De novo expression of nonhepatocellular cytokeratins in Mallory body formation

 Mallory bodies (MBs) are eosinophilic cytoplasmic inclusions observed predominantly in alcoholic liver disease. Although linked to disease activity, their pathogenesis is still unclear. Since intermediate filaments (cytokeratins) are major components of MBs, their cytokeratin polypeptide composition was analysed with monospecific antibodies for cytokeratins 7, 8, 14, 18, 19, and 20 by immunohistology. MBs were identified by light microscopy and ubiquitin immunostaining. All MBs were positive for cytokeratins 8 and 18. A significant percentage of the MBs was strongly positive for cytokeratins 19 and/or 20, which are not detectable in hepatocytes of normal liver and, in the case of cytokeratin 20, in hepatocytes of diseases devoid of MBs. MBs were essentially negative for cytokeratins 7 and 14. De novo expression of cytokeratins 19 and 20 was independent of the aetiology, occurring in all MB-associated diseases analysed, and seemed to precede MB formation, since in some hepatocytes a cytoskeletal-type staining pattern for these cytokeratins was present. In hepatocellular carcinomas cytokeratins 19 and 20 were frequently detected, but their cellular distribution was less closely associated with MBs. The ectopic expression of cytokeratins 19 and 20 appears to be related to MB formation and may take part in the derangement of the intermediate filaments during MB formation. Received: 21 May 1997 / Accepted: 10 September 1997     

Cytokeratin expression in hepatocellular carcinoma: an immunohistochemical study

Normal human hepatocytes express cytokeratins no. 8 and 18, whereas bile duct cells contain the same cytokeratins and, in addition, cytokeratins no. 7 and 19. This cytokeratin pattern is believed to be preserved during neoplastic transformation. Thirty-four cases of hepatocellular carcinoma (11 well differentiated, 16 moderately differentiated, 7 poorly differentiated) were studied on frozen sections using monoclonal antisera directed against individual cytokeratins no. 7, 8, 18, and 19 in an immunoperoxidase procedure. In 17 of 34 cases, tumor cells showed only reactivity with monoclonals anticytokeratin no. 8 and 18. However, 17 of 34 cases showed an aberrant pattern in that a variable number of tumor cells were stained with anticytokeratins no. 7 and/or 19 in addition to no. 8 and 18. Only three of 11 well-differentiated cases displayed an unexpected cytokeratin pattern, whereas an aberrant pattern was present in all seven of seven poorly differentiated cases. These results are in conflict with previously published data obtained by two-dimensional gel electrophoresis and immunohistochemistry. They indicate that the cytokeratin pattern might not always be preserved during neoplastic transformation. The implication of this finding for the differential diagnosis of metastatic gastrointestinal carcinomas is discussed.     

A cytokeratin immunohistochemical study of alcoholic liver disease: evidence that hepatocytes can express ''bile duct-type'' cytokeratins

A cytokeratin immunohistochemical study was performed on 40 liver biopsies diagnosed as alcoholic liver disease to further investigate the cytoskeletal changes occurring in this disease. On paraffin sections of 29 cases, a variable number of hepatocytes were reactive with a polyclonal antiserum that normally stains only bile ducts. Using monoclonal antibodies specific for a single cytokeratin polypeptide on cryostat sections, a variable number of hepatocytes were immunoreactive for cytokeratin no. 7 in 23 cases and also for cytokeratin no. 19 in seven cases. Both these polypeptides are restricted to bile duct cells in the normal liver. The number of hepatocytes positive for bile duct-type cytokeratins increased and their location changed with the severity of the disease. Mallory bodies were reactive with monoclonal antibodies CAM 5.2 and anti-cytokeratin no. 18 but unreactive with anti-cytokeratin no. 8. except in one case. In two cases, Mallory bodies reactive with both monoclonal antibodies anti-cytokeratin no. 7 and anti-cytokeratin no. 19 were found. These results clearly indicate that hepatocytes in alcoholic liver disease can express immunoreactivity for bile duct-type cytokeratins. Our data also demonstrate heterogeneity in the composition of Mallory bodies. Whether hepatocytes expressing bile duct-type cytokeratins are the precursors of Mallory body-containing cells is not clear at present.     

Initiation of apoptosis in hepatocytes during prolonged arterial hypotension and postresuscitation period

An increase in activity of Ca²⁺, Mg²⁺-dependent endonucleases on the second hour of hypotension coincided with the presence of DNA fragments in agarose gel. A correlation was established between the duration of hypotension, Ca²⁺, Mg²⁺-dependent endonuclease activity, and intensity of nuclear DNA fragmention. Apopotosis of hepatocytes is triggered during ischemia and develops during reperfusion. Translated fromByulleten' Eksperimental'noi Biologii i Meditisiny, Vol. 125, No. 3, pp. 285–288, March, 1998     

Ultradian rhythms in cell population. Problem of synchronization

The studies demonstrating cooperativity of cells in synchronization of their activity are reviewed. The total ultradian rhythm in a cell culture is taken as a marker of synchronization. Self-synchronization of interacting oscillators has been demonstrated in experiments. Special attention is paid to the mechanisms underlying formation of ultradian rhythms of protein synthesis in a culture of rat hepatocytes. Formation of populational rhythm in these cells is a function of cell density and time of culturing without replacing growth medium (conditioning). The addition of some individual exogenous gangliosides to the culture medium simulates the effects of conditioning. Immunocytochemical studies showed that intracellular expression of ganglioside determinants is enhanced during the conditioning of culture medium. Exchange of gangliosides between cells and their intracellular accumulation may be the first step in synchronization of cell activity. Synchronization of the protein synthesis oscillations was demonstrated in hepatocytesin situ in a denervated liver. From these observations and literature data it is concluded that self-synchronization of cellular activity is a fundamental regulatory mechanism of organ functioning which operates in line with regulatory systems acting at the organism level. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 12, pp. 604–609, December, 1997     

Expression of hepatic estrogen sulfotransferase in hepatocytes transplanted into the spleen of syngeneic animals

An immunohistochemical assay showed patchy expression of liver-specific androgen-dependent estrogen sulfotransferase in hepatocytes of male rats transplanted into the spleen of castrated syngeneic males during 4–5 months after transplantation. Hepatocyte islets not arranged in hepatic lobules were found in the spleen. No regular distribution of cells with the maximum and minimal expression of estrogen sulfotransferase was revealed. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 7, pp. 45–47, July, 1999     

Expression of an unusual estrogen-binding protein in the liver of androgenized adult female rats during liver regeneration after carbon tetrachloride poisoning

The expression of an unusual estrogen-binding protein induced in the liver of ovariectomized adult rats by androgen was greatly reduced after their pericentrally located hepatocytes were poisoned with carbon tetrachloride, but was partially restored in the course of subsequent liver regeneration. It is suggested that the androgen program established in the periportal hepatocytes of postpubertal animals is defective. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 7, pp. 28–31, July, 1996     

Apoptosis: Decrease of hepatocyte population in mice after hyperthermia

Hyperthermia caused hemodynamic disorders in the liver and degenerative and necrobiotic changes in hepatocytes of CBA mice. Total hepatocyte count decreased during restitution, this decrease being most pronounced 30 min after exposure. The number of binucleated cells also markedly decreased. The absence of necrotic changes in hepatocytes during the entire restitution period indicated their apoptotic death and elimination by macrophagal resorption. Under these conditions liver regeneration at the cellular level occured mainly via division of binucleated hepatocytes. On the other hand, proliferation of oval cells in the portal zones and their differentiation into hepatocytes were observed at certain stages of reparative regeneration of the liver. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 9, pp. 346–350, September, 2000     

Structural and functional morphology of acute phase reaction in the liver in experimental hypercholesterolemia

Synthesis of acute phase transmitters, migration and degranulation of pseudoeosinophils in hepatocytes, and damage to hepatocytes are observed during, the first few weeks of atherogenic diet. It is hypothesized that the acute phase reaction determines disturbances in the synthesis and catabolism of lipoproteins in the liver. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 4, pp. 460–464, April, 1998     

Characterization of specific binding sites for high-density lipoproteins on rat hepatocytes: Effects of estradiol and testosterone

Two types of binding sites for high-density lipoproteins (HDL): P₁ and P₂ K_dl=20 and K_d2=2.5μg/ml, N₁=130 and N₂=35 ng/mg cell protein) are identified on the surface of rat hepatocytes. Conditions for predominant determination of P₂ are created by employing radiolabeled lipoproteins (¹²⁵I-HDL) with a high specific activity (1000 dpm) and the differences in the kinetics of the P₁-and P₂-¹²⁵I-HDL complex formation. P₂ predominate on hepatocytes from females. The addition of estradiol to a culture of hepatocytes from males increases the content of P₂, while the addition of testosterone to hepatocytes from females decreases the content of P₂ to the levels determined in males. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 12, pp. 629–634, December, 1996     

Ultrastructure of rat liver cells after exhausting physical training

In rats, running of the maximum intensity caused death of some hepatocytes, an increase in the number of phagosomes in Kuppfer cells, and the emergence of connective tissue fibers in the space of Disse. Ultrastructural investigation of hepatocytes showed delayed release of bile products into bile capillaries, decrease in glycogen content, increase in the number of mitochondria (many of them were divided by the cristae), and irregular distribution of ribosomes in the rough endoplasmic reticulum. Accumulation of erythrocytes in the sinusoids, fragments of dead hepatocytes, Kuppfer cells with numerous phagosomes, and connective tissue fibers in the space of Disse were observed in rat liver after exhausting swimming. Study of hepatocyte ultrastructure revealed intense protein synthesis (as evidenced by increased number of ribosomes and unchanged mitochondria and cisternae of the rough endoplasmic reticulum), separation of cytoplasmic fragments with ribosomes into sinusoids, absence of glycogen, and lipid accumulation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 7, pp. 101–104, July, 1998     

Effect of the hydra peptide morphogen on posthypoxic disorders in rats exposed to prenatal hypoxia

Lipid peroxidation in the lungs and blood are activated while DNA synthesis in the tracheal epithelium and hepatocytes is inhibited during the first five days of postnatal life in rat pups after severe prenatal hypoxia. Intraperitoneal injection of the undecapeptide pGlu-Pro-Pro-Glu-Glu-Ser-Lys-Val-Ile-Leu-Phe, a peptide morphogen isolated from the hydra, before hypoxia normalizes lipid peroxidation in the lungs and blood of the five-day-old pups. A compensatory activation of DNA synthesis occurs in tracheal epithelium and hepatocytes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 3, pp. 269–272, March, 1997.     

Effect of bromocriptine on the expression of prolactin receptors in liver cells after common bile duct ligation

Immunoperoxidase method combined with cytofluorimetry showed that in contrast to hepatocytes, enhanced expression of prolactin receptors on rat cholangiocytes induced by common bile duct ligation cannot be suppressed by the prolactin secretion inhibitor bromocriptine. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 7, pp. 52–55, July, 1998     

Liver cells in a comparative morphological series of animals: Ultrastructural features and their significance

A detailed ultrastructural analysis of liver cells in a comparative morphological series of animals from various classes has provided insight into how liver structure was perfected in the course of evolution. Specific features of the adaptive responses acquired by hepatocytes in different animal classes for maximizing their many functions in the framework of the existing organ structure are identified. It is shown that the adaptations found in lower animals also normally occur in more highly organized animals, and that the same adaptations developed by hepatocytes during phylogeny are used in responding to various influences exerted upon the organism. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, No. 6, pp. 604–609, June, 1996     

Growth and differentiation in culture of clonogenic hepatocytes that express both phenotypes of hepatocytes and biliary epithelial cells.

A cell fraction containing small hepatocytes and nonparenchymal cells was isolated from the adult rat liver and was cultured in the presence of vitamin C. epidermal growth factor, nicotinamide, and dimethylsulfoxide. All of the small hepatocytes that had attached to a dish expressed hepatocytic phenotypes such as albumin, transferrin, and cytokeratin (CK)8 and CK18 but not biliary markers such as BD1, CK7, and CK19. Single hepatocytes started to proliferate and grew into colonies. The growth potential was variable among the cells, the highest case being that a single cell produced a colony containing over 100 cells in 10 days. The hepatocytes in the colony developed through a proliferation phase and then a differentiation phase. They showed very high bromodeoxyuridine labeling indexes during the first 7 days (proliferation phase), which gradually decreased thereafter. Phenotypic alterations took place at 7 to 10 days, and some hepatocytes started to express mature hepatocyte markers and biliary markers (differentiation phase). The presence of cells that coexpress albumin and biliary markers (CK7 and CK19) was demonstrated by double immunocytochemistry. In addition, cells were identified that ceased to express albumin and in turn were positive for CK19 or CK7. Therefore, the colony was considered to contain liver progenitor-like cells that can differentiate during culture into cells expressing phenotypes of mature hepatocytes or biliary epithelial cells.     

A crucial role of hepatocyte nuclear factor-4 expression in the differentiation of human ductular hepatocytes

Ductular structures are suggested to be bipotential progenitor cells that may differentiate into hepatocytes or biliary epithelial cells (BEC). To better understand the differentiation process, we studied the expression of hepatocyte nuclear factor (HNF) in ductular structures. Matured hepatocytes in normal liver expressed HNF-1, HNF-4alpha, HNF-3beta, and C/EBPalpha in the nucleus. Normal BEC expressed HNF-1 but did not express HNF-4alpha, suggesting an important role of HNF-4alpha in maintaining the phenotype of matured hepatocytes. Ductular structures were classified into ductular cells and ductular hepatocytes. Ductular cells showed glandular or bile duct-like appearance and strongly expressed cytokeratin-7. Ductular hepatocytes showed features between BEC and hepatocytes and heterogeneously expressed cytokeratin-7. Both ductular cells and ductular hepatocytes expressed HNF-4alpha, but the nuclear localization of HNF-4alpha was more prominent in ductular hepatocytes. The expression of HNF-4alpha mRNA in ductular hepatocytes was demonstrated at the single cell level by laser capture microdissection. Regenerative hepatocytes strongly expressed all HNFs in the nucleus, whereas residual hepatocytes in massive necrosis showed low or cytoplasmic expression. These results suggest that HNF-4alpha plays an important role in the differentiation and maintenance of the matured hepatocyte phenotype and that nuclear localization of HNFs is implicated in the accomplishment of their function.     

Stability of minisatellite loci in the genome of cultured hela cells

The stability of minisatellite loci representing DNA with the tandem organization of repeats is studied. DNA profiles of 194 HeLa clones are identical to the parent culture. They exhibit 36 bands detectable by the minisatellite probe Red4. Since the method ensures the record of changes in minisatellite loci appearing during the first two divisions of parent cell, mutations are assessed in 27,936 locus tests. Hence, the incidence of mutations in the culture is not higher than 3.5×10⁻⁷ per locus/per mitosis, providing that the number of generations in parental homogenous culture is taken into account. Translated fromByulleten' Eksperimetal'noi Biologii i Meditsiny, Vol. 122, No. 9, pp. 311–313, September, 1996     

Cytogenetic properties of spleen lymphoid cells in mice after bilateral nephrectomy

It is shown that mouse spleen lymphoid cells isolated 4 h after bilateral nephrectomy enhance mitotic activity of renal tubular epithelium in syngeneic recipients, but have no effect on hepatocytes and little effect on the proliferation of reticuloendothelial cells in these recipients. The cells isolated at the same times postoperation from sham-operated or unilaterally nephrectomized animals are ineffective. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 7, pp. 109–110, July, 1997     

Induction of prolactin receptors in cholangiocytes of male and female rats after ligation of the common bile duct

Intense expression of prolactin receptors in cholangiocyte nuclei, nuclear membranes, and cytoplasm was demonstrated by the indirect immunoperoxidase method in intact and gonadectomied male and female rats after ligation of the common bile duct. The intensity of staining increased during cholangiocyte proliferative response to the intervention and, in contrast to hepatocytes, did not depend on animal sex and the presence of the gonads. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 1, pp. 66–70, January, 1998     

Primary cell cultures isolated from Penaeus monodon prawns

We have devised a cell culture system for Penaeus monodon prawn cells that uses a defined synthetic medium. Organs were removed from adult prawns ranging in size from 13--19cm rostrum-to-telson length. Cultures consisted of either a blend of hematopoietic and lymphoid cells or ovarian cells. The cells divide rapidly in culture, doubling on average once per week for 5 to 6 weeks. These cultures continue to survive for at least 5 months but the rates of cell division are low after the first 5--6 weeks. In the literature, unicellular eukaryotic marine organisms such as chytrids may contaminate marine cell cultures. In some cases these eukaryotic contaminants may be difficult to distinguish from prawn cells unless detailed ultrastructure or characteristic developmental stages such as zoospores can be observed. Alternatively, we prepared molecular probes from repeated DNA sequences 100--400 bp in length in the P. monodon genome. These species-specific probes were hybridised to genomic DNA from cell culture to confirm proliferation of P. monodon cells in our cultures.