5 +/- 2.0 (range, 23.5 to 73.2) and 6.3 +/- 1.0 (range, 4.1 to 8.5) micromol/g wet weight, respectively. The mean activity +/- SEM of the K+-dependent 3-0-MFPase or the Na, K-ATPase was calculated by subtracting the activity of the basal-form from that of the total-3-0-MFPase, which was 113 +/- 21 (range, 11 to 177) nmol/g wet weight/minute. The activity of the Na, K-ATPase showed a significant correlation with muscle K-content (r = 0.52, p<0.001) and Mg content (r = 0.45, p<0.002). Though the external oblique muscles of renal stone patients in our study, as compared to data from other sources, had a considerably low concentration of K and Mg, they exhibited a good correlation with membrane-Na, K-ATPase activity. Our results, therefore, support previous observations made by other investigators.

Immunoblot evaluation of the specificity of the 29-kDa antigen from young adult female worms Angiostrongylus cantonensis for immunodiagnosis of human angiostrongyliasis.

The antigenic components of Angiostrongylus cantonensis young adult female worm somatic extract (FSE) were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The sera tested were from patients with proven angiostrongyliasis, other parasitic diseases, and healthy adults. Both the sera and cerebrospinal fluid (CSF) were tested from patients with clinical angiostrongyliasis. The CSF from patients with other neurological diseases were also included. Using SDS-PAGE, we found that the FSE comprised more than 30 polypeptides. Immunoblot analysis revealed at least 12 or 13 antigenic bands in patients with proven or clinical angiostrongyliasis, respectively. The patterns of reactivity recognized by the serum and CSF antibodies against FSE were similar. These antigenic components had molecular masses ranging from less than 14.4 to more than 94 kDa. The prominent antigenic band of 29-kDa might serve as a reliable marker for the diagnosis of angiostrongyliasis. The sensitivity, specificity, positive and negative predictive values of immunoblot analysis in this antigenic band were 55.6%, 99.4%, 83.3% and 97.4%, respectively.

Gnathostoma spinigerum: analysis of protein patterns by two dimensional gel electrophoresis.

The protein extracts from male (MS) and female (FS) adults and advanced third-stage larvae (LS) of Gnathostoma spinigerum were separated by high resolution two-dimensional gel electrophoresis (2-DE). The polypeptide spots, as detected by silver staining, were subsequently identified. The spot patterns of LS, MS and FS were highly complex and consisted of more than 75, 44, 52 prominent spots, respectively. In addition, the stage-specific protein patterns were identified. This 2-DE database should provide an important reference for future biological and biochemical studies of G. spinigerum.

Antigenic components of Gnathostoma spinigerum recognized by infected human sera by two-dimensional polyacrylamide gel electrophoresis and immunoblotting.

Antigenic components of Gnathostoma spinigerum larval extract were revealed by two-dimensional gel electrophoresis (2-DE) and immunoblot analysis using sera from patients with 6 proven cases of gnathostomiasis, 5 presumptive cases of gnathostomiasis, 3 proven cases of angiostrongyliasis, 3 proven cases of cysticercosis, and pooled sera from healthy adults. By the 2-DE, the larval extract was highly complex and consisted of more than 75 polypeptides. Immunoblotting analysis of this larval extract after reaction with each of 6 proven gnathostomiasis sera revealed various numbers of antigenic spots ranging from 30 to 70 spots at the approximate molecular masses of less than 14.4 to more than 94 kDa with isoelectric points (pI) of less than 4.65 to 9.6. Antigenic spots at the approximate molecular mass of more than 30 kDa were recognized with the proven angiostrongyliasis, proven cysticercosis and healthy control sera but these sera did not react with the spots at approximate molecular masses of 23-25 kDa with pI of 8.3-8.5. The reacted spots, which consisted of at least 1 to 2 spots, were unique for the recognition of gnathostomiasis sera. Five out of 6 (83.3%) proven and 4 out of 5 (80%) presumptive gnathostomiasis sera reacted with these specific spots.

Fasciola gigantica-specific antigens: purification by a continuous-elution method and its evaluation for the diagnosis of human fascioliasis.

Immunodominant antigens of an approximate molecular mass of 27 kD were obtained from an excretory-secretory product of adult Fasciola gigantica by a continuous-elution method. An indirect ELISA using the antigens obtained by this relatively simple procedure was developed for detecting specific antibodies from patients infected with F. gigantica. Sera from patients with other parasitic infections, healthy volunteers, and cholangiocarcinoma were also analyzed. The sensitivity, specificity, and positive and negative predictive values for this ELISA using the fractionated antigens were 100%. The data indicated a possible correlation of antibodies to F. gigantica with cholangiocarcinoma.

Indirect haemagglutination test using monoclonal antibody-affinity purified antigens for diagnosis of human paragonimiasis due to Paragonimus heterotremus.

An indirect haemagglutination test (IHA) using antigens purified by monoclonal antibody-affinity chromatography was developed for the diagnosis of human paragonimiasis caused by Paragonimus heterotremus. Sera from patients with paragonimiasis (n = 30) were evaluated, along with sera from other parasitic infections (n = 92), pulmonary tuberculosis (n = 18) and healthy controls (n = 30). The sensitivity, specificity as well as positive and negative predictive value of the IHA, calculated at the prevalence of disease at 17.6%, were all 100%.

Excretory-secretory antigenic components of adult Fasciola gigantica recognized by infected human sera.

The immunogenic components of Fasciola gigantica excretory-secretory (ES) products were revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting technic using sera from patients with F. gigantica infection, from patients with clinical suspected fascioliasis, from patients with other illness and from healthy adults. By SDS-PAGE, it was found that the ES products comprised more than 6 polypeptides. Immunoblotting analysis revealed 12 components which were strongly recognized by fascioliasis antisera. These antigenic components had a molecular mass ranging from less than 14.4 to 38 kDa. One antigenic band of 27 kDa was found to give a consistent reaction with fascioliasis antisera (100% sensitivity and 98% specificity). The present findings suggest that the 27 kDa components are sensitive and specific for the diagnosis of human F. gigantica infection.

Detection of Paragonimus heterotremus in experimentally infected cat feces by antigen capture-ELISA and by DNA hybridization.

An antigen capture enzyme-linked immunosorbent assay (antigen capture-ELISA) and DNA hybridization technique were developed and evaluated for their application in the detection of Paragonimus heterotremus infection in experimentally infected cats. An IgG fraction prepared from serum of a rabbit immunized with P. heterotremus excretory-secretory (ES) products was used as the capture antibody. An IgG1 monoclonal antibody specific to the 22- and 31.5-kDa ES products of P. heterotremus was used as the antigen probe. As little as 0.24 ng of the ES products could be detected by this technique. A specific P. heterotremus DNA probe derived from the P. heterotremus genomic DNA library containing 1,500 base pairs was used in a dot-blot hybridization assay for the detection of parasite DNA. The radioactively labeled probe could detect DNA released from as few as 2 P. heterotremus eggs. Both ELISA and DNA hybridization were found to have 100% specificity, with sensitivities of 73.7% and 100%, respectively.

Monoclonal antibodies to Paragonimus heterotremus and their potential for diagnosis of paragonimiasis.

Monoclonal antibodies (MAbs) specific to the lung fluke (Paragonimus heterotremus) were produced against the soluble metabolic products (excretory-secretory antigen). Three hybrids secreting MAbs specific for P. heterotremus antigens were identified by an indirect enzyme-linked immunosorbent assay (ELISA) against a panel of homologous and 24 heterologous parasite antigens and Mycobacterium tuberculosis. Of the three specific clones, clone 10F2, which was IgG1 producing and which gave immune complex bands with 31.5-kD and 22-kD polypeptides by gel electrophoresis and immunoblotting, was selected for further characterization and evaluation of its possible diagnostic potential. The result obtained from an indirect immunofluorescent antibody test suggested that MAb 10F2 reacted with mucosa and contents of the worm's intestine. The antibody could be readily used to prepare an affinity-purified antigen for use in an indirect ELISA that was highly sensitive and specific for the detection of circulating antibody in sera of paragonimiasis patients.

Growth and development of Gnathostoma spinigerum early third-stage larvae in vitro.

Early third-stage larvae of Gnathostoma spinigerum were cultured in RPMI-1640 supplemented with 25mM NaHCO3,100 units/ml penicillin G, 100 microg/ml of streptomycin, 5 microg/ml of amphotericin B and 10% foetal calf serum at 37 degrees C under 5% CO2 in air for 60 days. After 3 days of cultivation, the larvae moulted. Body sizes increased from 0.49 +/- 0.09 x 0.07 +/- 0.01 mm in length and width to 4.08 +/- 0.48 x 0.32 +/- 0.04 mm after 60 days of cultivation. The maximum body length and width of these larvae were 4.94 mm and 0. 35 mm, respectively. The survival rate (67.5 %) of the worms was observed at the end of cultivation. The addition of foetal calf serum was found to be essential for growth and development.

Specific monoclonal antibodies to Fasciola gigantica.

Monoclonal antibodies (mAbs) were produced against soluble metabolic products (excretory/secretory; ES) of the liver fluke, Fasciola gigantica. The ES antigen was obtained from spent culture medium in which the adult flukes had been maintained in vitro. From two cell fusions, the mAbs produces were exclusively associated with either IgG or IgM isotypes. When screened against a panel of homologous and heterologous parasite antigens by indirect ELISA, two mAbs were highly specific for F. gigantica. The remainder cross-reacted extensively with other parasites. Results from immunoblotting and immunofluorescence exhibited two patterns of reactivity. The first group of mAbs which recognized the multiple bands between > 14.4-27.5 kDa gave extremely bright fluorescence over all major muscular systems, vitelline gland, testes and intestinal caeca. The second group which reacted with the 185 kDa band showed a bright fluorescence over a thinner muscular layer underlying the tegument, intestinal caeca and testes.

A dot-ELISA test using monoclonal antibody-purified antigens for the diagnosis of paragonimiasis caused by Paragonimus heterotremus.

A dot enzyme-linked immunosorbent assay (dot-ELISA) using antigens purified by monoclonal antibody-affinity chromatography was developed for detecting antibodies to Paragonimus heterotremus in four groups of subjects. They consisted of 30 patients with P. heterotremus infection, 93 patients with other parasitic infections, 18 patients with pulmonary tuberculosis and 30 normal, healthy controls. Sensitivity, specificity, as well as positive and negative predictive values of the test were 100, 97, 88, and 100%, respectively.

Antigenic components of somatic extract from adult Fasciola gigantica recognized by infected human sera.

The antigenic components of Fasciola gigantica somatic extract were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting technique using sera from patients with F. gigantica infection, patients with clinically diagnosed fascioliasis, patients with other infections/illness and healthy adults. By SDS-PAGE, it was found that the somatic product comprised more than 22 polypeptides. Immunoblotting analysis revealed at least 13 components which were strongly recognized by sera of patients with fascioliasis. These antigenic components had molecular weights ranging from less than 14.4 to more than 94 kDa. One antigenic component, i.e. 38 kDa was found to give a consistent reaction with sera of patients with fascioliasis (100% sensitivity and 96.7% specificity). The finding suggests that the 38 kDa components may be a potential diagnostic antigen for fascioliasis.