Sándor Paku

Origin and Structural Evolution of the Early Proliferating Oval Cells in Rat Liver

We have analyzed the histological changes in rat liver after 2-acetylaminofluorene (AAF) administration. The data demonstrate that AAF-induced oval cells were preferentially generated by proliferation of the terminal biliary ductules that we suggest constitute the primary hepatic stem cell niche. The oval cells formed ductular structures, representing an extension of the canals of Hering. This histological organization provides continuous bile drainage of the hepatocytes and uninterrupted blood flow in the sinusoids. The oval cell ductules are surrounded by a continuous basement membrane that is intermittently disrupted by processes of stellate cells that form direct cell-cell contact with the oval cells. Although both AAF treatment and bile duct ligation results in proliferation of biliary epithelial cells, the mechanism(s) responsible for the proliferation of the biliary epithelium seems to differ in the two models. In contrast to the biliary proliferation stimulated by bile ligation, AAF-induced oval cell proliferation as well as the capacity of these cells to differentiate into hepatocytes, bile epithelial cells and possibly other cell lineages can be blocked by administration of dexamethasone.

Claudin-1, -3 and -4 proteins and mRNA expression in benign and malignant breast lesions: a research study.

IntroductionWe compared levels of protein and mRNA expression of three members of the claudin (CLDN) family in malignant breast tumours and benign lesions.MethodsAltogether, 56 sections from 52 surgically resected breast specimens were analyzed for CLDN1, CLDN3 and CLDN4 expression by immunohistochemistry. mRNA was also analyzed using real-time PCR in 17 of the 52 cases.ResultsCLDNs were rarely observed exclusively at tight junction structures. CLDN1 was present in the membrane of normal duct cells and in some of the cell membranes from ductal carcinoma in situ, and was frequently observed in eight out of nine areas of apocrine metaplasia, whereas invasive tumours were negative for CLDN1 or it was present in a scattered distribution among such tumour cells (in 36/39 malignant tumours). CLDN3 was present in 49 of the 56 sections and CLDN4 was present in all 56 tissue sections. However, CLDN4 was highly positive in normal epithelial cells and was decreased or absent in 17 out of 21 ductal carcinoma grade 1, in special types of breast carcinoma (mucinous, papillary, tubular) and in areas of apocrine metaplasia. CLDN1 mRNA was downregulated by 12-fold in the sample (tumour) group as compared with the control group using GAPDH as the reference gene. CLDN3 and CLDN4 mRNA exhibited no difference in expression between invasive tumours and surrounding tissue.ConclusionsThe significant loss of CLDN1 protein in breast cancer cells suggests that CLDN1 may play a role in invasion and metastasis. The loss of CLDN4 expression in areas of apocrine metaplasia and in the majority of grade 1 invasive carcinomas also suggests a particular role for this protein in mammary glandular cell differentiation and carcinogenesis.

2-acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: Confocal and electron microscopic studies

The 2‐acetylaminofluorene (AAF)/partial hepatectomy (PH) model is one of the most extensively studied experimental systems for oval cell proliferation and differentiation. We have previously described the oval cells as forming ductular structures surrounded by basement membrane, representing extensions of the canals of Hering. Herein we analyze the differentiation of oval cells into hepatocytes after varying degrees of liver damage induced by AAF. At a low dose of AAF, most oval cells synchronously differentiate into small hepatocytes by 6 days after the PH, resulting in complete restoration of the liver structure in 10 days. Higher doses of AAF delay the differentiation process and the new hepatocytes form foci, in contrast to what is observed at the low dose. Qualitatively, the differentiation process seems to be identical at the cellular level under both conditions. The transition from the expanding oval cell population into hepatocytes was correlated with the upregulation of hepatocyte nuclear factor 4 and the disappearance of the basement membrane. Also, the differentiation of oval cells into hepatocytes coincided with the loss of alpha‐fetoprotein and OV‐6 staining, and the replacement of the biliary cell‐specific α6 integrin and connexin 43 with the hepatocyte‐specific α1 integrin and connexin 32. In addition, bile canaliculi form between the new hepatocytes. In conclusion, these results indicate the rate of oval cell differentiation into hepatocytes is context dependent and suggest that, under favorable conditions, oval cells can complete this process much faster than previously appreciated. (HEPATOLOGY 2004;39:1353–1361.)

Immunohistochemical analysis of cytokeratin 7 expression in resting and proliferating biliary structures of rat liver

Cytokeratins are the largest subfamily of intermediate filament proteins and include more than 20 different gene products, which are expressed in an epithelial tissue‐specific manner. We studied by immunohistochemistry and confocal microscopy the distribution of cytokeratin subtypes in the biliary system of adult rat liver. A cytokeratin (CK)19+/7− cholangiocyte population was observed in the smaller branches of the biliary tree including the canals of Hering. They proliferated after 2‐acetaminofluorene (AAF) administration, although later the typical oval cells expressed CK7. This observation suggests that cholangiocytes with this cytokeratin phenotype may harbor adult hepatic stem cells. The CK19+/7− cholangiocytes were not present in the rat liver at birth, but developed postnatally. Similar cell populations were not observed in human livers. In conclusion, we propose that the CK19+/7− phenotype may be characteristic for adult hepatic stem cells in rat liver and that these cells are generated de novo after birth. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:863–870.)

Current concepts of tumor-induced angiogenesis

Tumor induced angiogenesis is responsible for the nutrition of the growing tumor and can also increase the probability of hematogenous tumor dissemination. Data obtained from morphological analysis of tumor angiogenesis can contribute to the development of new anti-angiogenic therapies. Based on in vitro and in vivo observations several models of angiogenesis were introduced, explaining the mechanism of lumen formation and the timing of basement membrane depositon. (1) Lumen is formed either by cell body curving or by fusion of intracellular vacuoles of nonpolarized endothelial cells. New basement membrane is deposited after lumen formation. (2) Slit-like lumen is immediately formed by migrating polarized endothelial cells. Basement membrane is continuously deposited during endothelial cell migration, only cellular processes of the endothelial cell migrating on the tip of the growing capillary are free of deposited basement membrane material. (3) Development of transluminal bridges in larger vessels - a process called intussusceptive growth - leads to the division of the vessels. These models, however, describe angiogenesis in tissues rich in connective tissue. Different processes of angiogenesis take place in organs - such as liver, lungs, adrenals, which are the most frequent sites of metastasis - having high vessel density without sufficient space for capillary sprouting. In the case of liver metastases of Lewis lung carcinoma the proliferation of endothelial cells was elicited only by direct contact between tumor and endothelial cells, leading to the development of large convoluted vessels inside the metastases. These vessels were continuous with the sinusoidal system, suggesting that these metastases have dual blood supply. This observation, among others, is in contrast to the generally accepted view that liver tumors have arterial blood supply. The increasing number of data demonstrating the dual or venous blood supply of liver metastases should be taken into consideration in the therapy of liver metastasis.

Expression of a decorin-like molecule in human melanoma

Decorin, a member of the family of small leucin-rich proteoglycans, has originally been described as a secreted proteoglycan component of the connective tissues, and has been implicated in the negative regulation of cell proliferation directly or via interactions with TGF-β. It was reported to be generally absent from tumor cells. Here we show that human melanoma cell lines express a decorin-like molecule. We detected decorin mRNA by RT-PCR in 7 out 7 human melanoma lines characterized by various metastatic potential. Using polyclonal antiserum against the core protein of decorin, the typical 80–120 kD glycanated form as well as a high molecular weight aberrant version (200–210 kD) of decorin were demonstrated by Western blot technique in the culture supernatants as well as in lysates of human melanoma cells. Finally, decorin epitope was also demonstrated immunohistochemically in human melanoma xenografts, as well as in tumor cells of surgically resected melanomas but not in melanocytes of nevi. The expression of this aberrant decorin did not inhibit thein vitro orin vivo growth of human melanoma cells, and it was independent of their metastatic potential. Human melanoma cell lines expressing aberrant decorin retained sensitivity to the antiproliferative and gelatinase-stimulatory effects of exogenous TGF-β.

Quantitative morphometric and immunohistochemical analysis and their correlates in cirrhosis – A study on explant livers

Abstract Background. Reproducible structural analysis was made on cirrhotic human liver samples in order to reveal potential connections between morphological and laboratory parameters. Material and methods. Large histological samples were taken from segment VII of 56 cirrhotic livers removed in connection with liver transplantation. Picro Sirius red and immunohistochemically (smooth muscle actin [SMA], cytokeratin 7 [CK7], Ki-67) stained sections were digitalized and morphometric evaluation was performed. Results. The Picro Sirius-stained fibrotic area correlated with the average thickness of the three broadest septa, extent of SMA positivity, alkaline phosphatase (ALP) values and it was lower in the viral hepatitis related cirrhoses than in samples with non-viral etiology. The extent of SMA staining increased with the CK7-positive ductular reaction. The proliferative activity of the hepatocytes correlated positively with the Ki-67 labeling of the ductular cells and inversely with the septum thickness. These data support the potential functional connection among different structural components, for example, myofibroblasts, ductular reaction and fibrogenesis but challenges the widely proposed role of ductular cells in regeneration. Conclusion.Unbiased morphological characterization of cirrhotic livers can provide valuable, clinically relevant information. Similar evaluation of routine core biopsies may increase the significance of this ‘Gold Standard’ examination.