Plasma IgG autoantibody against actin-related protein 3 in liver fluke Opisthorchis viverrini infection.

Opisthorchiasis secondary to Opisthorchis viverrini infection leads to cholangiocellular carcinoma through chronic inflammation of the bile ducts and possibly inducing autoimmunity. It was hypothesized that plasma autoantibodies directed against self-proteins are biomarkers for opisthorchiasis. Plasma from patients with opisthorchiasis was tested using proteins derived from immortalized cholangiocyte cell lines, and spots reacting with plasma were excised and subjected to LC-MS/MS. Seven protein spots were recognized by IgG autoantibodies, and the highest matching scored protein was actin-related protein 3 (ARP3). The antibody against ARP3 was tested in plasma from 55 O. viverrini-infected patients, 24 patients with others endemic parasitic infections and 17 healthy controls using Western blot and ELISA. Immunoreactivity against recombinant ARP3 was significantly more prevalent in opisthorchiasis compared to healthy controls at Western blotting and ELISA (P < 0.05). Plasma ARP3 autoantibody titres were also higher in opisthorchiasis compared to healthy individuals (P < 0.01) and other parasitic infections including Strongyloides stercoralis (P < 0.001), echinostome (P < 0.05), hookworms (P < 0.001) and Taenia spp. (P < 0.05). It was further characterized in that the ARP3 autoantibody titre had a sensitivity of 78.18% and specificity of 100% for opisthorchiasis. In conclusion, it may be suggested that plasma anti-ARP3 might represent a new diagnostic antibody for opisthorchiasis.

Overexpression of microRNA-21 regulating PDCD4 during tumorigenesis of liver fluke-associated cholangiocarcinoma contributes to tumor growth and metastasis.

MicroRNA, an endogenous noncoding RNA modulating gene expression, is a key molecule that by its dysregulation plays roles in inflammatory-driven carcinogenesis. This study aimed to investigate the role of oncomiR miR-21 and its target, the programmed cell death 4 (PDCD4) in tumor growth and metastasis of the liver fluke Opisthorchis viverrini-associated cholangiocarcinoma (CCA). The expression levels of miR-21 and PDCD4 were analyzed using the TaqMan miRNA expression assay and immunohistochemistry in liver tissues of both O. viverrini plus N-nitrosodimethylamine (NDMA)-treated hamsters and human CCA samples (n=23 cases). The functional assay for miR-21 was performed in CCA cell lines by the anti-miR-21 and pre-miR-21 transfection procedures. The peak of miR-21 levels were reached at 2 (hyperplastic lesions) and 6 (CCA) months of the O. viverrini plus NDMA-induced group and had a reverse response with its target PDCD4 proteins. In human CCA, miR-21 was overexpressed in tumor tissues when compared with nontumor tissues (P=0.0034) and had a negative correlation with PDCD4 protein (P=0.026). It was also found that high expression of miR-21 was significantly correlated with shorter survival (P<0.05) and lymph node metastasis (P=0.037) of CCA patients. Transient transfection of pre-miR-21 reduced the PDCD4 level and resulted in an increase of M213 CCA cell growth and wound-induced migration ability. These results indicated that miR-21 plays a role in the carcinogenesis and metastasis of O. viverrini-associated CCA by suppressing the function of PDCD4. Modulation of aberrantly expressed miR-21 may be a useful strategy to inhibit tumor cell phenotypes or improve response to chemotherapy.

Liver fluke-associated and sporadic cholangiocarcinoma: an immunohistochemical study of bile duct, peribiliary gland and tumour cell phenotypes.

AIM: To compare cell phenotypes displayed by cholangiocarcinomas and adjacent bile duct lesions in patients from an area endemic in liver-fluke infestation and those with sporadic cholangiocarcinoma. METHODS: 65 fluke-associated and 47 sporadic cholangiocarcinomas and 6 normal livers were studied. Serial paraffin-wax sections were stained immunohistochemically with monoclonal antibodies characterising a Brunner or pyloric gland metaplasia cell phenotype (antigens D10 and 1F6), intestinal goblet cells (antigen 17NM), gastric foveolar apomucin (MUC5AC), a gastrointestinal epithelium cytokeratin (CK20) and the p53 protein. RESULTS: 60% of the 112 cholangiocarcinomas expressed antigen D10, 68% MUC5AC, 33% antigen 17NM and 20% CK20; 37% showed overexpression of p53. When present together in a cholangiocarcinoma, cancer cells expressing D10 were distinct from those displaying 17NM or MUC5AC. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinomas displayed 17NM and p53 expression. Most cases of hyperplastic and dysplastic biliary epithelium expressed D10 strongly. Pyloric gland metaplasia and peribiliary glands displayed D10 and 1F6, with peribiliary gland hyperplasia more evident in the livers with fluke-associated cholangiocarcinoma; goblet cells in intestinal metaplasia stained for 17NM. No notable association of expression between any two antigens (including p53) was found in the cancers. CONCLUSIONS: Most cases of dysplastic biliary epithelium and cholangiocarcinoma display a Brunner or pyloric gland cell phenotype and a gastric foveolar cell phenotype. The expression of D10 in hyperplastic and dysplastic epithelium and in cholangiocarcinoma is consistent with a dysplasia-carcinoma sequence. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinoma display an intestinal goblet cell phenotype and overexpress p53, indicating differences in the aetiopathology of the cancers in the two groups of patients.

Association between genetic polymorphisms of CYP1A2, arylamine N-acetyltransferase 1 and 2 and susceptibility to cholangiocarcinoma.

Human CYP1A2 and arylamine N-acetyltransferases, which are encoded by the polymorphic CYP1A2 and NAT genes respectively, have been shown to have wide interindividual variations in metabolic capacity and may be potential modifiers of an individual's susceptibility to certain types of cancers. The present study aimed to evaluate the relationship between CYP1A2, NAT1 and NAT2 polymorphisms and cholangiocarcinoma (CCA), the most prevalent cancer in the north-east of Thailand. A total of 216 CCA patients and 233 control subjects were genotyped by polymerase chain reaction with restriction fragment length polymorphism based assays. Two CYP1A2 alleles (CYP1A2*1A wild-type and *1F), six NAT1 alleles (NAT1*4 wild-type, *3, *10, *11, *14A and *14B) and seven NAT2 alleles (NAT2*4 wild-type, *5, *6A, *6B, *7A, *7B and *13), which are the major alleles found in most populations, were analysed. Although CYP1A2*1A allele, NAT1*10 allele, and the NAT2 slow acetylator alleles were not associated with CCA risk, among the male subjects, the genotype CYP1A2*1A/*1A conferred a decreased risk of the cancer (adjusted odds ratio (OR) 0.28, 95% confidence interval (CI) 0.08-0.94) compared with CYP1A2*1F/1*F. Frequency distributions of rapid NAT2*13 and two slow alleles (*6B and *7A), but not the other major alleles, were associated with lower CCA risk. Adjusted OR of the genotypes consisting of at least one of these alleles significantly decreased the cancer risk compared with none of them (OR 0.26, 95% CI 0.15-0.44). This study suggests that the NAT2 polymorphism may be a modifier of individual risk to CCA.

Strong promoting effect of Opisthorchis viverrini infection on dimethylnitrosamine-initiated hamster liver.

Continuous administration of dimethylnitrosamine (DMN) to Syrian hamsters infected with the liver fluke, Opisthorchis viverrini (OV) results in a 100% incidence of cholangiocellular carcinomas. In a two-stage experiment, however, dosing with liver flukes caused only a few lesions to develop (Flavel, D.J. and Lucus, S.B. (1983) Carcinogenesis, 4, 927]. To clarify this anomaly, Syrian hamsters were initiated with 20 mg/kg DMN injected i.p. 19 days prior to 80 OV metacercaria infection. At 45 weeks, the animals receiving both DMN and the parasite demonstrated a 44% incidence of cholangiocarcinomas, a 93% incidence of cholangiofibrosis, a 35% incidence of mucous cystadenomas and a 98% incidence of hepatocellular nodules with an average number of 9.1 +/- 4.1 per animal. Animals receiving DMN alone developed 85% hepatocellular nodules with an average number of only 3.0 +/- 2.7 and no bile duct lesions. In the parasite alone group, only cholangiofibrosis was detected in a few animals and no lesions were encountered in untreated controls. These results thus demonstrate that the post-initiation influence of Opisthorchiasis is indeed effective in promoting the development of both cholangiolar and hepatocellular lesions initiated by DMN.

Different ras gene mutational frequencies in thyroid papillary carcinomas in Japan and Thailand.

The incidence and pattern of ras oncogene mutations in human malignancies demonstrate geographic and racial differences. For example, specificity of alterations is found in cholangiocellular carcinomas in Thai patients with a different etiology from those in Japanese patients. In the present study, a comparison of ras gene mutations in thyroid papillary carcinomas from Japanese and Thai patients was performed using single-strand conformation polymorphism and direct sequencing analyses. The incidence of ras mutation differed markedly in Japanese (two of 24 carcinomas, 8.3%) and Thai (five of 10 carcinomas, 50%) patients. In addition, all but one ras mutation occurred at codon 12 of the K-ras gene in the Thai cases. These results suggest that thyroid cancers in Thailand may be due to specific genetic and/or environmental factors.

Association of antibodies to Opisthorchis viverrini with hepatobiliary disease in northeastern Thailand.

The association between Opisthorchis viverrini infection and hepatobiliary disease was studied in northeastern Thailand. Positive rates of O. viverrini infection and antibody titers to O. viverrini adult worm extracts, which were determined by enzyme-linked immunosorbent assay (ELISA), were compared among four groups: Thai patients with cholangiocarcinomas (CHCA), those with calculus cholecystitis (CCLT), endemic area victims of traffic accidents, and Japanese individuals used as negative controls. While no difference was observed between cases with CCLT and the accident victims, the CHCA group showed a significantly higher positivity rate for fluke infection and higher antibody titers than the other groups. Comparison of the positivity rates and ELISA titers among intrahepatic and extrahepatic CHCA subgroups showed that only females with intrahepatic tumors had a low positive rate and significantly lower titers, with no other significant variation being evident. From these observations, a strong association between high intensity of past and/or present O. viverrini infection and the genesis of CHCAs was apparent in the majority of cases. However, in addition to the fluke infection, other unknown exogenous or endogenous factor(s) were suggested in the female cases.

Quantitative post-mortem study of Opisthorchis viverrini in man in north-east Thailand.

The results of an autopsy study of Opisthorchis viverrini infection in 181 accident subjects in northeast Thailand are presented. The age-prevalence profiles were similar whether determined by faecal examination for parasite eggs or by recovery of worms from the liver. The prevalence increased rapidly with age and reached a plateau at 70-80% in adults. The overall prevalence estimated by faecal examination was 69.2%, while that measured by worm recovery was 79.2%. The average intensity of infection, as indicated by eggs per gram of faeces, rose with age and became relatively stable in groups over 20 years of age. Worm burden also increased rapidly with age, reaching a peak in the 31-40 year groups and slightly declining in the 2 oldest age groups. Fluke distribution between individual subjects was highly dispersed; most people harboured no or low worm burdens (0-200 worms) while a small group of subjects (16.5%) carried 66% of the total worms recovered. The distribution of flukes within the liver appeared to be homogeneous. The results of the present study were similar to those from field studies, and possible mechanisms that generate these observed patterns of infection are discussed.

Ultrastructural and immunohistochemical analysis of cholangiocarcinoma in immunized Syrian golden hamsters infected with Opisthorchis viverrini and administered with dimethylnitrosamine.

Utilizing the experimental model in Syrian golden hamsters, we explored the role of immunization in carcinogenesis. The animals, which were infected with liver flukes (Opisthorchis viverrini), and administered a subcarcinogenic dose of dimethylnitrosamine, developed cancer. Pre-immunizing with a crude somatic antigen did not reduce cancer development, but accelerated carcinogenesis. Histopathological analysis of the cancer tissues was done once at week 30 and again at week 39 using H and E staining, immunostaining for the p53 tumor suppressor phosphoprotein, and electron microscopy. Thirty weeks after immunization, the immunized hamsters developed tubular adenocarcinoma at a higher rate (71.43%) than the non-immunized group (20.00%). This rate (20.00%) increased to 63.64% by week 39. The small foci cancer in the non-immunized group decreased in frequency from 80.00% (at week 30) to 36.36% (by week 39), suggesting the small foci cancer progressed to tubular adenocarcinoma during the 9-week interval. Most of the observed tubular adenocarcinoma was well differentiated. Nearly all hamsters that tested positive for cancer also tested positive for p53 immunostaining in the epithelia of the small bile ducts. The positive reaction for p53-immunostaining was localized in the rough endoplasmic reticulum, Golgi apparatus and perinuclear membranes. The electron micrographs of these positive p53-immunostained cells showed characteristics of early cancer. The detection of p53 in early cancer development makes it a candidate as a tumor marker.

Rhinocerebral mucormycosis: a report of eleven cases.

Rhinocerebral mucormycosis (RCM) is a rare, fulminant fungal infection that usually occurs in diabetic or immunocompromised patients. The mortality rate has been reduced recently with the advent of amphotericin B combined with aggressive surgery. Eleven RCM patients have been treated over the past five years at Srinagarind Hospital. Eight had underlying diabetes, five had renal failure and three of them had both. In eight patients, the diagnosis was established by KOH preparation before histological confirmation. Only two cases revealed positive cultures for Rhizopus spp and Cunninghamella spp. All patients underwent surgical treatments (extensive debridement, 8 cases; sphenoidectomy, 7 cases; ethmoidectomy 8 cases; maxillectomy 5 cases and orbital exenteration, 6 cases). Amphotericin B was administered to all patients as soon as the diagnosis of RCM was made. Only three patients survived. Early diagnosis and cooperation among ophthalmologist, otolaryngologist and physician are the most important factors for the survival of patients with mucormycosis.

Comparative in vitro sensitivity to metronidazole of Trichomonas vaginalis with isolates and clones.

Isolates of Trichomonas vaginalis from a group of Thai women in Khon Kaen, Thailand were studies for their in vitro sensitivity to metronidazole. Forty-five isolates of T. vaginalis and further 226 laboratory clones of these isolates were examined for drug sensitivity under anaerobic conditions using the CPLM (Cysteine-Peptone-Liver infusion Maltose) medium. The in vitro sensitivity of T. vaginalis to metronidazole, recorded as MLC values revealed highly variable sensitivity profiles Greater than 90% of the trichomonads studies were susceptible to relatively low concentrations of metronidazole in the range of 1-10 micrograms/ml. The remaining fraction of trichomonads, appeared to be less sensitive and required higher concentration of drug to attain the MLCs. The highest recorded MLC was 60 micrograms/ml. The geometric mean MLC of the 45 isolates was 7.99 micrograms/ml while that of The influence of the individual clones of subpopulations of T. vaginalis on the observed sensitivity of the isolate was also examined. A positive linear relationship was observed between the MLC of the isolated and the MLC of the least sensitive clone of the same isolate (p less than 0.05). This suggests that the most resistant clone present within an isolate of T. vaginalis plays an important role in determining the overall drug sensitivity of the isolate.

Molar pregnancy--associated ectopic decidua: report of a case and review of the literature.

Ectopic or extrauterine decidual transformation is regularly associated with pregnancy. This phenomenon has been observed most often in the ovary and cervix. Sporadic reports have documented decidua formation of peritoneal surfaces, lymph nodes and other organs in pelvic and abdominal cavity. Molar pregnancy-associated ectopic decidua has never been documented. We report the case of ectopic decidua at posterior surface of uterus, near the cul de sac, in a 45-year-old woman with molar pregnancy. The article review reveals that it is the first report of molar pregnancy-associated ectopic decidua.

The study of discriminant values of dyspeptic symptoms for identifying the etiology of dyspepsia.

This prospective study aims to determine whether specific symptoms or group of symptoms could positively discriminate the etiology of patients who present with dyspepsia. Two hundred and eight patients were studied and 111, 55, 35 patients were classified as non-ulcer dyspepsia, peptic ulcer disease and hepatobiliary disease, respectively. All patients completed a structured history questionnaire by personal interview and completed investigation with complete blood count, stool examination, liver function test, HBsAg, HBsAb, ultrasonography of the abdomen and endoscopy. Variable of interest and variables of statistical significance by univariate analysis were put into discriminant function of logistic model for discrimination. The results suggest that anorexia and no periodicity of epigastric pain significantly discriminated non-ulcer dyspepsia from peptic ulcer disease and hepatobiliary disease, pain occurring before a meal or when the patient was hungry and nocturnal epigastric pain significantly discriminated peptic ulcer disease from hepatobiliary disease.

Human pythiosis in Srinagarind Hospital: one year's experience.

We have reported four cases of human pythiosis arteritis from Srinagarind Hospital, Khon Kaen, Thailand. This unusual human infection occurring perhaps exclusively in thalassemia and hemoglobinopathy patients, should be noted by physicians, who work in areas with a high incidence of hemoglobinopathy, and for patients who present with unexplained arterial insufficiency. As our reported cases occurred within only one year, this condition may be more common than originally suspected and found more frequently if actively searched for.

Cell death/proliferation roles for nc886, a non-coding RNA, in the protein kinase R pathway in cholangiocarcinoma.

We have recently identified nc886 (pre-miR-886 or vtRNA2-1) as a novel type of non-coding RNA that inhibits activation of protein kinase R (PKR). PKR's pro-apoptotic role through eukaryotic initiation factor 2 α (eIF2α) phosphorylation is well established in the host defense against viral infection. Paradoxically, some cancer patients have elevated PKR activity; however, its cause and consequence are not understood. Initially, we evaluated the expression of nc886, PKR and eIF2α in non-malignant cholangiocyte and cholangiocarcinoma (CCA) cells. nc886 is repressed in CCA cells and this repression is the cause of PKR's activation therein. nc886 alone is necessary and sufficient for suppression of PKR via direct physical interaction. Consistently, artificial suppression of nc886 in cholangiocyte cells activates the canonical PKR/eIF2α cell death pathway, suggesting a potential significance of the nc886 suppression and the consequent PKR activation in eliminating pre-malignant cells during tumorigenesis. In comparison, active PKR in CCA cells does not induce phospho-eIF2α nor apoptosis, but promotes the pro-survival nuclear factor-κB pathway. Thus, PKR has a dual life or death role during tumorigenesis. Similarly to the CCA cell lines, nc886 tends to be decreased but PKR tends to be activated in our clinical samples from CCA patients. Collectively from our data, we propose a tumor surveillance model for nc886's role in the PKR pathway during tumorigenesis.

Expression profiles of oncomir miR-21 and tumor suppressor let-7a in the progression of opisthorchiasis-associated cholangiocarcinoma.

Altered miRNA expression could be a determinant of cancer development and/or progression. We aimed to study the role of oncomir miR-21 and tumor suppressor let-7a in the genesis of Opisthorchiasis-associated cholangiocarcinoma (CCA). The results showed that miR-21 was up-regulated while let-7a was down-regulated during cholangiocarcinogenesis in the hamster model and also in human CCA samples. The expression level of miR-21 had an inverse correlation with the mRNA level of its target RECK, a metastasis suppressor, in human CCA. Knockdown of miR-21 of KKU100 CCA cells significantly increased the mRNA level of RECK and suppressed the wound-induced migration of CCA cells. Our data suggest that miR-21 is one key molecule playing crucial roles in the CCA growth and metastasis. Manipulation of miRNA expression offers a potential avenue of CCA therapy.

Comparative clinicopathological study of resected intrahepatic cholangiocarcinoma in northeast Thailand and Japan.

Northeast Thailand has a very high incidence of intrahepatic cholangiocarcinoma (ICC), which is closely linked to infestation by the liver fluke, whereas the etiology of ICC in Japan remains to be clarified. This study compared the clinicopathological features, the expression of p53 and c-erbB-2 proteins, and the proliferative activity of ICC in 19 Thai and 23 Japanese patients with ICC who were treated by hepatic resection. The average age of the Thai patients (55.8 years) was lower than that of the Japanese (61.3 years). All Thai patients presented with symptoms, whereas 8 Japanese patients were asymptomatic. There were no significant differences in preoperative liver function test values. Tumors were less likely to be located in the right lobe in the Japanese (34.8%) than in the Thai patients (63.2%). Peribiliary fibrosis and adenomatous hyperplasia in noncancerous hepatic tissues were much more frequently found in the Thai patients (P = 0.0010; P<0.0001). No significant differences in the expression of p53 protein or c-erbB-2 protein were found between the two series of patients, but proliferative activity, evaluated on the basis of mean MIB1 labeling index, was significantly higher in the Thai patients (P<0.001). The present study suggested a higher proliferative activity of ICC in Thai patients than in Japanese patients.

Intrahepatic cholangiocarcinoma in Thailand.

Intrahepatic cholangiocarcinoma is defined as adenocarcinoma originating from bile ductules and segmental and lobar intrahepatic ducts. Four types of surgical pathology have been identified in the Khon Kaen endemic area in Thailand: peripheral, type I; intermediate, type II; central, type III; and diffuse, type IV. We report our experience with intrahepatic cholangiocarcinoma with emphasis on the surgical pathology, operative procedure, and associated survival time. We reviewed the records of patients treated for cholangiocarcinoma at Srinagarind Hospital from January 1, 1992 to February 28, 1997. There was a total of 411 patients, and 138 were intrahepatic and non-jaundiced. Tumors in the proximity of the gray zone i.e., portal, periportal with jaundice, were excluded. Patient profiles, surgical pathology, operative procedure, postoperative morbidity, and mortality were recorded. The data were analyzed using Kaplan-Meier survival curves. Of the 138 patients with intrahepatic disease who were non-jaundiced, 116 had type I, 10 had type II-III, and 12 had type IV. The wear ages of the patients were: 53.0, SE 9.2 years in type I; 57.1, SE 4.6 years in type II-III, and 50.2, SE 9.2 years in type IV. The male-to-female ratios in the three groups were 1.4 : 1, 1.5 : 1, and 5 : 1, respectively. The mean survival times in the three groups were 556, SE 63 days 374, SE 149 days and 97, SE 35 days. Most of the surgical procedures were tumor excisions (108/138). Right hepatectomy was performed in 63 patients, extended right hepatectomy in 8, left hepatectomy in 18, and extended left hepatectomy in 1. Palliative procedures were performed in the other patients because tumors were in both lobes. The mean survival time was 582 days (SE, 75), for right lobe surgery; 458 days (SE, 89) for left lobe surgery; and 127 days (SE, 58) for the other procedures. Mean survival time was 1039 days (SE, 201) in tumor stage III, 773 days (SE, 123) in stage IVa, and 382 days (SE, 60) in stage IVb. There were no significant differences in survival time according to age or sex. The results of surgery in type I and type II-III were better than the results in type IV. Survival time after right hepatectomy was better than that after left hepatectomy, although without statistical significance, but survival time was significantly better after both operations than after palliative procedures. The results of surgery according to pathological staging showed that survival time in stage III was better than that in either stage IVa or IVb, but only the difference from stage IVb was significant.