Establishment and characterization of a human ovarian neoplastic cell line, DO-s.

A permanent human neoplastic cell line, DO-s, was established from ascites of a patient with a well-differentiated mucinous cyst-adenocarcinoma of the ovary. This cell line grew as vermiform, floating colonies of epithelial cells in culture. The karyotype of DO-s was of a human female; the chromosome number ranged from 54 to 66 with several abnormalities, mainly trisomy. Epithelial-like character was confirmed by transmission electron microscopy and by the presence of cytokeratin. Inoculation of DO-s cells i.p. or s.c. in athymic nude mice resulted in, respectively, ascites and xenografts. Light and electron microscopical analysis of cultured cells and xenografts demonstrated that the cell line was derived of a mucinous adenocarcinoma biopsy. Tumor-associated antigens, cancer antigen 125 (CA 125), human milk fat globulin, and human placental alkaline phosphatase were expressed by cells in culture and in xenografts. Modulation of the antigens, CA 125 and human milk fat globulin, occurred in DO-s cells growing in athymic mice. Biochemical, immunohistochemical, and histochemical analysis showed that more than 50% of the alkaline phosphatase isoenzymes present in DO-s cells had the characteristics of human placental alkaline phosphatase and placental alkaline phosphatase-like alkaline phosphatase (AP), but fractions of intestinal AP and nonspecific AP (bone-liver-kidney) were also present. The expression of AP isoenzymes could be induced by an enhancement of the serum supplement in the culture media, and by dexamethasone, sodium butyrate, and bromodeoxyuridine. This line will be a valuable tool in studying the therapeutic effects of antibodies to tumor-associated antigens or other agents for ovarian cancer.

Human placental alkaline phosphatase in benign and malignant ovarian neoplasia.

In benign and malignant ovarian tumor patients, human placental alkaline phosphatase (HPLAP) was determined in serum and extracts from surgical tumor biopsies using a highly specific enzyme-antigen immunoassay based on a mouse monoclonal antibody (E6) to HPLAP. Serum HPLAP levels greater than or equal to 0.1 unit/liter were found in 58% of ovarian cancer patients. Serum carcinoembryonic antigen levels were positive (greater than 5.4 ng/ml) in 17% of these patients. HPLAP was detected in extracts from 13 of the 14 tumors investigated (range, 2.4 to 557 milliunits/g). Only the mixed heterologous Müllerian sarcoma was negative. The highest HPLAP content of normal ovarian tissue was 1.1 milliunits/g. The amount of heat-stable and L-p-bromotetramisole-insensitive alkaline phosphatase was in all cases much higher than the fraction recognized by E6. The neoplastic origin of HPLAP was confirmed immunohistochemically on paraffin sections by an indirect avidin-biotin-peroxidase staining procedure using E6. The staining pattern was compared to the histochemical distribution of total alkaline phosphatase on adjacent sections. A consistency was found between the amount of HPLAP in tissue extracts and its immunohistochemical distribution. In all the tumors, staining for HPLAP was observed mainly on the plasma membranes of carcinoma cells. In 9 of the 10 carcinomas, the histological distribution of HPLAP and also of total alkaline phosphatase was heterogeneous. HPLAP staining, present in one of five normal ovaries, was restricted to germinal inclusion cysts. The present results support the hypothesis that serous ovarian tumors originate from these cysts.

Immunohistochemical localization of placental alkaline phosphatase, carcinoembryonic antigen, and cancer antigen 125 in normal and neoplastic human lung.

Human placental alkaline phosphatase (HPLAP), carcinoembryonic antigen (CEA), and cancer antigen 125 (CA 125) were localized immunohistochemically in paraffin sections of normal lung tissue from 16 patients, using monoclonal antibodies and an indirect avidin-biotin-peroxidase staining procedure. HPLAP and CEA were present in epithelial cells of respiratory bronchioli and alveolar type I pneumocytes. CEA was also observed in the tracheal, bronchial, and bronchiolar epithelium. CA 125 was present in the tracheal, bronchial, bronchiolar, and terminal bronchiolar epithelium; in the tracheal and bronchial glands; and in the pleural mesothelium. Normal and hyperplastic type II pneumocytes were negative for HPLAP, CEA, and CA 125 but were histochemically positive for nonspecific alkaline phosphatase. Fetal lung tissue between 11 and 15 weeks of gestation was negative for HPLAP, CEA, and CA 125. The fetal tracheal and bronchial epithelium, tracheal glands, and pleural mesothelium were positive for CA 125. For ten malignant pulmonary tumors investigated, HPLAP staining was observed in five, CEA in nine, and CA 125 in seven. The localization of HPLAP, CEA, and CA 125 in apparently normal constituents of all pulmonary specimens is in disagreement with the concept that the expression of these substances in the lung is indicative of abnormal cellular activity.

The cytosolic glutathione S-transferase isoenzymes in the dog kidney cortex as compared with the corresponding MDCK renal cell line.

Cytosolic glutathione S-transferase (GST) (EC 2.5.1.18) isoenzymes of dog kidney and MDCK (an established dog renal cell line) were purified and studied. Specific GST activity was 248 and 317 nmol/min/mg protein, for dog and MDCK, respectively. Cytosolic GST was only partially purified by glutathione affinity chromatography, a substantial amount (43% and 84% for dog kidney and MDCK, respectively) of the GST activity was found in the flow-through fraction. Affinity bound GST was separated into 6 and 3 isoenzymes by anionic chromatofocusing for dog and MDCK, respectively. Flow-through GST was purified by gel filtration, anion exchange chromatography and anionic chromatofocusing showing only one GST isoenzyme, with distinct features from the affinity bound GST, for both dog and MDCK. The isoenzymes were characterized by their kinetic properties, subunit composition, specific substrates and inhibitors and immunoblot. The major dog GSTs (DII, DIV and DVI) correspond to the MDCK isoenzymes (MI, MII and MIII). Comparable pI values, a comparable affinity towards GSH and comparable sensitivities towards the inhibitors N-ethylmaleimide (NEM), triphenyltin chloride, cibacron blue and hematin were observed for the corresponding isoenzymes: DII and MI, DIV and MII, DVI and MIII. Co-electrophoresis showed that the subunit composition was identical for DII and MI, and for DIV and MII. Inhibitor and substrate sensitivities showed that the affinity bound GSTs belong to class pi and mu, the presence of class pi was confirmed by immunoblot analysis. One homodimeric GST isoenzyme was observed in the dog kidney and MDCK flow-through. Both dog and MDCK isoenzyme have a nearly neutral pI, a high affinity towards CDNB and an equal sensitivity towards triphenyltin chloride, cibacron blue and hematin. However, based on inhibitor studies and immunoblot, this isoenzyme could not be attributed to an identified GST class. The overall isoenzyme pattern of dog and MDCK affinity bound and flow through GST is comparable. The dog and MDCK affinity bound GSTs have similar characteristics and all belong to class mu or pi.

Volumetric control of arginine vasotocin and mesotocin release in the frog (Rana ridibunda).

Adult male frogs (Rana ridibunda) were subjected to several volumetric and osmometric stimuli and the influence on circulating concentrations of arginine vasotocin (AVT) and mesotocin was studied by the use of highly specific radioimmunoassays. During progressive blood withdrawal (haemorrhage group) urine flow decreased to zero, whereas no change occurred in the plasma and urine osmolality. Control levels of 34.3 +/- 7.3 pmol AVT/1 gradually increased up to 638.3 +/- 179.1 pmol/1 (P less than 0.001) after a blood loss of up to 50-60% of the blood volume. Plasma mesotocin concentrations also increased from 42.4 +/- 9.2 to 70.8 +/- 12.0 pmol/1 (n = 7). Hypervolaemia, produced by the repeated intravenous injection of isotonic Ringer solution, increased the urine flow and osmolality compared to controls but had no influence on the plasma levels of AVT and mesotocin. Hypernatraemia without volume change profoundly increased the urine osmolality but the urine flow was not affected; the plasma concentrations of AVT and mesotocin remained at the control level. Finally, during a 1-h immobilization stress a pronounced antidiuresis occurred in the presence of a constant plasma and urine osmolality and control plasma levels of AVT and mesotocin. It is concluded that the release of AVT and, to a smaller extent, of mesotocin is under volumetric control.

Effect of dehydration, haemorrhage and oviposition on serum concentrations of vasotocin, mesotocin and prolactin in the chicken.

Serum concentrations of arginine vasotocin (AVT), mesotocin and prolactin were determined by radioimmunoassay in Rhode Island Red chickens during and after dehydration, haemorrhage and oviposition. During dehydration increased circulating levels of AVT, mesotocin and prolactin were found. As water deprivation proceeded, marked differences were observed. After an initial rise in serum AVT, mesotocin and prolactin levels during mild and moderate dehydration, concentrations of both AVT and prolactin tended to normalize during continued water deprivation, while those of mesotocin remained high throughout the whole dehydration experiment with the highest at the end of the water-deprivation period. Removal of 5 ml blood at intervals of 10 min during six consecutive time-periods did not affect serum osmolality and circulating levels of AVT and prolactin, but slightly increased mesotocin. These results suggest an osmoregulatory role for AVT and prolactin, whereas mesotocin may be involved in volume control. Finally, 1 min after oviposition, control values of 19.5 +/- 3.4 pmol AVT/1 (n = 9) were raised more than sevenfold to 142.9 +/- 12.5 pmol/l (n = 11). Thereafter, a decline occurred with a half-life for AVT of 13 min with raised serum levels up to 31 min after oviposition. In contrast, the serum concentrations of mesotocin and prolactin remained unaffected by oviposition.

Human intestinal versus tissue-nonspecific alkaline phosphatase as complementary urinary markers for the proximal tubule.

The availability of early biological markers of renal damage is important for the identification of risk factors and for starting therapeutic intervention in the reversible phase of renal pathology. The usefulness of such markers relies upon their capacity to detect alterations in distinct nephron segments. Using specific monoclonal antibodies against the intestinal isoenzyme of alkaline phosphatase (IAP) and against the tissue-nonspecific isoenzyme (TNAP), we demonstrated that IAP expression in the human kidney is restricted to the straight part of the proximal tubule (the S3 segment), whereas TNAP is expressed mainly in the proximal convoluted tubule (the S1 and S2 segments) but also in the S3 segment. This complementarity opens perspectives for IAP and TNAP as distinct proximal tubular markers, particularly for IAP, since there are no other markers available that are specific for the S3 segment. Based on these monoclonal antibodies, specific and easy to use enzyme-antigen immunoassay (EAIA) procedures were developed to detect IAP and TNAP in human urine samples. The detection limits are below the lowest enzyme activities found in the urine of normal subjects, the intra- and inter-assay variability is low, the analytical recovery approaches 100%, and EAIA enzyme activity values correlate with ELISA immunoreactivity values. Furthermore, easy urine sample preconditioning allows antigen preservation over an extended time period at 4 degrees and -80 degrees C. Using these assays, it could be demonstrated in more than 20 occupationally and environmentally exposed cohorts and clinical patient groups that urinary IAP is indeed a marker of early alterations in the S3 segment, and that it behaves largely independently from urinary TNAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Human fetal intestinal alkaline phosphatase: molecular heterogeneity and immunological detection in amniotic fluids.

Human fetal intestinal alkaline phosphatase (fIALP) is present in amniotic fluids as free dimers (Mr 140,000) or membrane-bound through phosphatidylinositol residues. Extraction of corresponding particulate material with Triton X-100, resulted in release of tetrameric high Mr fIALP forms (Mr 380,000). In individual amniotic fluids, as well as in meconeum, both dimeric and tetrameric fIALP are sialylated to various extents. When measured by a double sandwich-ELISA, up to 10-fold higher fIALP antigen levels were found in amniotic fluids than when determined by an enzyme antigen immunoassay, based upon fIALP enzyme activity measurements. Frequency analysis of fIALP antigen levels, showed a more symmetrical distribution than analysis of fIALP enzyme activities; likewise, the lower 95% confidence limit, calculated for the fIALP antigen distribution curve, overlapped less with the bulk of values. In cystic fibrosis amniotic fluids, measurements of fIALP antigen levels resulted in a lower false-negativity outcome than fIALP enzyme activity measurements, whereas in amniotic fluids of trisomy pregnancies fIALP enzyme activities and fIALP antigen levels were equally unpredictive.

Neutrophils and acute ischemia-reperfusion injury.

Leukocyte infiltration in response to I/R injury is a well-known but poorly understood phenomenon. The contribution of neutrophils in this process is still controversial. Despite numerous data, little is known about exact numbers of infiltrating neutrophils. The role of monocytes/macrophages in this process is even more unclear. The role neutrophils in the kidney and other organs was reviewed. The variability in models and methods for neutrophil quantification were examined, along with carrying out a critical overview of depletion and anti-adhesion approaches. Nevertheless, the exact role attributed to neutrophils in the I/R process remains unclear.

Cyclosporine nephrotoxicity: comparative cytochemical study of rat kidney and human allograft biopsies.

The aim of this study was to detect early renal changes in the rat. Female Wistar rats received oral doses of cyclosporine (12.5, 25 or 50 mg/kg daily). The duration of the experiment was 1, 2, and 3 weeks. Controls received the vehicle only (olive oil). The following alterations were seen by light microscopy: Hypertrophy of the juxtaglomerular apparatus (PAS stain). Cytoplasmic droplets of neutral fat (Oil Red 0) in clusters of cortical tubules, probably belonging to the same nephron. Both the above phenomena increased with dosage and duration of treatment and were absent in controls. In the fat containing tubulus (FCT) brush border staining (alkaline phosphatase) was decreased or absent. Since after PAS the brush border was visualized in many FCT, it is concluded that many FCT were proximal tubulus (PT) of which the brush border has been damaged. In FCT mitochondrial staining (Cytochrome oxidase activity) was strongly decreased or absent. Mean lysosomal volume (acid phosphatase and dipeptidase II) is increased in the PT; in some cyclosporine animals, lysosomes were enlarged, while in others they were comparable to controls. Electron microscopy showed in some PT cells an increased number of empty vacuoles and focal alteration of mitochondria. Normal mitochondria were present next to grossly altered mitochondria. Autophagocytosis of mitochondria was clearly present. The lysosomes appeared swollen and contained electron dense material, not organised in the typical 50 A pattern of myeloid figures. These morphological changes suggest a defect of mitochondrial metabolism, leading to lipid deposition in PT. The mitochondrial metabolism can be disturbed by a direct toxic effect of cyclosporine or indirectly via ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevalence and clinical expression of HCV-genotypes in haemodialysis-patients of two geographically remote countries: Belgium and Saudi-Arabia.

Hepatitis C virus is the leading cause of acute and chronic liver disease in hemodialysis patients. There are at least six major HCV-genotypes, with a well documented geographical distribution in the general population. Moreover, HCV-genotype is one of the major determinants of the therapeutic response to Interferon Alpha in affected patients. Since the therapeutic outcome in HCV-positive hemodialysis patients, especially with regard to the different HCV-genotypes, is of interest, a multicentre epidemiologic study was performed in HCV-antibody positive hemodialysis patients of two geographically remote countries, i.e. in Flanders (Belgium) and in Saudi-Arabia. 184 chronic hemodialysis patients, with a positive second or third generation Elisa assay for HCV, were tested for HCV-viremia and HCV-genotype, using a 5' untranslated region (UR) nested PCR for the detection of HCV-RNA and subsequently type-specific probes to hybridize with HCV-RNA (Inno-Lipa). Additionally, clinical data were collected by means of a standardized questionnaire, thoroughly completed by the nephrologist in charge of each respective patient. Viremia was present in 79% of the patients (146 out of 184). The prevalence of HCV-genotypes differed significantly between Belgian and Saudi-Arabian dialysis-patients. In Belgian dialysis patients HCV-genotype 1b was most prevalent (i.e. 62%), while in Saudi-Arabian patients HCV-genotypes 4, 1b, and la were present in respectively 36,4%, 31,7%, and 25,8% of the HCV-PCR positive patients. Although there were significant differences between Belgian and Saudi-Arabian dialysis patients, no clinical data showed any significant correlation with the HCV-genotype. Transaminases, determined over a six months period, showed normal average values. Doubling of the transaminases, in at least one out of six measurements over a six monthly period, occurred only in 14% (alanine aminotransferase, ALT) and 10% (aspartate aminotransferase, AST) of the patients. In Belgian dialysis patients, HCV-genotype 4 (or HCV-genotype 5) significantly correlated with a more recent start of dialysis treatment. We conclude that there is a significant different geographical prevalence of HCV-genotypes in HCV-affected hemodialysis patients. None of the different HCV-genotypes shows any particular clinical expression. Transaminases are not a sensitive marker for ongoing HCV-replication in hemodialysis patients. In Belgian dialysis patients, a changing pattern of HCV-infection is suggested, with an increasing prevalence of HCV-genotype 4 (or HCV-genotype 5) in more recent years. These data suggest possible implications for the therapeutic strategy in dialysis patients.

Effects of foetal calf serum on cell viability, cytotoxicity and detoxification in the two kidney-derived cell lines LLC-PK1 and MDCK.

Foetal calf serum (FCS) dependent cell viability, cytotoxicity and detoxification were investigated in MDCK and LLC-PK1 cells. FCS was used at 0-10% (v/v). Viability and cytotoxicity were measured by neutral red uptake and by the MTT test. Viability of LLC-PK1 was strongly dependent on the FCS concentration, but that of MDCK cells only to a very limited extent. For both cell lines the cytotoxicity of HgCl(2) was FCS concentration dependent: lower toxicity was observed with 5-10% FCS than with 0-1% FCS. This effect was not observed for paracetamol. The results could not be explained by altered glutathione or glutathione S-transferase. The optimal FCS concentration of 1%, necessary to retain cell viability, had a limited influence on cytotoxicity. FCS concentration must be taken into consideration when cytotoxicity data from different studies are compared.

Isolation and characterisation of the class alpha, mu and pi glutathione transferases in LLC-PK1 and pig kidney.

Glutathione S-transferase (GST) isoenzymes from pig kidney cortex and LLC-PK1 (an established cell line derived from the pig proximal tubule) were purified by affinity chromatography, anionic and cationic chromatofocusing. Purification revealed nine isoenzymes in the pig kidney cortex and five isoenzymes in the LLC-PK1 cell line. SDS-polyacrylamide gel electrophoresis showed that the pig kidney cortex isoenzymes were homo- or heterodimeric; LLC-PK1 isoenzymes, however, were homodimeric. Isoenzymes from pig and LLC-PK1 showed a higher affinity towards glutathione. The isoenzymes were further characterised and divided into the different GST classes by studying specific inhibitors, specific substrates and immunological properties. Pig GSTs belong to class alpha, mu and pi. The GSTs in LLC-PK1 cells, on the other hand, belong to class pi and mu. The isoenzyme pattern in LLC-PK1 cells indicates the dedifferentiation of this particular cell line compared with the pig kidney cortex.

Effect of blocking T4-monodeiodination on hatching in chickens.

Chick embryos were injected with iopanoic acid (IOP) on either day 17 or 18 of incubation, and radioimmunoassays of triiodothyronine (T3) and thyroxine (T4) in serum and thyroid glands were performed from day 19 on through pipping and hatching and 1 day after hatching. The IOP was able to block T4 to T3 conversion in chick embryos from day 19 of incubation. Blocking T4 conversion did not delay hatching significantly, nor did it affect embryonic mortality significantly. Yolk sac retraction was not affected at hatching. A rise in serum reverse T3 (rT3) and T4 was observed after IOP administration. The rise in T4 could not be explained solely by a decreased T4 conversion. The results indicated that peripheral monodeiodination occurs in the late chick embryo.

EGF and TGF-alpha in the human kidney: identification of octopal cells in the collecting duct.

UNLABELLED: Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are well-known mitogens expressed in the kidney. Their human renal cell origin has not been conclusively identified. The distribution of EGF and TGF-alpha was investigated immunohistochemically in the adult human kidney in comparison with the monkey and rodent kidney. In humans, as in the monkey, two variants of EGF immunoreactivity were detected. One was present along the apical cell surfaces and diffusely in the cytoplasm of the thick ascending limb (TAL), co-localizing with Tamm-Horsfall protein, and in the distal convoluted tubule (DCT). The other occurred as overall membranous staining in the connecting tubule and cortical collecting duct (CD), and mainly as basal staining in the rest of the CD. The EGF stained cells in the cortical and outer medullary CD reached a diameter of 40 mu and were identified as intercalated or dark cells; they displayed a peculiar octopus-like shape, bearing long lateral extensions that stretched underneath and between 20 surrounding smaller negative cells. Cytoplasmic TGF-alpha staining appeared in the DCT and decreased further on. IN CONCLUSION: (1) the normal human distal nephron displayed EGF and TGF-alpha immunoreactivity in a partly complementary segmental and subcellular distribution pattern, partly differing from that in rodents. (2) EGF immunostaining revealed the presence of long lateral projections on CD intercalated cells; this peculiar morphology suggests a modulatory role within the CD epithelium, possibly involving the EGF immunoreactivity on their surface.