Modifications in integrin expression and extracellular matrix composition in children with biliary atresia.

BACKGROUND: The aetiology of biliary atresia (BA) is still unresolved. The study's aim was to investigate the distribution of extracellular matrix proteins and cellular adhesion molecules in children with BA compared to other cholestatic liver disease (CLD) and normal liver architecture (NLA). PATIENTS: Liver biopsies were obtained from children with BA (n=13), CLD (n=6) and NLA (n=8). METHOD: We systematically analysed ultra thin frozen sections from the liver hilum stained with 25 monoclonal antibodies for cellular characterisation, extracellular matrix proteins and adhesion molecules. RESULTS: 2 changes were specifically found in BA: laminin beta1 was reduced in children with BA vs. NLA and CLD. Conversely, integrin alpha 3 was increased in BA vs. NLA and CLD (p<0.05). Furthermore, we detected changes in a similar pattern for both BA and CLD vs. NLA: in BA and CLD perlecan was increased. On the contrary, integrin beta1 and entactin were decreased vs. NLA (p<0.05). DISCUSSION: Extracellular matrix proteins and adhesion molecules mediate cellular polarity and integrity, development of tubular structures, and proliferation. Therefore, our findings can be important for the understanding of the genesis of BA. CONCLUSION: The composition of extracellular matrix proteins and adhesion molecules in children with BA differs from NLA and other CLD in distribution of laminin beta1 and integrin alpha 3, which may have implications for genetic, immunologic and environmental associations in BA.

Assessment of biochemical liver markers, physical activity, fitness and body mass index for a cardiometabolic risk model in childhood.

AIM: The aim of this study was to investigate clustered cardiometabolic risk scores in healthy 10- to 12-year-olds using anthropometric characteristics, measurements of cardiorespiratory fitness (CRF) and physical activity and blood markers of metabolic disease. We also evaluated how including markers of liver cell injury would affect the clustered cardiometabolic risk assessment model. METHODS: This cross-sectional study focused on 99 children aged 10-12 years. The main outcome included assessing participants with increased and low cardiometabolic risk factors using a clustered risk score model that incorporated markers implicated in metabolic syndrome pathogenesis. Two clustered risk scores were calculated, one incorporating markers of liver cell injury. RESULTS: Children classified as 'increased risk' exhibited significantly lower CRF and higher body mass index Z-scores than their 'low-risk' peers. No significant differences in physical activity were observed. This trend remained unchanged when markers of liver injury were included in the clustered risk assessment model. CONCLUSION: The clustered risk score model is a scientifically robust method of cardiometabolic risk assessment, which reiterates the importance of weight reduction and CRF promotion in childhood. Our study did not show a significant contribution of liver injury markers, and further research is needed to evaluate their effect on cardiometabolic risk stratification in childhood.

Disseminated adenovirus infection with respiratory failure in pediatric liver transplant recipients: impact of intravenous cidofovir and inhaled nitric oxide.

Adenoviruses (AdV) are opportunistic pathogens that can lead to severe infections and respiratory failure (acute respiratory distress syndrome, ARDS) with high mortality in immunosuppressed patients. Cidofovir (CDV) has been used in adenoviral disease in bone marrow transplant recipients. Two pediatric liver transplant recipients with disseminated adenoviral disease and ARDS were treated with reduction of immunosuppression, CDV, and inhaled nitric oxide (iNO). CDV 1 mg/kg was given three times per week intravenously with intravenous hydration and oral probenecid. Viral suppression and clinical improvement was achieved. AdV hepatitis did not occur, and graft function was preserved, although acute rejection occurred in both patients. Adverse effects were mild and transient not requiring dose modification. Severe hypoxemia was reversed with iNO 10-20 p.p.m. CDV treatment of AdV infections in organ transplant recipients and other immunocompromised patients should be further investigated in prospective studies.

[Lectin-reactive alpha-fetoprotein in tyrosinaemia type I]. / Lektin-reaktives Alpha-Fetoprotein bei Patienten mit Tyrosinämie Typ I.

UNLABELLED: Despite the introduction of NTBC into the treatment of tyrosinaemia type I (TT1) and a considerable improvement in the outcome of these patients, the principal risk of developing hepatocellular carcinoma (HCC) in this metabolic disorder remains mainly in those children with late introduction of NBTC after the second year of life. Serial total alpha-Fetoprotein (AFP) levels are used to evaluate the individual risk to develop malignant changes. A failure of AFP to decrease on adaequate treatment or a secondary increase after a period of falling levels have been an indication for liver transplantation. Lectin-reactive alpha-Fetoprotein is a recently described marker to distinguish hepatocellular carcinoma from benign liver disease in adult cirrhotic patients. AIMS: To investigate if the analysis for Lectin-reactive alpha-Fetoprotein would lead to earlier detection of HCC compared to a judgement based on the evolution of standard total AFP alone. PATIENTS: We report the analysis of 12 patients with TTI and histologically proven HCC. There of 5 were diagnosed under one year of age, but NTBC treatment was started between 2 years 3 month and 7 years of age except in one case in which NTBC was introduced when the diagnosis of TTI was made. The remainder of the patients cover up to the age of 15 years. All patients had been treated with NTBC. METHODS: Lectin containing agarose gel for AFP electrophoresis leads to AFP separation according to different affinities of the varying carbohydrate chains of AFP to lectins. RESULTS: AFP subfractions could be identified in all 12 patients. In 6 patients the L3-AFP rose before the total AFP. In 3 patients the rise in L3-AFP was consistent with the rise of the total AFP and in 3 patients the L3-AFP was raised after the total AFP or did not increase at all. DISCUSSION: We were able to identify 6 out of 12 patients who had an early increase of L3-AFP before they developed a change in total AFP levels. The clinical significance of these early changes need to be determined. Lectin-affinity electrophoresis may have a potential role as an additional tool that may help to discriminate benign liver disease from HCC in TTI. CONCLUSIONS: We suggest the further evaluation of lectin-reactive AFP in TTI.

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells.

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell-endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 microm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 microm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy.

Ductular morphogenesis and functional polarization of normal human biliary epithelial cells in three-dimensional culture.

BACKGROUND/AIMS: The understanding of the physiology and function of human biliary epithelial cells (hBEC) has been improved by studies in monolayer culture systems. The aim was to develop a polarized model to elucidate the mechanisms of ductular morphogenesis and functional differentiation of hBEC. METHODS: The morphological, phenotypic and functional properties of hBEC cultured as three-dimensional aggregates in collagen gel were assessed in medium supplemented with (or without) human hepatocyte growth factor (hHGF) and foetal bovine serum. RESULTS: In the absence of added mitogens and serum, cells maintained as morphologically polarized aggregates, organized around a central lumen, were positive for phenotypic markers of biliary epithelium and negative for markers of other cell types. Functional markers, gamma-glutamyl-transferase, anion exchanger-2, responses to gamma interferon and forskolin induced secretion, were preserved. hHGF increased both the size and number of aggregates and induced hBEC to invade the gel and lumena forming anastomosing networks of cells. CONCLUSIONS: Collagen gel culture in the absence of added growth factors and serum provides a model for analysis of the polarized functions of hBEC. The formation of poorly organized cords of cells in response to hHGF suggests that collagen gel culture may provide a model for the investigation of atypical ductular morphogenesis of the human biliary tract.

Morphogenesis of primary human biliary epithelial cells: induction in high-density culture or by coculture with autologous human hepatocytes.

Although the control of biliary ductular morphogenesis has received some attention particularly using isolated rat biliary epithelial cell models, the regulation of human bile duct formation is not well defined. In the present study, using a 3-dimensional culture model comprising primary human biliary epithelial cells (BECs) and coculture with primary human hepatocytes, we have sought to define the factors involved. We have shown that primary human BECs can be expanded on collagen gels in the absence of growth factors or serum. When plated in high density in double collagen gels, BECs established 3-dimensional structures that subsequently developed into well differentiated polarized luminal ducts. This morphogenic response occurred in the absence of hepatocyte growth factor (HGF) and epidermal growth factor. Strikingly, the addition of growth factors (in the presence of serum) resulted in loss of polarity although the cells retained growth responses to both factors. Coculture of BECs with autologous human hepatocytes enhanced the ability of low-density BECs to undergo ductulogenesis. This effect was mimicked by addition of conditioned medium from previous hepatocyte-BEC cocultures. These findings indicate that for human biliary ductular morphogenesis, epithelial cell-cell interactions are required but that mesenchymally derived factors such as HGF may not be important.

Maintained function of primary human hepatocytes by cellular interactions in coculture: implications for liver support systems.

The application of primary hepatocytes in hybrid artificial liver systems has been hampered by the gradual loss of differentiated morphology and function in vitro. Therefore, we have established a coculture model of autologous human hepatocytes and biliary epithelial cells (BEC) in collagen gel in the presence of hepatotrophic growth factors. Furthermore, we examined the effect of hepatocyte cell perfusion in a women multicompartment capillary membrane system. Normal hepatocytes isolated from human liver produced albumin for more than 2 weeks in serum-free media, and were further stimulated by conditioned medium. When cocultured with BEC, albumin secretion was greatly enhanced, suggesting that cellular interactions promote tissue-specific differentiation. When perfused in bioreactors, albumin levels were maintained at steady state for longer than 2 weeks. These data indicate that differentiation of primary hum hepatocytes can be maintained by coculture interactions and three-dimensional hybrid organ devices, providing appropriate growth factors and matrix for tissue regeneration.

Dedifferentiation of human hepatocytes by extracellular matrix proteins in vitro: quantitative and qualitative investigation of cytokeratin 7, 8, 18, 19 and vimentin filaments.

BACKGROUND/AIMS: Liver cirrhosis and carcinogenesis are accompanied by an alteration in extracellular matrix material. Histological studies reveal upregulation of the intermediate filaments cytokeratins 8 and 18 and de novo synthesis of vimentin, and cytokeratin 7 or 19 in hepatocytes. The aim of this study was to investigate how these two processes are linked. METHODS: Human hepatocytes were seeded: (i) on the matrix components collagen I, IV, laminin, or fibronectin; (ii) on stoichiometrically different complete matrices, derived from human placenta (matrix I) or the Englebreth-Holm-Swarm tumor (matrix II), and (iii) inside a three-dimensional collagen I sandwich. Filament expression and assembly were measured by cytofluor analysis or confocal laserscan microscopy. RESULTS: The matrix components or complete matrices triggered enhancement of cytokeratins 8 and 18 and de novo synthesis of cytokeratins 7, 19 and vimentin in a characteristic way. Confocal images demonstrated a dense and uniform network of cytokeratin 18 in freshly isolated cells, which was "replaced" by a few, thick protein bundles within 20 days. Interestingly, newly synthesized cytokeratin 19 structurally resembled the cytokeratin 19 organization in biliary epithelial cells. Marked cytokeratin alterations could be partially prevented when hepatocytes were grown in a three-dimensional collagen sandwich. CONCLUSIONS: Pathological alterations to the chemical composition, molecular structure, or spatial arrangement of the liver matrix lead to specific changes in the intermediate filament pattern in human hepatocytes. We assume that degradation of the matrix results in pathological alterations to the hepatocyte-receptor matrix-ligand ratio, followed by a switch from physiological to pathological cell-activation.

Historical development of surgical instruments exemplified by hemostatic forceps.

We describe the historical development of surgical instruments exemplified by hemostatic forceps, starting with antique and medieval forceps used for the arrest of bleeding to modern atraumatic hemostatic forceps used for vascular reconstruction. Their development proceeded mainly in three steps: (1) development of ligature forceps directly for hemostasis (Celsus, first century AD; Paré, 1582; L. Heister, 1743; (2) development of atraumatic forceps, which facilitate vascular reconstruction or anastomosis creation by temporarily clamping vessels (Höpfner, 1903; Stich, 1907; Jeger, 1913; (3) construction of tangential forceps, which, by partially clamping vessels with partial maintenance of blood flow, allow the reconstruction or anastomosis of large vessels in a side-to-side technique. The first tangential occlusion clamp was developed by Friedrich Trendelenburg (1844-1924) in 1908, when he established the operative treatment for embolism of the pulmonary artery. This clamp was later modified by A. W. Meyer (1927) and V.P. Satinsky (ca. 1950).

[Use of normal human hepatocytes in a hybrid organ system]. / Einsatz von normalen humanen Hepatozyten in einem hybriden Organsystem.

Using the bioreactor model developed by J. Gerlach, we examined the potential of normal human hepatocytes for application in bioartificial liver devices. From normal human donor livers 1.5 x 10(8) hepatocytes were isolated. Hepatocytes were perfused in a woven multi-compartment capillary system in serum-free culture medium containing ammoniachloride over a period of 2 weeks. These cells demonstrated a well differentiated ultrastructure with formation of junctional complexes and bile canaliculi between adjacent cells. During reactor run, a constant albumin synthesis with levels above 11 mg/ml and maintenance of urea production and lignocaine metabolism (MEGX-test) were detected. These initial results indicate that normal human hepatocytes express typical morphology and ultrastructure and are able to keep differentiated functions in suitable perfusion models. Combination of the distinct human liver cell populations might enable promotion of further specific functions (clotting factors) and induction of liver cell proliferation.

The immunological role of biliary epithelial cells in human liver transplant rejection.

From histopathological analyses after liver transplantation it is evident that the biliary epithilium is an important target for leucocytes of the graft recipient. Besides clinical and histopathological investigations undertaken by several authors it was also endeavoured to determine the immunological impact of the biliary epithelial cells (BEC) in vitro. As for the intrahepatic BEC, in vitro studies proved to be restricted owing to difficult isolation procedures and the limited number of cells yielded from transplanted organs. Therefore, studies on cultured extrahepatic BEC served as a model for the immunological features of the biliary epithelium in transplantation. Herein, in vivo and in vitro studies dealing with BEC and immunologically mediated hepatic disorders are reviewed in order to understand better the pathogenesis after liver transplantation. Furthermore, possible underlying mechanisms of BEC-directed immunity with regard to BEC-leucocyte interactions are discussed.

Establishment and immunological characterization of cultured human gallbladder epithelial cells.

Biliary epithelial cells are a primary site of damage in liver allograft rejection and in immunologically mediated diseases such as primary biliary cirrhosis. Human leukocyte antigens and adhesion molecules on the biliary epithelium are associated with T-lymphocytic binding, recognition and destruction. To investigate relevant cellular immunological mechanisms under standard conditions, we have established an in vitro model using human gallbladder epithelial cells. Although not directly affected in these aberrations, gallbladder epithelial cells are excellent objects for immunological investigations. More than 10(8) highly purified cells were isolated and cultured longer than 6 wk in confluent monolayers. Cell growth was routinely established on uncoated plastic culture dishes, and serum-free media could be applied for immunological experiments. Cell characterization was performed by means of specific monoclonal antibodies typical for biliary epithelial cells. Cytokeratins 1 through 8, 18 and 19 and human epithelial cell antibody 125 always showed strong positive staining. Antigen patterns were examined before and after treatment with interferon-gamma by use of immunohistochemical staining methods. Untreated human gallbladder epithelial cells expressed human leukocyte class I antigens but few or no class II antigens. After stimulation with interferon-gamma induction of human leukocyte antigen-DR, -DP and -DQ was detected. In addition, intercellular adhesion molecule 1 was induced on these gallbladder epithelial cells. Therefore an immunological competence similar to that of biliary epithelial cells could be demonstrated. In vitro cell cultures of gallbladder epithelial cells offer a promising tool for subsequent investigations concerning intrahepatic biliary epithelial cells and their interactions with cells of the immune system.

Cytomegalovirus- and interferon-related effects on human endothelial cells. Cytomegalovirus infection reduces upregulation of HLA class II antigen expression after treatment with interferon-gamma.

Cultured human umbilical vein endothelial cells (HUVEs) were infected with human cytomegalovirus (HCMV) strain AD169. Up to 50% HUVEs proved to be positive for HCMV early nuclear antigens 24 hours after inoculation with virus. Following infection kinetics of surface expression of HLA class I and II, intercellular adhesion molecule (ICAM-1) and endothelial lymphocyte adhesion molecule (ELAM-1) on HUVEs were investigated by means of flow cytometry. A slight increase in HLA class I expression was observed, whereas expression of HLA class II (DR, DP, DQ) antigens was not induced by infection with HCMV. Furthermore, when compared with uninfected cells treated with interferon-gamma (IFN-gamma), reduced enhancement of HLA-DR expression was conspicuous in HCMV-infected cells treated with IFN-gamma. There is evidence that only a portion of HUVE is affected in its ability to upregulate HLA class II antigens. While expression of ICAM-1 was found to be enhanced between 8 and 20 hours after infection with a maximum at 12 hours after infection, no modulation of ELAM-1 was seen.

A rapid non-radioactive fluorescence assay for the measurement of both cell number and proliferation.

Measuring the incorporation of radioactive thymidine into the cell nucleus gives important information as to cell activation and proliferation. In this study the DNA-intercalating fluorochromes, Hoechst 33342 and Hoechst 33258, were tested as an alternative to the classical [3H]thymidine assay. Mitogen and alloantigen stimulated lymphocytes as well as FK 506 and CsA inhibited lymphocytes were treated with the two dyes, and the cell number and proliferation rates by means of measured fluorescence values. Of these tested fluorochromes H33342 appears to be an appropriate alternative to the [3H]thymidine assay. It mirrors the cell number in a fast and convenient manner without any pretreatment of the cell suspension which can remain in the culture plates. The complete assay procedure including data analysis can be performed rapidly and the standard deviations are small. This dye may also prove to be of value in other assay procedures, e.g., adhesion experiments.