Infection of murine hepatocyte cultures by herpes simplex virus (HSV) 1 and 2

A study was undertaken of the interaction between liver cells and Herpes Simplex Virus (HSV) in vitro. Hepatocytes were obtained from HSV-resistant (C57/B16) and from HSV-susceptible (BALB/c, A/J, C3H) mouse strains and cultured according to standard methods. Each culture was infected with several strains of HSV-type 1 or of HSV-type 2, respectively. The multiplicity of infection was 5. The cytopathic effect was evaluated by light- and electron-microscopy. The number of infectious particles was determined using rabbit kidney or Vero cell cultures. All evaluations were made at different time intervals after infection. No difference concerning the replication rate of HSV-1 and 2 in isolated h…

Monoklonale Gammopathie bei HIV-Infektion

A lambda-light chain-IgA plasmocytoma, accompanied by a changing clinical picture of fever, nocturnal perspirations and weight loss, developed in a 46-year-old homosexual male with AIDS, stage IV (classification according to the Centers for Disease Control). He had been suffering from recurrent Salmonella septicaemia. Serum protein electrophoresis demonstrated marked elevation of the beta- and gamma-fractions (44% and 24%, respectively). There were 15% plasma cells in the differential blood count and in the bone marrow smear. Immunoelectrophoresis demonstrated free lambda-light chains. The IgA concentration in cerebrospinal fluid was raised to 202 mg/l, and there was an IgA paraproteinaemia…

Abstracts from the sixth meeting of the international association of pancreatology, November 2–4, 1994, Chicago, IL

Expression of the gene of the alpha-smooth muscle-actin isoform in rat liver and in rat fat-storing (ITO) cells.

Fat storing cells (FSCs) in the liver represent the main site of vitamin A deposition in the body. These cells are considered to play an important role during scar formation and fibrogenesis in the liver. The putative descent of FSCs from the fibroblastic or from the myofibroblastic system have not been determined yet by morphological or immunohistochemical studies. To further define the origin of these liver cells, we analysed the pattern of expression of three structural proteins: vimentin, desmin and the α-smooth muscle (SM)-actin isoform in FSCs of the rat liver, in smooth muscle cells (SMCs) from the aorta and in rat skin fibroblasts. FSCs were studied by immunohistochemical methods im…

Sinusoidal endothelial liver cells in vitro release endothelin — Augmentation by transforming growth factorβ and Kupffer cell-conditioned media

Endothelin is the most potent vasoconstrictor peptide known today. Using a radioimmunoassay for endothelin, we measured immunoreactive endothelin in culture media of guinea pig sinusoidal endothelial liver cells and human umbilical vein endothelial cells. A time-dependent release of immunoreactive endothelin by confluent sinusoidal endothelial liver cells in culture was found. Sinusoidal endothelial liver cells produced similar amounts of immunoreactive endothelin as umbilical vein endothelial cells, about 900 pg/microgram DNA per 24 h. In the presence of transforming growth factor beta a dose-dependent increase of immunoreactive endothelin release was measured. The maximal increase of 50% …

Cultured Ito cells of rat liver express the alpha 2-macroglobulin gene.

Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize a2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized a2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis and fluorography. a2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5 - 11 days after the isolation of the cells, increasing amounts of a2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled a2-macroglob…

Human immunodeficiency virus infection mimics autoimmune hepatitis--a case report.

The stellate cell (Ito-cell, fat-storing cell, lipocyte, perisinusoidal cell) of the liver. New insights into pathophysiology of an intriguing cell.

Ultrastructural localization of interferon-producing cells in the livers of patients with chronic hepatitis B

Cells expressing alpha- and gamma-interferon were localized in the liver tissue of patients with chronic hepatitis B by means of light and electron microscopy using monoclonal antibodies. Interferon-positive cells were regularly seen in the infiltrating mononuclear cells, and the number showed a good correlation with the degree of the necroinflammatory activity of the disease. In chronic persistent hepatitis and in normal livers, they were infrequent or virtually absent. alpha-Interferon was shown to be positive in lymphocytes, polymorphonuclear leukocytes and fibroblasts, Kupffer cells and, weakly, in the cytoplasm of a few hepatocytes in cases of active hepatitis, whereas gamma-interferon…

Fat storing cells (FSC) of rat liver synthesize and secrete fibronectin

Summary Fat storing cells (FSC) of rat liver were isolated and kept in culture for up to 2 weeks. Freshly isolated cells and cells in culture were characterized functionally (vitamin A content) and morphologically. Their synthetic capacity for fibronectin was studied quantitatively by ELISA-measurement and qualitatively by biosynthetic labeling, immunoprecipitation and SDS-PAGE analysis. The synthesis product and the kinetics of the synthesis and secretion were compared with those obtained from culture of hepatocytes. FSC were shown to synthesize and secrete fibronectin under the culture conditions used. No fibronectin synthesis was detected during the first 3 days of culture. Thereafter th…

Serum hyaluronate and type III procollagen aminoterminal propeptide concentration in chronic liver disease. Relationship to cirrhosis and disease activity.

. To analyse the relationship between the presence of liver cirrhosis and hepatic inflammation and the serum concentrations of the aminoterminal propeptide of procollagen type III (P-III-NP) and of hyaluronic acid (HA) in chronic liver disease, we measured P-III-NP and HA concentrations in paired serum samples from 133 patients with various chronic liver diseases, from 22 patients with acute hepatitis and from 50 healthy age-matched controls. In 24 (of the 133) patients with autoimmune chronic liver disease, follow-up determination was performed during therapeutic treatment with immunosuppressive drugs. Compared with controls P-III-NP concentrations (medians) were significantly elevated in …

Coexpression of extracellular matrix glycoproteins undulin and tenascin in human autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common entity of cystic diseases of the kidney leading to end-stage renal insufficiency. Changes in extracellular matrix (ECM) are regarded to be an important pathogenic factor connected with the genes assumed to be responsible for human ADPKD. In order to assess the biological significance of altered expression and deposition of ECM glycoproteins for human ADPKD at molecular levels fresh-frozen tissue from ADPKD kidneys, fetal kidneys and normal adult kidneys were comparatively tested by immunohistochemistry for the presence of multifunctional ECM glycoproteins undulin, tenascin and fibronectin, interstitial collagen types I,…

Fat-storing cells of the rat liver synthesize and secrete C1-esterase inhibitor; modulation by cytokines.

During liver fibrogenesis, fat-storing cells transform into myofibroblast-like cells and produce increasing amounts of extracellular matrix proteins. Because fat-storing cells produce α2-macroglobulin, an important serine protease inhibitor (serpin), we investigated whether fat-storing cells also synthesize C1-esterase inhibitor, another important serpin. C1-esterase inhibitor synthesis was studied in rat fatstoring cells at day 0, 3 and 7 after isolation by biosynthetic labeling, immunoprecipitation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Messenger RNA was examined by Northern-blot analysis. C1-esterase inhibitor gene expression and synthesis were detectable in fresh…

The gene of hepatocyte growth factor is expressed in fat-storing cells of rat liver and is downregulated during cell growth and by transforming growth factor-β

Hepatocyte growth factor (HGF) has been detected in non-parenchymal cells but not in hepatocytes. We performed Northern blot analysis of total RNA extracted from rat hepatocytes, Kupffer cells, endothelial cells and fat-storing (Ito-) cells. Total RNA was extracted from fat-storing cells at different times after isolation and from cells treated with different amounts of transforming growth factor beta. The RNA was hybridized with HGF, fibronectin-, and alpha-actin-specific cDNA probes, consecutively. We found an abundant amount of HGF mRNA in freshly isolated fat-storing cells, but not in other liver cells. The amount of the HGF transcripts decreases significantly in FSC during the time of …

Hepatic transferrin receptors in hereditary hemochromatosis.

Localization and mRNA steady-state level of cellular fibronectin in rat liver undergoing a CCl4-induced acute damage or fibrosis.

Abstract In an attempt to investigate cellular fibronectin synthesis and deposition during acute liver damage and fibrogenesis, we used the presence of the additional type III-related ED-A domain of cellular fibronectin as a characteristic for distinguishing it from the plasma form. Using site-specific antibodies, we localized cellular fibronectin deposition in the necrotic pericentral areas of acutely damaged liver tissue after a single CCl 4 -gavage, whereas in control liver only trace amounts of cellular fibronectin were detectable along the sinusoids. Upon several CCl 4 -administrations leading to liver fibrosis, cellular fibronectin deposits were accumulated in the fibrotic septa. Nort…

Rat hepatocytes in primary culture synthesize and secrete cellular fibronectin

Fibronectins, involved in cell-matrix interactions and cell attachment, are glycoproteins which show a remarkable heterogeneity, due to alternative splicing. The type III-related domains, ED-A and ED-B, are present in cellular fibronectin in a variety of ratios whereas they are absent in circulating plasma fibronectin. Fibronectin synthesis by hepatocytes which are accepted as suppliers of plasma fibronectin was studied in primary cultures during a 6-day culture period. Using site-specific antibodies we demonstrate that rat hepatocytes are also able to synthesize and secrete fibronectin bearing the ED-A domain from Day 3 on after inoculation. By immunocytological characterization of the hep…

Liver membrane autoantibodies in chronic active hepatitis

Summary Target antigens relevant for immune reactions in inflammatory liver diseases should be expressed on the hepatocellular membrane. Using mechanically or enzymatically isolated rabbit hepatocytes, we evaluated the influence of cell integrity on the detection of membrane-expressed antigens by sera from patients with chronic hepatitis and by murine monoclonal antibodies. Our results provide evidence that target antigens of liver membrane autoantibodies (LMA) as well as liver kidney microsomal antibodies (LKM) are not expressed on the hepatocellular membrane of viable and intact isolated rabbit hepatocytes. However, LMA were detected in the sera of 56% of patients with autoimmune chronic …

Chronifizierte paranoid-halluzinatorische Psychose als Erstmanifestation einer HIV-Infektion?

In a 36-year-old patient an acute onset of psychosis occurred, probably due to HIV infection. For one year HIV-infection with reduced T4/T8 ratio had been known without clinical manifestation (stage IV B of the CDC-classification). He developed chronic delusional hallucinations, which persisted for more than one year in spite of adequate psychoactive drug therapy. So far AIDS-related dementia has not become evident. Focal lesions caused by opportunistic infections or tumour were excluded by computed tomography and magnetic resonance imaging. The latter revealed several small lesions and the brain scan showed a nonhomogeneous pattern of cerebral blood flow. CSF-examination disclosed a mild l…

Liver cell damage caused by monoclonal antibody against an organ-specific membrane antigen in vivo and in vitro

Summary Monoclonal antibodies have been raised against different antigenic determinants of normal rabbit hepatocytes. One antibody (2D3) recognized a liver-specific 43 kDa protein displayed exclusively on the basolateral portion of the hepatocellular membrane. Purified monoclonal antibodies were injected intravenously into rabits. Following the injection of antibody 2D3, a dose-dependent increase of liver enzyme activities in sera was observed. Within 8 h, marked morphological alterations of the hepatocytes, including multiple cell necroses, could be demonstrated by light and electron microscopy. When isolated vital rabbit hepatocytes in culture were used as targets, cytotoxic effects of th…

Biosynthesis of Lipopolysaccharide-Binding Protein in Rabbit Hepatocytes

Studies reported here show that the recently discovered acute-phase protein, lipopolysaccharide-binding protein (LBP), is synthesized by hepatocytes. For these studies, explanted rabbit hepatocytes were grown in the presence of 35S-methionine. Biosynthetically labelled LBP in the cells and supernatant was identified using immunoprecipitation with rat anti-rabbit LBP antibody. This antibody immunoprecipitates both the LBP polypeptide and the glycosylated protein. With a cell-free translation system a comparison of RNA from normal rabbit liver with that isolated from acute-phase rabbit liver indicated that a translatable LBP message is only found in the RNA from acute-phase liver. Studies wit…

Differential expression of collagen types I, III, and IV by fat-storing (Ito) cells in vitro

It has been observed that Ito cells in vitro undergo phenotypical changes ("activation") similar to those noted in vivo during the development of liver fibrosis. Because conflicting data have been published on the amount and different types of collagens synthesized by Ito cells in vitro, collagen biosynthesis was studied at different "activation" stages on both the protein and RNA levels. Immunoprecipitation of endogenously labeled collagen showed that freshly isolated ("resting") Ito cells synthesize mainly collagen type IV. Collagen type I was hardly detectable in the earlier stage of primary culture, but it clearly increased starting 5 days after isolation. Compared with the basal rates …

Binding properties of mechanically and enzymatically isolated hepatocytes for IgG and C3.

— The presence of Fc and C3 receptors was studied on mechanically and enzymatically isolated rabbit, mouse and rat hepatocytes as well as on hepatocytes derived from primary cultures. The same cell preparations were used for studying the uptake of an antibody against nuclear antigens. Mechanically isolated hepatocytes were able to bind AIgG and activate complement. However, the same cells were not able to form rosettes with EA or with EAC. Enzymatically isolated hepatocytes did not bind AIgG or activate complement nor did they form rosettes with EA or with EAC. The mechanically isolated cells and the nonviable fraction of the enzymatically isolated cells showed a nuclear fluorescence when i…

Tenascin gene expression in rat liver and in rat liver cells. In vivo and in vitro studies.

Tenascin is a major glycoprotein constituent of the extracellular matrix with a strong affinity to fibronectin; its distribution is believed to be temporarily and spatially limited. Tenascin gene expression is increased during wound healing processes. As repair mechanisms in chronic liver diseases resemble wound healing we studied tenascin gene expression in rat liver and in isolated rat liver cells. In normal rat liver a tenascin specific antiserum stains sinusoidal cells with fiber-like prolongations, which at the same time are desmin-positive (ITO-cells). In the CCl4-acutely-damaged liver a strong tenascin staining is detected in cells located among the mononuclear cells of the inflammat…

Alternative splicing products of the tenascin gene distinguish rat liver fat storing cells from arterial smooth muscle cells and skin fibroblasts

Abstract Fat storing-(Ito-)cells (FSC) transform into a myofibroblast-like cell type during liver fibrogenesis. A similar development can be observed in cell culture. At the moment, a definite marker to differentiate transformed FSC from smooth muscle cells (SMC) is not available. We recently found that FSC, SMC and skin fibroblasts (SF) synthesize tenascin, a novel matrix protein. As it is reported that various tissues express different tenascin forms by the mechanism of alternative pre-mRNA splicing, we analyzed the tenascin transcripts in these cell types. Total RNA extracted from cultured FSC, SMC and SF, analyzed by Northern blot hybridization, showed a 7.2 kb transcript in FSC, a 8.7 …

Sinusoidal endothelial cells from guinea pig liver synthesize and secrete cellular fibronectin in vitro.

Endothelial liver cells were obtained from guinea pig by enzymatic digestion and centrifugal elutriation. Cells were cultured on gelatin and fibronectin pretreated culture vessels. Endothelial cells were characterized by phase-contrast microscopy, electron microscopy and the presence of Factor VIII-related antigen. Fibronectin secretion was determined in cell-free supernatants by a sensitive and specific ELISA and localized on fixed cultured cells by immunofluorescence. [35S]Methionine endogeneously labeled fibronectin was immunoprecipitated from supernatants and cellular lysates and displayed on sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. After attachment to the culture…

Prostanoid release of cultured liver sinusoidal endothelial cells in response to endotoxin and tumor necrosis factor

Abstract Vascular endothelial cells release prostanoids, especially prostacyclin, when properly stimulated. In addition to short acting stimuli like thrombin and histamine an increased prostanoid release occurs in the presence of endotoxin, interleukin 1 or tumor necrosis factor (TNF). The response of sinusoidal endothelial liver cells to such stimuli — probably important in hepatic inflammatory disease — is unknown. Sinusoidal endothelial liver cells from the guinea pig were isolated by centrifugal elutriation and investigated as confluent monolayers. Their prostanoid release in response to endotoxin and human recombinant TNF was determined by radioimmunoassays and compared to that obtaine…

Guinea pig Kupffer cells can be activated in vitro to an enhanced superoxide response

Summary In the preceding paper it was shown that Kupffer cells isolated by digestion of the liver and purified by centrifugal elutriation can be activated in vitro by lipopolysaccharide and muramyl dipeptide to an enhanced superoxide response upon zymosan phagocytosis. Lipopolysaccharide and muramyl dipeptide also led to a strongly increased prostaglandin E 2 release during the phagocytosis of zymosan. This activation was accompanied by an increased production of prostaglandin E 2 during the incubation with the stimuli. Prostaglandin E 2 synthesis was inhibited by the cyclooxygenase inhibitor indomethacin, reduced by dexamethasone, but only slightly decreased by the lipoxygenase inhibitor n…

Regulation of cytochrome P450 IID by acute phase mediators in C3H/HeJ mice.

Abstract Cytochrome P450 IID6 is a drug metabolizing enzyme and the major target antigen in LKM-1 antibody positive chronic active hepatitis. The histological hallmark of chronic active hepatitis is a lymphocytic infiltrate in the liver. It is unknown whether and how cytokines produced and secreted by these tissue infiltrating mononuclear cells regulate the cellular expression of cytochrome P450 IID6. To study the effect of interleukin 1, tumor necrosis factor and interleukin 6 on the hepatocellular RNA expression of cytochrome P450 IID, we injected each of the cytokines in C3H HeJ mice. We found a time-dependent suppression of the cytochrome in the liver. Six hours after the intraperitonea…

Electron microscopic observations on primary hepatocyte cultures infected with herpes simplex virus Types I and II

The replication cycle of the Herpes simplex virus (HSV) strains I and II has so far been described mainly in established proliferative cell cultures. Most of the biochemical data and ultrastructural cell changes regarding the virus-cell interaction have been obtained from ‘permissive’ cells which allow almost unrestricted viral multiplication. It seems obvious, however, that the in vivo viral infections are not represented adequately by these experiments. In order to achieve a more realistic view of the ultrastructural events during HSV infection of adult tissue, cell cultures were prepared from adult mouse and rat livers and infected with several HSV strains. Established ‘permissive’ cell …

Treatment of Patients with Chronic Type B Hepatitis and Concurrent Human Immunodeficiency Virus Infection with a Combination of Interferon Alpha and Azidothymidine: A Pilot Study

Six patients with chronic type B hepatitis and concurrent infection with the immunodeficiency virus were treated with 600 mg azidothymidine (AZT)/day and 3 X 10(6) units of interferon-alpha (IFN-alpha) every other day for a total of 4 months. None of the patients treated lost the hepatitis B virus (HBV). HBV-DNA concentrations were not significantly influenced by this treatment. Human immunodeficiency virus (HIV) infection was also not affected except for a transient rise in CD 4-positive cells in 2 individuals, who had initially low CD 4-positive cells. Treatment did not influence the presence of HIV-Ag in the serum. In conclusion, a combination therapy of IFN and AZT does not seem to be b…

Functional spectrum of sinusoidal endothelial liver cells

Lymphocytes from hepatic inflammatory infiltrate kill rat hepatocytes in primary culture

In the last few years it has become possible in the liver to isolate lymphocytes from inflammatory infiltrates and to culture them in vitro. Most of the lymphocyte clones obtained are CD 8 + cytotoxic cells, but interactions between these lymphocytes and hepatocytes in primary culture have not been analysed previously. In this study, cloned human T lymphocytes from liver biopsies and from the peripheral blood of patients with chronic hepatitis B or primary biliary cirrhosis, after phenotypical and functional characterization into CD 8+ or CD 4+ cytotoxic lymphocytes, were activated in an antigen-independent fashion by adding either anti CD 3 or anti CD 2/R-3 monoclonal antibodies to the cel…

Demonstration of antibodies to the surface (anti-p41) and core proteins (anti-p24) of the human immunodeficiency virus (HIV) in individuals positive for anti-HIV.

Diagnosis of infection with the human immunodeficiency virus (HIV) relies on the demonstration of antibody to this virus. Occasionally, the combined analysis of sera using ELISA and western blot reveals false-positive results. We have compared a newly developed test to detect antibodies to the core (anti-p24) and surface (anti-p41) proteins of HIV with the established tests described above. Anti-p24 and anti-p41 were negative in three individuals positive for anti-HIV by ELISA and immunoblot; they had a low risk to acquire HIV infection and were clinically and immunologically normal and suspected false positive previously. In 62 individuals at risk, anti-p41 was always positive while anti-p…

Expression of Ia-antigens on guinea pig Kupffer cells

Summary The expression of the Ia-antigen on guinea pig Kupffer cells was studied employing two monoclonal antibodies against two different determinants of the Ia-molecule. The study was performed in situ on liver sections and on isolated highly purified Kupffer cells kept in culture up to 6 days. The influence of guinea pig hepatocyte culture supernatant and of supernatants of phytohemoagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) on the Ia expression was measured. Immunofluorescence staining of cryostat sections revealed that the monoclonal antibodies used are able to detect Ia-antigens on liver macrophages in situ. The in vitro studies strongly suggest that all Kupf…

Alpha- and gamma-interferon (IFNα, IFNγ) but not interleukin-1 (IL-1) modulate synthesis and secretion of β2-microglobulin by hepatocytes

Soluble serum beta 2-microglobulin has been thought to result from membrane shedding by activated T-lymphocytes. This hypothesis could explain the increase of beta 2-microglobulin serum levels during virally induced mononucleosis, but not elevated levels as observed in other virally induced and in malignant diseases. In this paper we demonstrate that beta 2-microglobulin is a true secretory protein, and that its synthesis in hepatocytes is modulated by IFNs but not by IL-1. While the 45,000 MW HLA antigen can be found only in cell lysates, beta 2-microglobulin is shown to be secreted also into the culture medium like other secretory proteins (e.g. albumin-factor B-complement C3). Furthermor…