Cholera toxin-induced fluid secretion in rat gut ligated loops: influence of bile from normal or cholera toxin-immunized rats.

Fresh normal rat bile premixed with cholera toxin (CT) did not significantly affect the CT-induced fluid accumulation in rat jejunal ligated loops. Bile from rats intrajejunally (i.j.) immunized three times with CT definitely inhibited CT-induced fluid secretion. Bile duct ligature (BDL) for 1-4 days in unimmunized rats, in contrast with mice, did not significantly affect subsequent CT-elicited fluid secretion in their ligated loops. BDL for 4 days in rats i.j. immunized with CT, only slightly decreased the CT-neutralizing ability of their gut loops. Passive transfer during 24 hr of bile from i.j.-immunized rats, but not from normal rats, into gut of normal recipient rats with BDL, significantly protected loops made in such recipients. The affinity-purified antibodies of immune bile, mixed with CT, neutralized its effect. Our data show that, unlike mice, rat bile acids are not required for expression of the CT effect in gut loops. In addition, bile from i.j.-immunized rats contains enough anti-CT antibodies to be protective on its own, but is not necessary for substantial gut protection against CT in i.j.-immunized BDL rats. Our results confirm a major and complementary role of both biliary and intestinal secretory IgA antibodies in protection of the rat gut mucosa against CT-induced fluid secretion.     

Proliferating cell nuclear antigen in normal and regenerating rat livers

Immunohistochemical analysis using proliferating cell nuclear antigen (PCNA) antibody shows a negligible number of cells stained in normal liver, but much higher numbers in regenerating liver 24 and 48 h after surgery. We also verified different results by biochemical analysis. Two forms of PCNA, L type (eluted at low concentrations of KCl from a phosphocellulose column) and H type (eluted at high KCl concentrations), were observed in the nucleoplasm of regenerating livers 24 and 48 h after surgery. Treatment of the H type fraction with nuclease caused the H type to disappear and the amount of L type to increase. PCNAs in the cytoplasm are P type (eluted in the pass through fraction) and L type. Surprisingly, the total amounts of P type and L type in cytoplasmic extracts are comparable to those of L type and H type in the nucleoplasm. These results suggest that newly synthesized PCNA is immediately converted into the P and L complex forms. The P type and some of the L type that lacks a nuclear localization signal remain in the cytoplasm; the rest of the L type with a nuclear localization signal is transferred into the nuclei. Then, some of the L type in the nucleoplasm forms the H type, which binds to DNA. These three types of PCNA are also found in significant amounts in the nucleoplasm and cytoplasm of normal rat liver despite its nonproliferating state.     

Characterization of a primary bile ductular cell culture from the livers of rats during extrahepatic cholestasis.

The establishment of novel bile ductular cell cultures was accomplished with the use of explants of a hyperplastic bile ductular tissue preparation obtained from rat livers at 10 to 15 weeks after bile duct ligation or a bile ductular cell fraction isolated from this tissue preparation by a procedure involving Percoll density gradient centrifugation. Observations made on these primary explant and monolayer bile ductular cell cultures were limited to the first 3 days of culture where the morphologic features of the bile ductular epithelium remained fairly well preserved, while fibroblast contamination was found to be very low. These cultured cells also retained over this period a high specific activity for the bile ductular cell marker enzyme gamma-glutamyl transpeptidase, as well as possessed measurable but decreasing specific activities for leucine aminopeptidase and alkaline phosphatase. Karyotypic analysis of the cultured monolayer cells further showed them to be diploid. In addition, preliminary transplantation studies demonstrated the presence of well-differentiated bile ductular-like structures following inoculation of the freshly isolated bile ductular cell fraction into the interscapular fat pads of recipient rats.     

The isolation and characterization of a population of extravillous trophoblast progenitors from first trimester human placenta

BACKGROUND: It is widely accepted that most if not all villous cytotrophoblasts from term placentae are committed to differentiate into syncytiotrophoblast, but that early in gestation villous cytotrophoblasts are bipotential and capable of differentiating into either extravillous trophoblasts (EVTs) or syncytiotrophoblast. In contrast, our previous work has suggested that two separate populations of cytotrophoblast exist in the first trimester, one committed to EVT differentiation and the other to syncytiotrophoblast differentiation. In this work, we have isolated and characterized the population of 'EVT progenitors'. METHODS: First trimester villous explants were cultured for 10 days then subjected to sequential trypsinization. Viable cells that adhered to Matrigel following trypsinization were cultured for up to 5 days and characterized by immunohistochemistry. RESULTS: The isolation protocol yielded >90% cytokeratin positive trophoblasts, which expressed markers characteristic of EVT progenitors. Over 5 days of culture, these isolated putative EVT progenitors did not syncytialize, but approximately 20% differentiated into HLA-G positive EVTs. CONCLUSIONS: It is likely that the isolated putative EVT progenitors are the population of EVT progenitors previously identified in vivo. The characteristics of these isolated putative EVT progenitors provides further evidence for separate progenitors of EVT and syncytiotrophoblast in the first trimester.     

Characterizations of and interactions between bile ductule cells and hepatocytes in early stages of rat hepatocarcinogenesis induced by ethionine.

Numerous hepatic cell lineage pathways have been proposed for the development of hepatocarcinogensis induced by chemical carcinogens in rats. The roles of bile ductule cells and hepatocytes in the development of carcinogenesis were investigated using light and electron microscopic procedures to detect differences in morphology and in the phenotypic expression of antigens that are associated with each cell type. In early stages of hepatocarcinogenesis (4-10 weeks after initiation of feeding of a choline-deficient ethionine containing diet), both bile ductulelike (BDL) cells and hepatocytes were seen in mitosis. At the light microscope level, BDL cells showed intense cytoplasmic pyronin (RNA) staining and were positive for the antigens defined by monoclonal antibody 270.38 (bile ductule cells and "oval" cell marker) and glutathione-S-transferase (Yp isoform), whereas hepatocytes were positive for the antigens defined by monoclonal antibodies 270.26 and 258.26 (liver parenchymal cell markers), catalase activity (peroxisome marker) and adenosine triphospatase activity (bile canalicular marker). The authors frequently encountered BDL cells and hepatocytes in close proximity. Ultrastructural examination showed extensive plasma membrane appositions between a subset of BDL cells and hepatocytes. Desmosome structures, tight junctions, microvilli interdigitations and ATPase-positive bile canalicularlike structures were present along the contiguous plasma membrane domains of BDL cells and hepatocytes. Many of the BDL cells attached to hepatocytes were also attached to other BDL cells that had retained a basal lamina. In many cases, BDL cells connected to both hepatocytes and other BDL cells were no longer completely surrounded by basal lamina and had acquired a dual polarity as a consequence of their sharing apical and lateral membrane domains with both BDL cells and hepatocytes. BDL cells showed increased numbers of microperoxisomes (catalase positive organelles) and numerous free ribosomes. Hepatocytes showed a prominent development of the smooth endoplasmic reticulum, a feature prominent in hepatocytes within hyperplastic nodules. Since BDL cells and hepatocytes proliferate and BDL cells and hepatocytes develop intercellular junction sites, the authors propose that both cell types in early stages of carcinogenesis have the capacity to enter the cell lineage pathway leading to the development of hepatocarcinoma. Furthermore, the finding that BDL cells and hepatocytes form multiple attachment sites at the level of the plasma membrane, suggests the possibility that at some stage convergence of separate hepatic cell pathways may occur.     

Isolation of bile ducts from rat liver: technique and preliminary ultrastructural characterization.

A technique is described for isolation from the liver of a fraction rich in bile ducts. Ultrastructurally the isolated bile ducts showed good overall cellular preservation. The staining of the isolated bile ducts with the ruthenium red procedure clearly demonstrates the surface coat on the luminal surface of biliary epithelial cells and the intracellular microfilaments. The technology presented in this paper should make possible a new approach to the investigation of the hepotobiliary system.     

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.     

Isolation and characterization of a stem cell population from adult human liver

Several studies suggested the presence of stem cells in the adult normal human liver; however, a population with stem cell properties has not yet been isolated. The purpose of the present study was to identify and characterize progenitor cells in normal adult human liver. By stringent conditions of liver cell cultures, we isolated and characterized a population of human liver stem cells (HLSCs). HLSCs expressed the mesenchymal stem cell markers CD29, CD73, CD44, and CD90 but not the hematopoietic stem cell markers CD34, CD45, CD117, and CD133. HLSCs were also positive for vimentin and nestin, a stem cell marker. The absence of staining for cytokeratin-19, CD117, and CD34 indicated that HLSCs were not oval stem cells. In addition, HLSCs expressed albumin, alpha-fetoprotein, and in a small percentage of cells, cytokeratin-8 and cytokeratin-18, indicating a partial commitment to hepatic cells. HLSCs differentiated in mature hepatocytes when cultured in the presence of hepatocyte growth factor and fibroblast growth factor 4, as indicated by the expression of functional cytochrome P450, albumin, and urea production. Under this condition, HLSCs downregulated alpha-fetoprotein and expressed cytokeratin-8 and cytokeratin-18. HLSCs were also able to undergo osteogenic and endothelial differentiation when cultured in the appropriated differentiation media, but they did not undergo lipogenic differentiation. Moreover, HLSCs differentiated in insulin-producing islet-like structures. In vivo, HLSCs contributed to regeneration of the liver parenchyma in severe-combined immunodeficient mice. In conclusion, we here identified a pluripotent progenitor population in adult human liver that could provide a basis for cell therapy strategies.     

Wild rat type C virus: isolation and characterization.

Type C virus has been isolated for the first time from a feral rat (Rattus rattus). The virus exhibits general properties of rat leukemia virus (RaLV) including the RaLV-specific p30 antigen. It shows low infectivity for rat cells and no infectivity for cells of other species. Virus neutralization and interference tests indicate that the new isolate is also antigenically closely related to other RaLV strains of laboratory rat origin.     

Coexpression of neural cell adhesion molecules and bcl-2 in intrahepatic cholangiocarcinoma originated from viral hepatitis: relationship to atypical reactive bile ductule

It has been shown that atypical reactive bile ductules (ARBD) display positive immunoreactivity of neural cell adhesion molecules (NCAM) and bcl-2. We investigated the clinicopathological features of intrahepatic cholangiocarcinoma (CC) arising in cases of viral hepatitis B or C (VHBC) and examined their relation to ARBD by means of immunohistochemical analysis. Sixty-eight surgical cases with CC were included in this study. The cause of the background liver disease was hepatitis B surface antigen (HBsAg)(+) in eight cases, antihepatitis C virus antibody (HCVAb)(+) in 13 cases, both HBsAg(+) and HCVAb(+) in one case, and both HBsAg(-) and HCVAb(-) in 46 cases. The average age of patients with CC arising in the HBsAg(+) group was significantly less than that of patients with CC in the HCVAb(+) group (P = 0.0192). Immunohistochemically, CC arising in the HBsAg(+) and HCVAb(+) groups was correlated with coexpression of NCAM/bcl-2 in the tumor cells (P = 0.0068 and P = 0.0382, respectively). Among the 12 cases of CC coexpressing NCAM/bcl-2, 11 were of mass-forming and peripheral type (P = 0.0437), and lymph node metastasis was a rare finding compared with CC showing negative coexpression of NCAM/bcl-2 (P = 0.0213). The tumor cells of CCs arising in VHBC have some characteristics of ARBD. In such tumors, because lymph node metastases were rarely seen and lymph node dissection did not improve patient's survival, lymph node dissection can be limited.     

The isolation and partial characterization of a herpesvirus from a case of bovine metritis

Summary A virus isolated from the uterine exudate of a cow with metritis had physical, chemical and cytopathogenic properties characteristic of a member of the herpesvirus group. Neutralization tests indicated that the virus was not related to any of the previously described bovine herpesviruses.     

Establishment and characterization of a cytotrophoblast cell line from normal placenta of human origin

A cell line has been established from human placentae at thefirst trimester of normal pregnancy. The cell line was obtainedby culture of purified cytotrophoblast cells in serum-free mediumsupplemented with epidermal growth factor, insulin, dexamethasoneand 0.1% bovine serum albumin. The cells can be subculturedfor >30 passages in one to three splits. All the cells weremononuclear epithelial-like cells positive to cytokeratin 18,gonadotrophin-releasing . hormone (GnRH), neuropeptide Y, neurotensin,leucine-enkephalin, dopamine and 5-hydroxytryptamine inununo-cytochemicalstaining. The cells secreted GnRH, progesterone and oestradiol(in the presence of testosterone) but little human chorionicgonadotrophin and no -endorphin. The cell line showed humankaryotypes and had a population doubling time of 48 h in serum-freemedium. However, the cells would stop growing in the mediumcontaining fetal bovine serum. A normal cytotrophoblast cellline established in serum-free medium will be particularly usefulin the study of cytotrophoblast cell proliferation and differentiation.     

The isolation and identification of Bacteroides ssp. from the normal human vaginal flora

The occurrence of gram-negative anaerobic bacilli in the normal vaginal flora was studied in 20 normal healthy women attending a family-planning clinic. A swab was taken from the cervix and posterior fornix and Bacteroides spp. were isolated on a selective medium from 13 (65%) subjects. A heavy growth of Bacteroides was obtained from 11 specimens but only a few colonies were isolated from two specimens. Where possible, 10 representative colonies from each subject were studied and 113 isolates were identified by conventional bacteriological tests. Most isolates (78%) belonged to the B. melaninogenicus/oralis group. The commonest species identified in this group were the B. bivius/disiens complex (42%), B. melaninogenicus ss. intermedius (22%) and ss. melaninogenicus (16%). Asaccharolytic strains were isolated in smaller numbers from 54% of subjects, but only five strains of the B. fragilis group were isolated from two subjects; fusobacteria were not detected.     

The isolation and identification of Bacteroides spp. from the normal human gingival flora

Gram-negative anaerobic bacilli were isolated on an enriched selective medium from specimens of subgingival plaque from 20 normal, healthy adults without periodontal disease. a heavy growth of Bacteroides and Fusobacterium spp. was obtained from all specimens and 10 representative colonies from each subject were selected for identification by conventional bacteriological methods. Results were obtained in these tests with 188 isolates; some strains of the B. melaninogenicus/oralis group would not grow in pure culture on solid media and showed satellitism in mixed culture, and some strains of fusobacteria did not survive repeated subculture. Most isolates (68%) belonged to the B. melaninogenicus/oralis group. The commonest species identified in this group were B. oralis (41%), B. melaninogenicus ss. melaninogenicus (26%) and ss. intermedius (17%). B. oralis was isolated from all subjects and at least one strain of B. melaninogenicus was isolated from 18 out of 20 subjects. Only eight strains of the B. fragilis group were detected and they probably represented transient carriage. Fusobacterium spp. and L. buccalis were isolated from 15 out of 20 subjects and accounted for 21% of the isolates studied, but most could not be identified specifically by the set of tests used. Asaccharolytic species of Bacteroides were isolated in small numbers from seven subjects.