Expression of MUC1, Thomsen-Friedenreich-related antigens, and cytokeratin 19 in human renal cell carcinomas and tubular clear cell lesions.

The expression of MUC1, MUC2, mucin-associated Thomsen-Friedenreich-related antigens (TF, sialosyl-TF, Tn, and sialosyl-Tn), and cytokeratin 19 (CK19) was systematically investigated in situ in 58 resected human kidney tumours, surrounding tissue of normal appearance, and two normal kidneys obtained at autopsy, using monoclonal antibodies. In kidney tissues of normal appearance, TF, s-TF, MUC1 and CK19 were positive in distal tubules and collecting ducts but negative in proximal tubules. In contrast, MUC2, Tn, and s-Tn were negative throughout the normal renal tubular system. Almost all renal cell carcinomas (RCCs) showed strong immunoreactivity for MUC1, but all were negative for MUC2. Some RCCs expressed TF, Tn, s-Tn, and CK19. In addition, the immunomorphological characteristics of the majority of clear-cell RCCs and clear/granular RCCs with anti-MUC1 and anti-CK 19 closely resembled those of the collecting duct and the distal tubule rather than the proximal tubule. In the renal tissue of otherwise normal appearance adjacent to clear-cell RCCs and clear/granular RCCs, clear cells with excessive storage of glycogen were often found in the collecting duct system, but only rarely in the proximal tubules. These results suggest that the majority of clear-cell RCCs and clear/granular RCCs may originate from the collecting duct system.

Graft-versus-leukemia reactivity involves cluster formation between superantigen-reactive donor T lymphocytes and host macrophages.

T-cell-mediated antitumor effects play an important role clinically in allogeneic graft-versus-leukemia (GvL) reactivity, whereas T-cell-mediated antihost effects are associated with a risk of developing graft-versus-host (GvH) disease. GvL and GvH were compared in an animal tumor model system after the systemic transfer of allogeneic antitumor immune T lymphocytes from B10.D2 [H-2d; minor lymphocyte-stimulating antigen (Mls)b] mice into ESb-MP tumor-bearing or normal DBA/2 (H-2d; Mls(a)) mice. Here we demonstrate that this T-cell-mediated therapy involves the formation of clusters of donor CD4 and CD8 T cells with host macrophages, in particular, with a subpopulation expressing the lymphocyte adhesion molecule sialoadhesin. DBA/2 mice and the derived tumor ESb-MP express viral superantigen 7 (Mls(a)), an endogenous viral superantigen that is absent from B10.D2 mice. To test the contribution of viral superantigen 7-reactive Vbeta6 donor T cells in the GvL-mediated eradication of liver metastases, we performed immunohistological and transmission electron microscopy studies. Vbeta6+ CD4 and CD8 T cells from B10.D2 donors formed tight clusters with host sialoadhesin-positive macrophages, and transmission electron microscopy pictures revealed direct membrane-membrane interactions between T cells and macrophages. Clusters were more abundant and consisted of more cells in tumor-bearing hosts (GvL model) than in non-tumor-bearing hosts (GvH model). In addition, Vbeta6 T cells within the clusters showed a strong proliferation activity, indicating stimulation. Moreover, in an in vitro tumor cytostasis assay, primed as well as nonprimed purified Vbeta6 T cells from donor mice were able to inhibit the proliferation of superantigen-expressing ESb-MP lymphoma cells. This suggests that the transferred superantigen-reactive Vbeta6 T cells contribute to the eradication of metastases. The observed cell clusters might be sites for antigen presentation and the activation of tumor-reactive T cells.

Cytokeratin expression is reduced in glycogenotic clear hepatocytes but increased in ground-glass cells in chronic human and woodchuck hepadnaviral infection.

Hepatocytes of normal adult liver express cytokeratins (CKs) 8/18, but bile duct cells additionally contain CK7/19. We have previously demonstrated the frequent occurrence of foci of altered hepatocytes in association with hepatic tumors in humans and provided evidence for a preneoplastic nature of the focal lesions. In this study, we investigated the CK expression in both the preneoplastic lesions and extrafocal parenchyma. Sixty-seven explanted livers with cirrhosis or advanced fibrosis harboring preneoplastic focal lesions, with or without hepatitis B virus (HBV) infection, as well as 9 livers with HBV-associated fulminant hepatitis, were studied for the expression of CK7/8/14/18/19. Five livers from woodchucks infected with the woodchuck hepatitis virus (WHV) were also investigated. Glycogenotic clear hepatocytes were negative or weakly positive for CK8/18, while amphophilic hepatocytes were strongly positive for these CKs, the changes being associated with marked reduction and increase, respectively, of highly organized membranous components in their cytoplasm. This allows the distinct recognition of the clear-cell and clear-cell-dominant preneoplastic lesions in the human and woodchuck livers. In ground-glass hepatocytes expressing viral antigens, an unusual accumulation of CK8/18 was observed, but there was no evidence of preferential necrosis of ground-glass hepatocytes. Many CK7- and CK19-positive ductular (oval) cells were found in extrafocal liver tissue, but only rarely were they present within focal lesions.

Xenomorphic hepatocellular precursors and neoplastic progression of tigroid cell foci induced in rats with low doses of N-nitrosomorpholine.

Tigroid cell foci (TCF) are a well-defined entity induced in rat liver by chemical carcinogens, their significance for hepatocarcinogenesis being controversial. Using cytomorphological, cytochemical and morphometric approaches, we studied the evolution and fate of TCF sequentially from 7 to 110 weeks in groups of 50 male Sprague-Dawley rats, which remained untreated or received N-nitrosomorpholine (NNM) orally at concentrations of 3 and 1 mg/kg body wt/day for 7 and up to 75 weeks, respectively. An increased incidence of hepatocellular neoplasms developed in exposed animals compared with controls, which was significant for adenomas at both dose levels, and for carcinomas (HCC) after the longer exposure to the lower dose level (P < 0.0001). TCF appeared frequently in addition to other types of proliferative foci of altered hepatocytes (FAH) including clear/acidophilic and mixed cell foci (MCF) in NNM-treated and rarely in untreated rats. Striking similarities in the cellular phenotypes of TCF and many hepatocellular neoplasms indicated the potential of TCF for progression to both adenomas and carcinomas. TCF emerged from xenomorphic cell foci (XCF), which consisted of hypertrophied hepatocytes typically presenting an enlarged nucleus, abundant glycogen, smooth and rough endoplasmic reticulum, altered activities of several enzymes of carbohydrate metabolism and an increased cell proliferation (P < 0.001) compared with the extrafocal parenchyma. TCF shared many features with XCF, but their basophilia and proliferative activity was higher. The number of FAH appearing at the two dose levels of NNM was similar but the average size of TCF and MCF was frequently higher at late time points in the group developing a significantly higher incidence of HCC, which suggests a pronounced acceleration of neoplastic conversion in established preneoplastic cell populations rather than the induction of additional FAH by sustained effects of low doses of carcinogens.

Effects of low doses of N-nitrosomorpholine on the development of early stages of hepatocarcinogenesis.

Male Sprague-Dawley rats received the hepatocarcinogen N-nitrosomorpholine (NNM) in the drinking water at low dose levels ranging from 6 mg/l to 60 mg/l for 6 and 12 weeks, respectively. Foci of altered hepatocytes (FAH) were demonstrated histochemically using changes in the activities of glucose-6-phosphate dehydrogenase and glycogen phosphorylase, and in the glycogen content as markers. Proliferating cells were detected by the immunohistochemical reaction for proliferating cell nuclear antigen (PCNA). The number and size of foci of altered hepatocytes increased in a time and dose-related manner. The dose-effect curves were non-linear with a slight positive slope at the low doses and a markedly increased slope at higher doses. The number of PCNA positive hepatocytes showed a dose-dependent increase. In addition to the granular distribution of PCNA in the nuclei, hepatocyte nuclei with homogeneously distributed PCNA occurred in animals exposed to 60 mg/l NNM. It is proposed that these cells are related to the occurrence of hepatocytes with higher ploidy induced by NNM and may be regarded as cells in the G2 phase of the cell cycle. The non-linear shape of the dose-response-curve of the FAH suggests that some mechanisms contribute to carcinogenesis over the whole dose range, whereas other mechanisms enhance carcinogenesis only at higher doses. The relevance of the non-linear dose-effect curve for the risk assessment of carcinogens is discussed.

Spongiotic pericytoma: a benign neoplasm deriving from the perisinusoidal (Ito) cells in rat liver.

Spongiosis hepatis has been known for some time to develop frequently in livers of rats and fish treated with hepatocarcinogens and was considered to derive from the perisinusoidal (Ito) cells (PSC). Using rat liver treated with N-nitrosomorpholine at different dose levels, we studied the cellular composition and origin as well as the proliferation kinetics of spongiosis hepatis by immunohistochemical demonstration of desmin, vimentin, and alpha-smooth-muscle actin, and by autoradiographic determination of [3H]-thymidine incorporation, respectively. The vast majority of the cells forming spongiosis hepatis were positive for desmin and vimentin but negative for alpha-smooth-muscle actin, confirming the cellular origin of spongiosis hepatis from PSC. In addition, immunohistochemical demonstration of desmin and vimentin revealed that spongiosis hepatis is an integral part of larger lesions consisting of focal PSC aggregates. These aggregates show a significantly increased incorporation of [3H]-thymidine compared with PSC in the extrafocal tissue and in the liver tissue of untreated control animals. In stop experiments, this increased labeling index was maintained many months after withdrawal of the carcinogen, in line with the earlier observation of a progressive behavior of spongiosis hepatis. We conclude that PSC may give rise to proliferative lesions appearing as PSC aggregates associated with more or less pronounced spongiosis hepatis. The persistence, the proliferative activity, and the slow expansive growth of these lesions suggest a benign neoplastic behavior. We therefore propose to classify these lesions as spongiotic pericytoma. Malignant tumors possibly originating from spongiotic pericytoma should consequently by classified as perisinusoidal (Ito) cell sarcomas.

Cell proliferation and cell death (apoptosis) in hepatic preneoplasia and neoplasia are closely related to phenotypic cellular diversity and instability.

Preneoplastic and neoplastic hepatic lesions were induced in male Sprague-Dawley rats by oral exposure to N-nitrosomorpholine (12 mg/kg body wt/day) for 7 weeks (stop model). Twelve, 23 and 34 weeks after withdrawal of the carcinogen, cell proliferation and cell death (apoptosis) were studied in defined phenotypes of preneoplastic foci of altered hepatocytes (FAH), hepatocellular adenomas (HCA) and carcinomas (HCC) by autoradiographic determination of the labelling index (LI) resulting from continuous administration of [3H]thymidine for 48 h and by simultaneous counting of apoptotic bodies respectively. Compared with the liver parenchyma of untreated controls and the extrafocal parenchyma of treated animals, the mean LI was elevated in all types of FAH, HCA and HCC, but the extent of this increase differed markedly between the diverse phenotypes. The increase in the LI was significant for clear/acidophilic, intermediate and mixed/basophilic cell foci, but remained insignificant for the relatively rare tigroid and amphophilic cell foci. The previously established progression-linked phenotypic instability in the predominant cell lineage leading to HCC was associated with a gradual increase in the mean LI showing four significantly different proliferative stages: (i) clear/acidophilic and intermediate cell foci excessively storing glycogen, (ii) mixed/basophilic cell populations in FAH and glycogen-storing HCA, (iii) glycogen-poor HCA and glycogen-storing HCC and (iv) glycogen-poor HCC. The inverse correlation between glycogen accumulation and cell proliferation during progression from glycogenotic FAH to glycogen-poor HCC indicates that the fundamental metabolic shift associated with the gradual disappearance of the glycogenosis is essential for the evolution of the malignant phenotype. The mean ratio of necrotic cells (RN) was somewhat higher in all types of FAH compared to the normal and extrafocal liver parenchyma, but this was not statistically significant. Only when HCA and HCC appeared was there a significant increase in the mean RN, proceeding with the progression of neoplastic development. Our results do not support the concept that cell death (apoptosis) plays a major role in counterbalancing cell replication in FAH, but rather suggest that cell death occurs more frequently in the course of hepatocarcinogenesis the more neoplastic development advances.

Preneoplastic foci of altered hepatocytes induced in rats by irradiation with alpha-particles of Thorotrast and neutrons.

Prestages of hepatocellular neoplasms induced in rats by continuous internal alpha-radiation of Thorotrast or by fractionated external radiation with neutrons were studied by cytomorphological, cytochemical and morphometric methods. Irradiation with both Thorotrast and neutrons resulted in a significant increase in the number and volume fraction of foci of altered hepatocytes (FAH), the occurrence of which at 14 months correlated well with the previously reported increased incidence of hepatocellular neoplasms appearing after long lag periods. The morphological and biochemical phenotypes of radiation-induced FAH were similar to those of preneoplastic lesions described earlier in hepatocarcinogenesis elicited by chemicals or viruses.

Over-expression of glucose transporter isoform GLUT1 and hexokinase I in rat renal oncocytic tubules and oncocytomas.

Renal oncocytomas, which have previously been shown to originate from the collecting duct system, were induced in male Sprague-Dawley rats by oral administration of N-nitrosomorpholine (NNM) for 7 weeks. The expression of glucose transporter isoforms GLUT1 and GLUT2, and of several enzymes involved in glucose metabolism [hexokinase (HK), pyruvate kinase (PK), lactate dehydrogenase (LDH), malate dehydrogenase (MDH)] were studied by cytochemical approaches in serial cryostat sections of the kidney 12, 23 and 34 weeks after withdrawal of NNM. Oncocytic tubules connected with collecting ducts were first observed 23 weeks, and oncocytomas 34 weeks after withdrawal. The cytochemical pattern of oncocytic tubules and oncocytomas was similar, but differed markedly from that of normal collecting ducts in nearly all variables studied; expression of GLUT1 and hexokinase I proteins were strongly increased; activities of HK, PK and MDH were elevated, while LDH activity was reduced. These results suggest that oncocytic transformation is associated with fundamental changes in energy metabolism which differ from those in cell lineages leading to other types of renal cell tumours, such as clear/acidophilic and basophilic cell tumours. The characteristic over-expression of GLUT1 may be used as a diagnostic criterion for the discrimination between oncocytes and acidophilic (granular) cells in clear/acidophilic renal cell tumours which show a reduced expression of this glucose transporter protein.

Expression of glucose transporter isoforms (GLUT1, GLUT2) and activities of hexokinase, pyruvate kinase, and malic enzyme in preneoplastic and neoplastic rat renal basophilic cell lesions.

Sequential changes in the expression of two glucose transporter isoforms (GLUT1, GLUT2), and in the activities of hexokinase, pyruvate kinase and malic enzyme during the development of rat renal basophilic cell tumors were studied using histochemical techniques. Early basophilic cell tubules are similar to proximal convoluted tubules (PCT) in their overall histochemical pattern, particularly in the expression of glucose transporters, suggesting that basophilic cell tubules and tumors derived from them arise from PCT. In comparison with PCT, basophilic cell tubules show slightly increased activities of all the enzymes studied. In basophilic cell tumors, markedly elevated hexokinase and pyruvate kinase activities are accompanied by a considerable reduction in the expression of GLUT2. GLUT1 expression is not found in basophilic cell tubules or PCT. Small basophilic cell tumors also do not express GLUT1, but GLUT1 is regularly expressed in several cell layers surrounding necrotic areas within large basophilic cell tumors. Our results indicate that increased glycolytic activity and reduced GLUT2 expression take place during the development of renal basophilic cell tumors.

Sequential changes in glycogen content, expression of glucose transporters and enzymic patterns during development of clear/acidophilic cell tumors in rat kidney.

Renal clear cell tubules and clear/acidophilic cell tumors were induced in male Sprague-Dawley rats by 7 weeks oral administration (stop model) of N-nitrosomorpholine (NNM) at a concentration of 12 mg/100 ml in the drinking water. Twelve, 23 and 34 weeks after withdrawal of NNM serial cryostat sections of the kidneys were histochemically analyzed for the following parameters: glucose transporter proteins (GLUT1, GLUT2), glycogen content and the activities of glycogen synthase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6Pase), glucose-6-phosphate dehydrogenase (G6PDH), hexokinase (HK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), alkaline phosphatase (ALP), acid phosphatase (ACP) and gamma-glutamyltransferase (GGT). Clear cell (glycogenotic) tubules first appeared at 23 weeks, and clear/acidophilic cell tumors at 34 weeks after withdrawal of the carcinogen. G6Pase, ALP, GGT and GLUT2 were absent in clear cell tubules, clear/acidophilic cell tubules, and clear/acidophilic cell tumors indicating a sequential origin of all these types of lesions from the collecting duct system, in line with previous morphological findings. In comparison to the collecting duct epithelium, glycogenotic tubules demonstrated an increased activity of PHO and reduced activities of glycolytic and mitochondrial enzymes, which were accompanied by a strongly reduced expression of GLUT1. Moderately increased activities of glycolytic and mitochondrial enzymes were observed in the clear cells of clear/acidophilic cell tubules and tumors compared with those in glycogenotic tubules. They had slightly increased activities of the glycolytic enzymes GAPDH and PK compared with normal collecting duct epithelium, while most of them were nearly lacking in GLUT1. Our findings suggest that glycogen storage is not due to an increased uptake of glucose from the blood, but results from a disturbance in intracellular flux of metabolites. The development of clear cell tubules from the normal collecting duct epithelium is accompanied by a markedly decreased expression of GLUT1 along with a reduction in glycolytic and mitochondrial enzymes. This reduction of enzyme activities is replaced by an increase in enzyme activities in clear/acidophilic cell tumors indicating a fundamental shift in carbohydrate metabolism during progression from preneoplastic to neoplastic lesions.

Altered transferrin gene expression in preneoplastic and neoplastic liver lesions induced in rats with N-nitrosomorpholine.

The expression of the gene for the iron transport protein transferrin was found to be altered in preneoplastic and neoplastic lesions induced in the rat liver by N-nitrosomorpholine. The total RNA of ten hepatocellular carcinomas (HCC) was investigated by Northern blot analysis using a cDNA-probe comprising 150 bp of the 3' region and compared with the total hepatic RNA in untreated rats. Seven hepatocellular carcinomas showed slight or pronounced reduction in transferrin expression. In situ hybridization of two additional hepatocellular carcinomas revealed marked reduction in the mRNA level for the transferrin gene compared with the surrounding tissue. In contrast, the majority of early preneoplastic lesions storing excess glycogen and tigroid cell foci expressed increased levels of transferrin mRNA. The loss of glycogen in mixed cell foci, which represent a later stage of hepatocarcinogenesis, was usually accompanied by a decrease in transferrin mRNA suggesting a close relationship between this change in gene expression and cellular dedifferentiation emerging during hepatocarcinogenesis.

Glycogen phosphorylase hyperactive foci of altered hepatocytes in aged rats.

In untreated 12- to 24-month-old rats, the enzyme histochemical pattern of 45 focal hepatic lesions was investigated in serial sections. In addition to previously characterized glycogen storage foci, a new type of enzymatically altered hepatic focus was found. The outstanding feature of this was an increased glycogen phosphorylase activity. The frequent appearance of glycogen phosphorylase hyperactive foci simultaneously exhibiting excessive glycogen storage suggests a close relationship to the well known glycogen storage foci representing an early stage in the sequence of cellular changes which lead to hepatic tumors.

Computerized ultrasound B-scan texture analysis of experimental diffuse parenchymal liver disease: correlation with histopathology and tissue composition.

By means of statistical pattern recognition procedures, a quantitative description of the ultrasound B-scan images of experimental diffuse liver disease has been carried out. Fatty livers, fatty fibrosis/cirrhosis, and cirrhosis without fatty infiltration of the liver were studied in female Wistar rats. Separation accuracies of more than 80% between the tissue classes "normal" vs "fatty infiltration," or "normal" vs "fatty cirrhosis," using only two statistical image parameters were found. A subclassification of the diffuse parenchymal liver disease was not possible. It is shown by multiple linear regression analysis that the image parameter "mean grey level" correlates better with total lipid content than with the amount of connective tissue. Furthermore it is demonstrated that connective tissue leads only to a weak increase in "mean grey level," whereas the addition of connective tissue to a given tissue lipid can lead to a reduction in image brightness.

Echographic tissue characterization in diffuse parenchymal liver disease: correlation of image structure with histology.

Seventy livers were examined in vitro using a computerized ultrasound B-mode data acquisition and analysis system. For tissue characterization, statistical parameters from pattern recognition algorithms describing image brightness and image structure were used. Reference classification based on histopathology as well as on chemical/morphometrical analysis led to the diagnostic classes of normal, fatty liver, fibrosis/cirrhosis and fatty fibrosis/cirrhosis. Comparing the two reference methods for ultrasound tissue characterization, reclassification based on chemical/morphometrical analysis resulted in a significant increase in diagnostic accuracy. The strong correlations between statistical ultrasound image parameters and morphometrical features reflect the relevance of our statistical approach to ultrasound tissue characterization.

Computerized ultrasound B-scan texture analysis of experimental fatty liver disease: influence of total lipid content and fat deposit distribution.

Statistical pattern recognition procedures allow a quantitative description of ultrasound-B-scan image texture. According to well-established animal models, different types of fatty liver disease were induced in female Wistar rats. For the correlation of the computerized ultrasound image with its underlying histology a variable tissue model based on histomorphological data, texture analysis of the histological image and biochemical measurements of total lipid, water and hydroxyproline content was created. Whereas a regional arrangement of large fat deposits leads to a significant increase in the "mean grey level" (measure of image brightness) of the ultrasound-B-scan image, there is no difference in image brightness between normal liver tissue and liver steatosis for the tissue model with diffuse homogeneous fatty infiltration. It is demonstrated by multiple linear regression analysis that the "mean grey level" of the ultrasound-B-scan image depends not only on total lipid content but even more on the histomorphological fat deposit distribution.