Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis.

High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG(-/-) mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG(-/-) mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG(-/-) mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-kappaB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG(-/-) mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.

Osteoprotegerin inhibits osteolysis and decreases skeletal tumor burden in syngeneic and nude mouse models of experimental bone metastasis.

Certain malignancies, including breast cancer, frequently metastasize to bone, where the tumor cells induce osteoclasts to locally destroy bone. Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor family, is a negative regulator of osteoclast differentiation, activation, and survival. We tested the ability of recombinant OPG to inhibit tumor-induced osteoclastogenesis, osteolysis, and skeletal tumor burden in two animal models. In a syngeneic model, mouse colon adenocarcinoma (Colon-26) cells were injected into the left ventricle of mice. Treatment with OPG dose-dependently decreased the number and area of radiographically evident lytic bone lesions, which, at the highest dose, were undetectable. Histologically, OPG also decreased skeletal tumor burden and tumor-associated osteoclasts. In a nude mouse model, OPG treatment completely prevented radiographic osteolytic lesions caused by human MDA-MB-231 breast cancer cells. Histologically, OPG decreased skeletal tumor burden by 75% and completely eradicated MDA tumor-associated osteoclasts. In both models, OPG had no effect on metastatic tumor burden in a panel of soft tissue organs. These data indicate that OPG may be an effective therapy for preventing osteolysis and decreasing skeletal tumor burden in patients with bone metastasis.

p53 in malignant and benign liver lesions.

p53 expression was studied by immunohistochemical methods in benign and malignant human epithelial liver lesions in 46 patients from Hungary. Positive immunostaining for p53 protein, indicating the overexpression or prolonged half-life of p53 protein, was detected in the nuclei of tumour cells of seven of the 16 hepatocellular carcinomas (HCC) (44%), including three HCC patients with hepatitis B virus infection. Immunostaining of p53 was seen in one of the seven hepatoblastomas, none of the 17 focal nodular hyperplasias, and none of the six hepatocellular adenomas. The detection of p53 in a relatively high percentage of the HCC cases in Hungary, a country in which aflatoxin contamination of the diet is rare, suggests that factors other than aflatoxin led to the accumulation or overexpression of p53 in these patients.

Inhibitory and promoting effects of carbon tetrachloride-induced liver cirrhosis on the diethylnitrosamine hepatocarcinogenesis in rats.

The modifying effect of the experimentally induced liver cirrhosis on the diethylnitrosamine (DENA)-hepatocarcino-genesis was investigated in male Fischer 344 rats. Cirrhosis was produced by either repeated intragastric doses of CCl4 for 3 months or by simultaneous administration of CCl4 and phenobarbital (PB) in drinking water for 6 weeks. The hepatocarcinogenic regimen consisted of multiple ip. administrations of DENA (10 mg/kg b.w. per dose, up to a total dose of 200 mg/kg b.w.). All the animals were killed 8 months after starting the experiment. The chronic CCl4-post-treatment exerted a strong promoting effect, while the established cirrhosis completely prevented the formation of hepatocellular carcinomas.

Overexpression of transforming growth factor-alpha in hepatocellular carcinoma and focal nodular hyperplasia from European patients.

The expression of transforming growth factor-alpha (TGF-alpha) was studied in 33 surgically excised human hepatocellular carcinomas (HCCs), focal nodular hyperplasias (FNHs), and the surrounding liver tissue from patients who were life-long residents of Hungary. Monoclonal antibodies were used to localize TGF-alpha and hepatitis B surface antigen (HBsAg) using an avidin-biotin-peroxidase immunohistochemical method on paraffin-embedded sections. Transforming growth factor-alpha was detected in the tumor tissue in 13 of 16 patients with HCC (81%) and in 12 of 17 patients with FNH (71%). Three patients with HCC were actively infected with hepatitis B virus, indicated by the detection of HBsAg in the serum and in adjacent nontumorous liver. Transforming growth factor-alpha was detected in the same nontumorous hepatocytes as HBsAg, often in areas of the hepatocyte cytoplasm, with a "ground glass" appearance. Bile duct cells were stained for TGF-alpha in the surrounding nontumorous liver tissue and a more intensive immunostaining was observed in the proliferating bile ducts in the FNH cases, which suggests that TGF-alpha may participate in the growth regulation of bile ducts.

Keratinocyte growth factor promotes healing of left-sided colon anastomoses.

BACKGROUND: Inadequate healing and consequent leakage from bowel anastomoses are a significant cause of postoperative morbidity and mortality. Systemic application of keratinocyte growth factor (KGF) has been shown to promote mucosal healing in models of colitis in rats and mice. The aim of the present study was to evaluate the effect of systemic KGF administration on healing of colonic anastomoses in rats. METHODS: Rats underwent laparotomy, division of the left colon, and sigmoido-sigmoidostomy. KGF (5 mg/kg) or vehicle were administered intraperitoneally in two groups (n = 30 per group) 12 hours prior to surgery, and then once daily until sacrifice (6 animals per group; 2, 4, 7, 12, and 21 days after surgery). Bursting pressure measurements, histologic evaluation, morphometric analysis, mucin and collagen staining, and hydroxyproline measurements of the anastomotic site were performed. RESULTS: Administration of KGF significantly increased anastomotic bursting pressure on postoperative days 2, 4, and 7 by 34%, 49%, and 19%, respectively. Histology, mucin staining, and measurements of the colonic crypt depth showed markedly less extended inflammation with an increased acidic mucin content and a significantly thickened mucosal layer in the KGF treated group when compared with vehicle-treated animals. CONCLUSIONS: KGF promotes healing of colonic anastomoses in rats during a 1-week postoperative period following large bowel surgery. KGF may be acting to accelerate host reparative processes as well as to enhance protection of the anastomotic wound bed by increased colonic epithelium proliferation, increased mucus production, and reduction of the inflammatory activity at the anastomotic site.

[Polymerase chain reaction and its application]. / A polimeráz lánc reakció és alkalmazási területei.

The polymerase chain reaction (PCR) is one of the most important new methods in molecular biology. It is widely used in genetic and anthropologic basic research, in oncology and virology, in all those fields, where molecular biologic methods can give answers to the questions raised. The procedure enables one to multiply with extreme precision targeted pieces of amounts as little as one target molecule of DNA or RNA by five to six logs, making them easy to be handled and examined by routine molecular biological methods. The method is presented through one possible application field, that is of great importance in the study of hepatocarcinogenesis. Sensitivity of PCR in detection of hepatitis B virus DNA is greater by four logs than animal inoculation, the last most sensitive method known.

[Local fibrinolytic treatment in subtotal pulmonary embolism]. / Lokális fibrinolyticus kezelés subtotalis tüdóembóliában.

Authors present an interventional radiological technique used for a successful treatment of 12 patients with massive pulmonary embolism. The method for rapid statement of diagnosis and the range of indications are discussed. In massive pulmonary embolism, after a diagnostic angiography, in 12 patient's mechanical thrombus drilling, in 3 cases thrombus aspiration, in 8 cases thrombus explosion and in other 1 balloon recanalisation was performed combined with a low dose (20,000-60,000 IU/h Streptokinase or the double dose of Urokinase) local fibrinolytic infusion. No major complication was observed and all the patients were successfully treated.

[Management of extensive, potentially fatal, pulmonary embolism]. / Klinikai halálhoz vezetö nagy kiterjedésü tüdöembóliák kezelése.

Extensive pulmonary embolisms were suspected in 11 patients with severe cardiogenic shock admitted to an intensive care unit. The urgently established diagnosis was always based on clinical symptoms and on a complex criteria system elaborated by the authors. The "blind" diagnosis of subtotal pulmonary embolism was confirmed by further noninvasive examinations in 10 cases. During the measures of complex resuscitation, the authors administered high doses of streptokinase in 10 cases and ultra-high dose of streptokinase for one patient. The emergency treatment was successful in four cases.

[Treatment possibilities for extensive pulmonary embolism as an alternative to the Trendelenburg operation]. / Nagy kiterjedésü tüdöembóliák kezelési lehetöségei, mint a Trendelenburg-mütét alternatívái.

The authors describe the clinical course of successfully treated patients with extensive, subtotal pulmonary embolism. After the diagnosis was confirmed by isotopic scan or pulmonary angiography, mechanical thrombus destruction was applied followed by low dose loco-regional thrombolysis in 11 patients by streptokinase. Five patients were treated with ultrahigh dose of streptokinase through peripheral vein, one patient via pulmonary artery catheter and one patient was treated with high dose urokinase by pulmonary catheter in combination with mechanical thrombus destruction by guide wire. All the treatment procedures were proved to be successful. After detailed case reports, the authors review the life saving thrombolytic treatment of acute subtotal pulmonary embolism as an alternative of Trendelenburg operation.

[Comparative study of verapamil and bisoprolol in the secondary prevention of myocardial infarction]. / A verapamil és bisoprolol összehasonlító vizsgálata szívinfarktuson átesett betegek szekunder prevenciójában.

The effect of Ca-antagonist, long-acting verapamil and the selective beta-1 blocking bisoprolol were investigated and compared in the secondary prevention after myocardial infarction. Eighty-seven patients were enrolled, 27 patients were not included because of the exclusion criteria, 30 patients were treated with verapamil and 30 patients with bisoprolol. During the 540 days of follow up period treadmill ergometry and dobutamine stress-test with SPECT investigation were performed two times. Both clinically and the data of our investigations the effect of the two drugs in the secondary prevention was good, and even at the 540th day the protective effect was still excellent.

[Lifesaving thrombolysis--in the light of contraindications]. / Eletmentö thrombolysis--a kontraindikációk tükrében.

The authors review the different methodological suggestions concerning thrombolysis, especially from the aspects of the absolute and relative contraindications of the treatment. They present case reports to prove that in patients with life threatening thromboembolic diseases some points of contraindications may be disregarded. They suggest a new strategy of absolute and relative contraindications be made considering the currently available recently introduced thrombolytic therapy.

CD4+CD45RBHi T cell transfer induced colitis in mice is accompanied by osteopenia which is treatable with recombinant human osteoprotegerin.

BACKGROUND AND AIMS: Transfer of CD4+CD45RBHi T cells into semi syngeneic immunodeficient mice represents a model of inflammatory bowel disease (IBD). As patients with IBD often suffer from osteopenia, we studied if this T cell transfer in mice results in osteopenia in addition to colitis, and if treatment with osteoprotegerin (OPG) has effects on the bone mineral density of T cell transferred mice. We also investigated whether osteopenia was due to malabsorption as a result of a dysregulated digestive tract or as a consequence of the inflammatory process. METHODS: CD4+CD45RBHi or CD4+CD45RBLo T cells (4 x 10(5)) were sorted from CB6F1 and transferred into C.B.17 scid/scid mice. Recipient mice were treated with human IgG1 Fc (control) or Fc-OPG three times per week in a prophylactic regimen as well as a therapeutic regimen (after 10% body weight loss) and were evaluated for osteopenia and colitis. RESULTS: Mice that received CD4+CD45RBHi T cells developed osteopenia (as indicated by decreased bone density accompanied by decreased osteoblasts and increased osteoclasts) and colitis (as indicated by histological changes in the large intestine). Mice that received CD4+CD45RBLo T cells developed neither osteopenia nor colitis. All animals consumed, on average, the same amount of food and water over the course of the study. Prophylactic treatment with Fc-OPG increased bone density in mice that received either CD4+CD45RBHi or CD4+CD45RBLo T cells but had no effects on the gastrointestinal tract. Fc-OPG treatment of osteopenic mice with established IBD caused the normalisation of bone density. Osteopenia in CD4+CD45RBHi T cell recipients was accompanied by hypoparathyroidism that was partially normalised by treatment with Fc-OPG. CD4+CD45RBHi T cell recipients also had a bone marrow inflammatory cell infiltrate expressing tumour necrosis factor alpha which was unaffected by treatment with Fc-OPG. CONCLUSIONS: CD4+CD45RBHi T cell transfer results in osteopenia in addition to colitis. Evidence suggests that this osteopenia was induced by inflammatory cell infiltration and not by malabsorption of calcium. Recombinant human osteoprotegerin effectively treated the osteopenia. OPG may be a useful therapeutic option for treating osteopenia in patients with IBD.

Cytokeratin patterns of liver carcinomas induced by diethylnitrosamine in monkeys.

BACKGROUND: Although the general conception is that hepatocyte- and bile duct-specific cytokeratin (CK) patterns are maintained throughout the neoplastic process, there is an increasing number of reports showing deviation from the rule. CK patterns have been found to be similar across species barriers, so it could be expected that studying the CK patterns of experimentally induced liver tumors may contribute to the elucidation of these controversies. EXPERIMENTAL DESIGN: A CK immunohistochemical study was carried out on histologic sections from hepatocellular carcinomas (HCCs) and preneoplastic lesions from 118 monkeys chronically dosed with diethylnitrosamine (DEN), using mAbs to CK 8, CK 18, CK 7, and CK 19. RESULTS: Normal monkey hepatocytes differed from human hepatocytes by displaying CK 19 in addition to the CK 8/CK 18 pairs, whereas the CK pattern of the bile duct epithelial cells was identical in monkey and human liver. In association with DEN-induced hepatocarcinogenesis, heterogeneity was observed in the CK expression, both in the HCCs and nontumorous parts of the livers. The majority of the HCC cases displayed one of the three CKs normally present in monkey hepatocytes, whereas positive expression of all three CKs (CK 8, CK 18, CK 19) and negative CK 7 was preserved in only 19.5% of the HCC cases. A so-called mixed staining pattern (negative and positive CK staining within the same tumor) was observed in approximately one-fourth of the cases. There was no correlation between the preservation of the hepatocyte-specific CK pattern and the degree of differentiation, tumor grade, or DNA ploidy of the HCCs. In approximately 10% of the primary tumors, CK 7 was expressed in the entire parenchymal cell compartment of the HCC nodules, whereas it was present in a mixed staining pattern in more than half of the cases. In lung metastases, CK 7 was less common, only expressed in approximately one-fourth of the cases. Alterations in the CK patterns were observed in the nonneoplastic hepatocytes of the tumor-bearing monkeys. These included mixed staining patterns in which the CKs appeared as positive and negative regenerating nodules side-by-side. As was observed in the HCCs, CK 7 was more commonly expressed in the nonneoplastic parenchyma in the form of mixed staining pattern than the other three CKs. Moreover, CK 7-negative HCCs occurred more frequently in CK 7-negative livers than in positive livers. Proliferation of CK 7- and CK 19-positive bile ductules and bile ductular-like (oval) cells was frequently associated with the DEN-induced liver injury and hepatocarcinogenesis. CONCLUSIONS: This is the first report on CK expression in monkey liver. The findings show that the hepatocyte specific pattern is not always preserved during DEN-induced hepatocarcinogenesis and may therefore not be useful in differentiating between HCCs and cholangiocarcinomas.

Connective tissue content of fibrolamellar carcinoma and other human liver tumors.

The connective tissue content of four different human liver tumors (Fibrolamellar carcinoma, FLC; hepatocellular carcinoma, HCC; focal nodular hyperplasia, FNH and hepatocellular adenoma, HCA) was investigated by computer aided morphometry. A significantly higher connective tissue content was found in FNH and FLC as compared to HCA and HCC. The distribution of the connective tissue was homogeneous in the cases of FNH, HCA and HCC, while a highly unhomogeneous distribution was observed in FLC cases.

Connective tissue content of fibrolamellar carcinoma and other human liver tumors.

The connective tissue content of four different human liver tumors (Fibrolamellar carcinoma, FLC; hepatocellular carcinoma, HCC; focal nodular hyerplasia, FNH and hepatocellular adenoma, HCA) was investigated by computer aided morphometry. A significantly higher connective tissue content was found in FNH and FLC as compared to HCA and HCC. The distribution of the connective tissue was homogeneous in the cases of FNH, HCA and HCC, while a highly unhomogeneous distribution was observed in FLC cases.

Influence of protective drugs on the EEG during short-term transient ischaemia.

Influence of drugs on the cessation time of the brain electrical activity (resistance time) during total cerebral ischaemia evoked by clamping of the aorta for 50 sec; on the duration of its reappearance during reperfusion (restitution time) and on the background activity of EEG were studied. The experiments were carried out in 7 groups. Each group contained 5 animals. Nine clampings with intermittent reperfusion periods of 10 min were performed in each animal. One group served as control. In the remaining ones after the first three clampings the animals were given Glyo-6, Nootropil, Verpamil, Epanutin, Inactin or Lidocain. The resistance and restitution times measured in the control group as well as the reproductibility of the power spectrum values of the EEG provided evidence for the stability of the model. Glyo-6 in a dose of 10 mg/kg, Nootropil in a dose of 100 mg/kg or Verpamil in a dose of 0.125 mg/kg did not alter the above-mentioned parameters. As an effect of the administration of Epanutin in a dose of 10 mg/kg, the resistance time increased slightly, whereas restitution time decreased significantly. Administration of Inactin in a dose of 15 mg/kg, or Lidocain in a dose of 100 mg/kg increased considerably resistance time for a period of about one hour. The results indicate that in the initial phase of ischaemic brain damage both the cessation of EEG activity and the restitution during reperfusion after short-term occlusion of the circulation can be influenced favourable with drugs which decrease cerebral metabolism, inhibit synaptic transmission and have membrane stabilizing effect.

Aflatoxin B1 formamidopyrimidine adducts in human hepatocarcinogenesis: a preliminary report.

The presence of aflatoxin-B1-formamidopyrimidine, a persistent aflatoxin-deoxyribonucleic acid (DNA) adduct, was investigated in vivo by immunohistochemical analysis in 14 paired hepatocellular carcinoma and nontumorous human liver tissue sections using a monoclonal anti-aflatoxin-B1-formamidopyrimidine antibody. Nuclear and cytoplasmic accumulations of adducts were found in 4 of 14 nontumorous specimens but in none of 14 tumorous tissues and in none of three normal control livers. In vitro adduct formation and cellular DNA was investigated with a modified DNA immunoblot assay. These studies revealed (a) no significant difference in the amount of adduct formed by DNA samples with or without integrated hepatitis B virus DNA, (b) no difference in the amount of adduct formed with DNA from either tumorous or nontumorous tissues from a given individual, and (c) remarkable and reproducible differences in the capacity of DNA from different individuals to form in vitro adducts. Our DNA immunoblot assay will facilitate further studies on the molecular role of aflatoxin-B1-form-amidopyrimidine adducts in human hepatocarcinogenesis.

Keratinocyte growth factor ameliorates dextran sodium sulfate colitis in mice.

Keratinocyte growth factor (KGF) is emerging as an important mediator of mucosal defense and repair in the colon. The aim of the present study was to evaluate and further characterize the effects of exogenous KGF administration utilizing the dextran sodium sulfate (DSS) model of colitis in mice. Colitis was induced via oral administration of DSS (5 g/100 ml) to Balb/c mice for eight days. Intraperitoneal administration of KGF (5 mg/kg, once daily) or vehicle (VEH) was initiated 1 hr prior to the induction of the colitis (N = 10, each group). Mucosal injury of the entire colon was histologically assessed and graded. An approximately fourfold reduction in the crypt damage score was noted in the KGF group when compared to controls (VEH) (2.8 +/- 1.03 and 11.4 +/- 0.78, respectively). The significant reduction of mucosal injury in KGF treated mice confirms that KGF is a key mediator maintaining the integrity of the colonic mucosa.