Establishment and characterization of cell lines from liver fluke-associated cholangiocarcinoma induced in a hamster model.

Cholangiocarcinoma (CCA) is a relatively rare tumor that occurs primarily in tropical countries and particularly in those with a high incidence of liver fluke infection. A hamster model for a liver fluke-associated CCA has been described previously. In the present study, hamster cholangiocarcinoma cell lines were established and characterized in order to obtain information regarding diagnostically useful tumor marker which could shed light for a future investigation for human cholangiocarcinoma. Two related cell lines, one from the original intrahepatic bile duct tumor and one from an allotransplanted tumor, were established. The established cell lines were found to have population doubling times of 31 and 26 hours respectively, and were maintained in Ham's F12 medium supplemented with 10% fetal bovine serum for over 80 passages. The cell monolayers were subjected to scanning and transmission electron microscopic study and found to have ultrastructural characteristics, including cytoplasmic lumens, consistent with those of adenocarcinoma cells of epithelial origin. An immunoperoxidase study using monoclonal antibodies (MAbs) specific for tumor antigens showed the cytoplasm and membrane of both cell lines to be positive. These antigens were also secreted in soluble form into the culture medium, judging from polyacrylamide gel electrophoresis in the presence of SDS and from immunoblot analyses. Different lines of evidence presented suggested that a 200 kDa glycoprotein produced and secreted by the tumor cell lines could be considered a cholangiocarcinoma-associated marker which has diagnostic potential.

A new method for factor VII deficient substrate preparation and coagulation studies in malaria.

A simplified technique using DEAE-cellulose chromatography for the preparation of factor VII deficient substrate was developed in order to reduce the high cost of individual factor VII assay in the routine coagulation laboratory. The substrate prepared from cryo-removed human and bovine plasma had a high correlation (r = 0.9929) with two of the most popular imported commercial substrates available (DADE, Ortho). When compared several other imported commercial substrates of equal quality, the prepared substrate was 3,000 to 6,000 times cheaper. Using the prepared factor VII deficient substrate along with other commercial substrates available, two hundred and fifty patients with malaria (fifty cases of P. vivax and two hundred cases of P. falciparum) were studied for coagulation and fibrinolysis abnormalities. Only P. falciparum infections showed prolonged PT and aPTT which correlated with the degree of parasitemia (r = 0.0972). Factors V, VII, and IX were the most sensitive parameters in the expression of coagulation defects and most coagulation abnormalities were due to liver involvement. Plasmin activity was normal in P. vivax patients but it was significantly increased in P. falciparum patients with > 5% parasitemia. Only two of the complicated cases of P. falciparum patients showed the evidence of DIC.

Heterogeneity of antibody response to glomerular basement membrane antigen in patients with different forms of glomerulonephritis.

Antibodies to collagenous and noncollagenous components of glomerular basement membrane (GBM) have been detected by immunoblotting in some sera from patients with various kinds of glomerulonephritis. A half proportion of patients with rapidly progressive glomerulonephritis (RPGN), chronic focal glomerulonephritis (CFGN), idiopathic membranous glomerulonephritis (MGN). IgA nephropathy and lupus nephritis (LE-GN) had IgG antibodies to heterogenous components in acid insoluble fraction of pepsin digested GBM. This acid insoluble fraction represented a complex of collagen and noncollagenous proteins of GBM. Following digestion of acid insoluble fraction with bacterial collagenase, the triple helical collagenous components of GBM were destroyed and released most likely of noncollagenous proteins. Antibodies to this noncollagenous proteins were found in only some patients with chronic glomerulonephritis (17.6%) and lupus nephritis (21.4%). Upon reaction with human placenta derived type IV collagen, different frequencies of antibody response were found in patients of different groups. However, all these reactive sera showed a similar immunoblotting pattern. The relationship between antibody response to antigenic components from human GBM or human placenta and pathogenesis of renal disease is unclear. However, the occurrence of spontaneous autoantibody response to some exposed GBM self antigens may mediate further renal destruction resulting in chronic ongoing stage of the disease.

Hematological and coagulation studies in malaria.

One hundred and twenty-six patients with malaria (30 cases of P. vivax and 96 cases of P. falciparum) were studied for evidence of hematological coagulation and fibrinolysis abnormalities. Anemia associated with malaria was observed only in P. falciparum infections and there was no correlation between the degree of anemia and the percentage of parasitemia. Decreased hematocrit levels were found to be statistically significant in P. vivax infected patients (P greater than 0.05). Thrombocytopenia was observed in both P. vivax and P. falciparum malaria patients (P less than 0.001) and correlated with the degree of parasitemia (r = 0.974). Plasmin activity was normal in P. vivax malarial patients but it was significantly increased in patients with a P. falciparum of more than 5 per cent parasitemia. Coagulation profiles showed normal PT, aPTT, and TT in P. vivax infected patients while prolonged PT and aPTT were observed in P. falciparum infection which correlated with the degree of parasitemia (r = 0.0992). Coagulation factors V, VII, and IX were the most sensitive parameters in the expression of coagulation defects and most coagulation abnormalities were due to liver involvement. However, 2 of 20 complicated cases of P. falciparum showed evidence of disseminated intravascular coagulation (DIC).

Wilms' tumor, male pseudohermaphroditism and glomerulonephritis: the Drash syndrome. First case report in Thailand and review the literatures.

The Drash syndrome, the first case in Thailand is reported. The patient had male pseudohermaphrodite, Wilms' tumor of the left kidney and mesangial proliferative glomerulonephritis. Metastasis of Wilms' tumor was noted in the liver, omentum, pelvic peritoneum and vertebral body. Chemotherapy (actinomycin D, vincristine and dexamethasone) and local irradiation were given. The patient developed pneumonia, diarrhea and ended up with Steven-Johnson syndrome from Cotrimoxazole hypersensitivity.

Rapidly progressive glomerulonephritis in Thai children.

Sixteen children with extensive crescentic glomerulonephritis and rapid renal deterioration were selected from 476 patients with glomerulopathy for study. The patients (1-14 yr, M:F = 5:11) presented with edema, oligoanuria, hypertension, gross hematuria and uremic symptoms in 81, 62, 62, 56 and 50 per cent, respectively. The mean Scr was 804 (+/- 436) micromole/L and BUN 38 (+/- 13.4) mmole/L. Anemia was found in 100 per cent, hematuria in 100 per cent, heavy proteinuria 75 per cent, hypoalbuminemia 40 per cent, hypercholesterolemia 38 per cent and low C3 40 per cent. The underlying causes of RPGN included idiopathic 9, PSAGN 6 and LE 1. Eight patients recovered with normal or slightly elevated Scr while the diseases progressed to ESRD in 8 patients. Idiopathic RPGN and extensive (greater than 80%) crescentic glomerulonephritis correlated with a poor prognosis.

Immunological and immunopathological studies of patients with burn scar.

Sera obtained from 5 cases of burn scars (patients group 1) and cases of burn scars with squamous cell carcinoma (patients group 2) of the skin are analysed for anticollagen antibodies. Anticollagen type VI was demonstrable in the serum of one out of 5 cases in group 1 and 4 out of 6 cases in group 2. Collagen type VI was demonstrable in the scar without carcinoma of one patient whose serum contained anticollagen type VI. It is suggested that collagen type VI in the scar and their immunobiologic reaction may be related to the regulation of tumor development in scar tissue of the skin.

Development of immune-complex glomerulonephritis and amyloidosis in Syrian golden hamsters infected with Opisthorchis viverrini.

Renal disease associated with Opisthorchis viverrini infection was investigated in Syrian golden hamsters. On the fourth week after infection with 100 viable metacercariae; anti-tegumental membrane antibodies were detected in the sera by immunofluorescence antibody technic and by enzyme-linked immunosorbent assay. Six weeks after infection tegumental and anti-tegumental membrane immune-complex and amyloid fibrils were found in the glomeruli. Amyloid was characterized to be AA protein. Acute proliferative glomerulonephritis associated with the brightest immune-complex deposits developed in week 8 after infection. Intensity of immune-complexes in all glomeruli were reduce gradually thereafter and replaced by amyloid. Progressive obsolescence of the glomeruli, tubular atrophy, interstitial inflammation and fibrosis associated with massive proteinuria and deterioration of renal function appeared in week 10 after infection toward the end of the experiment in week 38 after infection.

Establishment and characterization of a cholangiocarcinoma cell line from a Thai patient with intrahepatic bile duct cancer.

A new human cholangiocarcinoma cell line (HuCCA-1) was established from cholangiocarcinoma (CCA) tissue fragments surgically removed from a Thai patient with intrahepatic bile duct cancer. The growth medium used for the primary cell culture was Ham's F12 supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml epithelial growth factor (EGF). Approximately one month later, the cells were subcultured in Ham's F12 supplemented with only 10% FBS. The population doubling time was approximately 55 hr. Staining of the cells for cytokeratin and mucin confirmed that the cells were mucin-secreting tumor of epithelial cell origin. The supernatant fluid secreted a number of non-specific tumor markers including CA125 and traces of MCA and AFP. The ability of the HuCCA-1 cell line to synthesize specific marker that may have potential in the diagnosis of cholangiocarcinoma is now being investigated.

Basement membrane antibodies in Alport's syndrome.

By the indirect immunofluorescent technique, sera from patients with Alport's syndrome showed a reaction with the basement membrane of the capillary wall of glomeruli and peritubular vessels and nearby interstitial tissue of normal monkey and mouse kidney as a substrate. It also revealed bright staining to the matrix surrounding the clusters of EHS tumor cells and stromal tissues. These reactions were caused by autoantibodies present in these sera. These findings were supported by the detection of antilaminin, nidogen and anti collagen type VI in the sera by ELISA method. These evidences suggest that the Alport's patients developed stage of autoimmunity. The exact causes were not so clear, but seemed to be due to multiple factors.

The primary nephrotic syndrome in children at Ramathibodi Hospital: clinical and clinicopathological study.

Mesangial proliferative glomerulonephritis (MesPGN) is the most common histopathological finding of primary nephrotic syndrome in Thai children. Prediction of glomerular morphology is possible by the clinical characteristics: the age at onset older than 6 years old, occurring mostly in males, with or without hematuria, no hypertension, normal serum creatinine level, response to initial corticosteroid therapy but frequent relapses. Epidemiological study pertaining to associated environmental factors and immunological response of Thai children is needed.