Downregulation of reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is associated with enhanced expression of matrix metalloproteinases and cholangiocarcinoma metastases.

BACKGROUND: Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) has been implicated in the attenuation of tumor metastasis by negatively regulating metalloproteinase (MMP) levels. RECK gene expression is downregulated in many solid tumors, with this downregulation being associated with poor prognosis. This study evaluated the role of RECK in cholangiocarcinoma (CCA). METHODS: The expression of RECK, MMP-2, and MMP-9 in paraffin sections of hamster and human CCA specimens was analyzed by immunohistochemistry. Functional analysis of RECK was performed in RECK small interfering (si) RNA knockdown CCA cell lines. The effect of aspirin on RECK status and function was evaluated using Western blotting, gelatin zymography, invasion and proliferation assays, and PhosphoELISArray analysis of Ras downstream mediators. RESULTS: Hamster tissues showed high RECK expression in hyperplastic biliary duct epithelia, low RECK expression in precancerous lesions, and no RECK expression in CCA. In human specimens, RECK was highly expressed in normal biliary cells, whereas intrahepatic CCA showed low levels of expression. Downregulation of RECK was correlated with tumor metastasis (P < 0.01) and shorter patient survival (P < 0.02). RECK expression levels were inversely correlated with MMP-2 and MMP-9 expression (P < 0.05). SiRNA RECK-depleted M139 CCA cells exhibited increased MMP-2/-9 gelatinase activities and invasiveness. Aspirin (500 µM) demonstrated myriad effects in human CCA cell lines, including growth suppression, reduced phosphorylation of Akt/Erk/c-Jun, elevation of RECK expression, inhibition of MMP-2/MMP-9 activity, and enhanced invasiveness. CONCLUSIONS: RECK functions as a metastasis suppressor in CCA; upregulation of RECK expression could provide a potential therapy to improve the prognosis of this type of cancer.

Circulating CD14(+) CD16(+) monocyte levels predict tissue invasive character of cholangiocarcinoma.

Chronic inflammation as a risk factor for cancer development is driven in part by monocyte/macrophages, which in many cancers exhibit pro-tumorigenic activity. In this study we identified elevation in CD14(+) CD16(+) , a minor blood monocyte subpopulation in cholangiocarcinoma (CCA) patients, compared to normal and biliary disease patient specimens. Tumour association was suggested by the observation that this elevated level decreased to normal after tumour resection. Moreover, the elevated level of CD14(+) CD16(+) monocytes in CCA patient blood correlated with degree of MAC387-positive (recent blood-derived macrophage migrant-specific marker) tumour-associated macrophage infiltration as determined by immunohistochemistry. These CD14(+) CD16(+) monocytes were suggested to enhance tumour progression as this subpopulation possesses (i) high expression of adhesion molecules (CD11c, CD49d, and CD54) and scavenger receptor (CD163), which enable them to adhere strongly to endothelial cells, and (ii) that peripheral blood monocytes from CCA patients express high levels of growth and angiogenic factor-related genes (epiregulin, VEGF-A and CXCL3). Elevation of peripheral CD14(+) CD16(+) monocyte levels was associated with features associated with poor prognosis CCA parameters (non-papillary type and high number of tissue macrophages). These data indicate that the CD14(+) CD16(+) monocytes from CCA patients with pro-tumorigenic characteristics may associate with rapid tumour progression and poor patient outcome. If confirmed in subsequent studies, the level of CD14(+) CD16(+) monocytes may serve as a marker for disease activity in CCA patients and serve as a target for pathogenic macrophage specific drug development.

Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes.

The present study compared the genetic variation among 14 different geographical isolates of Opisthorchis viverrini sensu lato from Thailand and Lao PDR using sequence data for 2 mitochondrial DNA genes, the subunit 1 of NADH dehydrogenase gene (nad1) and cytochrome c oxidase gene (cox1). Four different nad1 haplotypes were detected among isolates, all of which were identical at the amino acid sequence level. Nucleotide sequence variation among 14 isolates ranged from 0 to 0.3% for nad1. Two different cox1 haplotypes were detected among isolates. These two haplotypes differed at 2 nucleotide positions, one of which resulted in a change in the amino acid sequence. Nucleotide sequence variation among isolates for cox1 ranged from 0 to 0.5%. Comparison of cox1 sequences of O. viverrini to those of other trematodes revealed nucleotide differences of 13-31%. A phylogenetic analysis of the cox1 sequence data revealed strong statistical support for a clade containing O. viverrini and 2 other species of opisthorchid trematodes; O. felineus and Clonorchis sinsensis.

Allelic loss on chromosome 5q34 is associated with poor prognosis in hepatocellular carcinoma.

PURPOSE: To identify and characterize novel genetic alterations in hepatocellular carcinoma (HCC). METHODS: DNA was extracted from 29 HCC and corresponding normal tissues and amplified with 59 different 10-base arbitrary primers. A 550 bp DNA fragment amplified using primer Q-9 and which was present in 19 of 29 cases (66%) was cloned, sequenced, and compared with known nucleotide sequences deposited in Genome database, and quantified by real-time PCR. RESULTS: DNA alterations were found on chromosomes 5q34, 6p25.2 and 8q12.1 in 11 of 29 cases (38%), 7 of 29 cases (24%), and 12 of 29 cases (41%), respectively. Multivariate analysis showed that the allelic loss on chromosome 5q34 was an independent prognostic factor for poor survival of HCC patients, with the median survival time of 19 weeks for allelic loss versus 109 weeks for no allelic loss (P = 0.001). CONCLUSIONS: This study indicates that allelic loss on chromosome 5q34 may be involved in the development of HCC and could be used as a prognostic indicator in HCC patients.

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.

Inhibitory effect of Streblus asper leaf-extract on adhesion of Candida albicans to denture acrylic.

This in vitro study aimed at determining the effects of various sublethal concentrations of Streblus asper leaf ethanolic extract (SAE) on adherence of Candida albicans to acrylic surface. A colorimetric tetrazolium assay using (2,3)-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide was used to make the quantitative determination. The SAE at a concentration equivalent to nystatin (6.24microg/ml) pinpointed the minimal exposure time of SAE in suppressing candidal adhesion to acrylic. Adhesion of Candida albicans to acrylic was determined after exposure to SAE for 1, 15, 30, 60, 120 and 180min. The minimum concentration of SAE that significantly reduced adherence (P<0.05) after a 4-h exposure was 31.25mg/ml. In addition, a significant reduction (P<0.01) of candidal adhesion to acrylic occurred after a 1min exposure to 62.5mg/ml of SAE. Pre-treatment of yeast with 62.5mg/ml of SAE for 1h before adhesion assay significant reduced the adherence as 20.54% compared to the untreated control, whereas the same treatment with acrylic strips did not show any effect. These findings indicate that exposure of Candida albicans to sublethal concentrations of SAE results in a reduction in the ability of the yeasts to adhere to denture acrylic, possibly preventative of denture stomatitis.

Novel DNA amplification on chromosomes 2p25.3 and 7q11.23 in cholangiocarcinoma identified by arbitrarily primed polymerase chain reaction.

PURPOSE: To detect and characterize amplified DNA sequences in cholangiocarcinoma (CCA). PATIENTS AND METHODS: We extracted DNA from tumor and corresponding normal tissues of 30 patients with CCA and amplified with 30 random ten-mer arbitrary primers by the arbitrarily primed polymerase chain reaction (AP-PCR) technique. RESULTS: Our results showed gains of genomic sequences at high frequency. Using the AX-11 arbitrary primer, we determined an amplified DNA fragment occurred frequently in the tumors analyzed. The DNA fragment was isolated and identified as two sequences mapped to chromosomes 2p25.3 and 7q11.23. Specific primers were designed employing these sequences and used for detecting amplification by real-time quantitative PCR. The amplification of the DNA sequences on chromosomes 2p25.3 and 7q11.23 was detected in 10 (33%) and 6 (20%) cases, respectively. Thirteen (43%) cases showed amplification on both or one of the chromosomes. In addition, amplification of the DNA on chromosome 2p25.3 was predominantly observed in poorly differentiated tumors. CONCLUSIONS: Our findings suggest that the novel amplified DNA on chromosomal regions at 2p25.3 and 7q11.23 might be involved in the development and progression of CCA.

In vitro inhibitory effect of Streblus asper leaf-extract on adhesion of Candida albicans to human buccal epithelial cells.

This in vitro study aimed at determining the effects of a sublethal concentration of Streblus asper Lour (Moraceae) leaf ethanolic extract on adherence of Candida albicans to human buccal epithelial cells (HBEC). The minimum concentration of Streblus asper leaf ethanolic extract (SAE) that significantly reduced adherence (P<0.05) after a 1-h exposure was 15.6 mg/ml. However, there was a significant reduction (P<0.05) of candidal adhesion to HBEC after 1-min exposure to 125 mg/ml of SAE. Pre-treatment of either Candida or HBEC, or both, with 125 mg/ml of SAE for 1h resulted in reduced adherence. SAE at concentrations of 125 and 250 mg/ml also showed 41 and 61% inhibition of germ tube formation, respectively, which might affect adherence. These findings indicate that the sublethal concentration of SAE may modulate candidal colonization of the oral mucosa thereby suppressing the invasive potential of the pathogen.

Increased cell proliferation of mouse fibroblast NIH-3T3 in vitro induced by excretory/secretory product(s) from Opisthorchis viverrini.

Infection by Opisthorchis viverrini is a strong risk factor for cholangiocarcinoma. However, the mechanism by which the parasite is involved in carcinogenesis is not clear. In addition to the direct damage of the bile duct epithelium via direct contact with O. viverrini, the excretory/secretory (ES) product(s) released from the parasites may play important roles in this process. We therefore investigated the responses of a fibroblast cell line, NIH-3T3, to ES product(s) released from O. viverrini by using a non-contact co-culture technique. In this culture system, the parasites in the upper chamber had no direct contact with the NIH-3T3 cells in the lower chamber of the culture plate. The results indicated a marked increase in NIH-3T3 cell proliferation in the non-contact co-culture condition with either 0% or 10% calf serum in the medium compared with that without parasites. ES product(s) increased cell proliferation by stimulating the expression of phosphorylated retinoblastoma (pRB) and cyclin D1, the key proteins in driving cells through the G1/S transition point of the cell cycle. This led to the induction of cells going into the S-phase of the cell cycle. ES product(s) also changed the morphology of NIH-3T3 cells to a refractive and narrow shape, which allowed the cells to proliferate in the limited culture area. For the first time, we have been able to demonstrate increased cell proliferation induced by the ES product(s) from O. viverrini; this finding may clarify how O. viverrini ES product(s) affect human bile duct epithelium during cholangiocarcinogenesis.

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.

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.

Selective activity of Streblus asper on Mutans streptococci.

The purpose of this in vivo one group time series design and single blind study was to determine the antimicrobial effectiveness of a mouthrinse containing Streblus asper leaf extract on Streptococcus mutans and total salivary bacteria following single 60 s rinse. Changing in salivary pH and buffer capacity during the experimental period were also measured. Thirty human subjects participated in this study. At each experimental session, a pretest saliva sample was taken. After giving the pretest samples, the subjects rinsed with S. asper leaf extract or distilled water control for 60 s, then the post-test saliva samples were collected at 0, 0.5, 1, 3, 5 and 6 h. The samples were used to determine the numbers of S. mutans and total salivary bacteria. The results indicated that S. asper leaf extract significantly reduced S. mutans counts compared with distilled water. However, the mean difference from baseline of total salivary bacterial counts was not significantly different between rinsing with S. asper leaf extract and distilled water. Moreover, S. asper leaf extract showed no effects in modifying the salivary pH and buffer capacity. It is concluded that of mouthrinse containing S. asper leaf extract can reduce S. mutans without changing an oral ecology.

Affinity electrophoretic determination of oligosaccharide specificity of Butea monosperma agglutinin.

The oligosaccharide specificity of newly isolated Butea monosperma agglutinin (BMA) was determined by two-dimensional lectin affinity electrophoresis of alpha-fetoprotein (AFP) with concanavalin A, lentil lectin, erythroagglutinating phytohemagglutinin and Allomyrina dichotoma lectin, of which the specificities to known AFP oligosaccharides had been established. Effects of neuraminidase treatment on the reactivities of AFP to the lectins were also studied. The results indicated that BMA had the highest affinity for the exposed Gal residues of nonreducing termini of biantennary complex-type oligosaccharides, and that the affinity was reduced to zero in the following order by the presence of monosialyl residue of the Man alpha1->3 arm, monosialyl residue of the Man alpha1->6 arm, monosialyl residue of the Man alpha 1->3 arm and bisecting G1cNAc, and disialyl residues. BMA did not recognize Neu5Ac alpha2->6 and Neu5Ac alpha2->3 substitutions of Gal. These characteristics of BMA were shown to be useful in identifying malignancy-associated alteration of AFP sugar chains.

Isolectins from seeds of Artocarpus lakoocha.

Two isolectins (ALA-I and ALA-II), were isolated from seed extracts of Artocarpus lakoocha by anion exchange chromatography on Q-Sepharose fast flow columns at pH 8.5 and 8.0 ALA-I was unbound to the column at pH 8.5 and moved towards the cathode in non-denaturing polyacrylamide gel electrophoresis, whereas ALA-II possessed opposite properties. The two A. lakoocha agglutinins appeared to be composed of two dissimilar subunits (alpha and beta of M(r) 14,000 and 17,200) bound non-covalently. The isolectins possessed several similar properties including: blood type agglutination; pH optimum; pH and temp stability; as well as binding specificity towards asialomucins.

Observations on the development of stunting in children of the Khon Kaen region of Thailand.

A cohort of children in North-East Thailand was followed from birth to 2 years of age in an attempt to throw light on factors determining the development of stunting in linear growth. By 2 years the group as a whole had an average deficit in height of nearly -2 standard deviations. Those index children whose sibs were stunted had larger deficits than those with normal sibs. Their mothers were also shorter and lighter. These findings suggest that it is possible to think in terms of stunted families. No differences were identified in socio-economic factors and the prevalence of infection was in general low. Dietary intakes estimated by 24-hour recall, supplemented at 1 and 2 years by 24 h weighing, were satisfactory for most nutrients except iron, calcium and niacin. Intakes of Ca and P were lower in the more stunted children. A number of variables were measured in urine and blood at 1 and 2 years but few relationships could be established with the degree of stunting. Excretions of calcium and phosphorus showed weak negative correlations with height. On average the serum concentration of calcium was satisfactory but that of phosphorus was somewhat low. Concentrations of somatomedin C, thyroxin and vitamin D were within reported normal ranges, with no relation to the degree of stunting. From a comparison of the linear growth of these children with the results of other reports from Thailand it is suggested that environmental factors have produced stunting in the cohort as a whole, but the cohort is essentially homogeneous, showing within it a normal range of genetic variation. If that is so, major differences in intake or biochemistry between the taller and shorter children would not be expected. The problem remains of why the group as a whole is stunted. This is the first systematic attempt to assess biochemical factors that may be related to stunting in Third World children; these results are essentially negative, although there are hints that point at a possible deficiency of calcium.