Biomedical subjects

Frank Dombrowski

Publications and source records attributed to Frank Dombrowski.

29 records · Page 2Linked to original sources

PubMed2004

Insulin receptor, insulin receptor substrate-1, Raf-1, and Mek-1 during hormonal hepatocarcinogenesis by intrahepatic pancreatic islet transplantation in diabetic rats.

Low-number transplantation of pancreatic islets into the livers of diabetic rats leads to transformation of the downstream liver acini into clear-cell foci of altered hepatocytes (FAHs). These FAHs correspond to the glycogen-storing (clear-cell) phenotype of hepatocellular preneoplasias and develop into hepatocellular adenomas (HCAs) and hepatocellular carcinomas (HCCs) within 6 to 24 months. In addition, they show metabolic alterations that resemble well-known insulin effects, most likely constituting the result of the local hyperinsulinemia. Thus, we investigated FAHs, HCAs, and HCCs for altered expression of insulin receptor, insulin receptor substrate-1 (IRS-1), Raf-1 and Mek-1. Light and electron microscopic immunohistochemistry revealed a translocation of insulin receptor from the plasma membrane (normal tissue) into the cytoplasm in clear-cell FAHs and an increase in insulin receptor expression in HCAs and HCCs. FAHs also showed an increase in IRS-1 gene expression, investigated by in situ hybridization and quantitative reverse transcription-PCR. IRS-1, Raf-1, and Mek-1 proteins were strongly overexpressed in FAHs and tumors, as compared with the unaltered liver tissue. These overexpressions were closely linked to the clear-cell phenotype of preneoplastic and neoplastic hepatocytes, because basophilic FAHs (later stages) and basophilic tumors showed no overexpressions. In this endocrine model of hepatocarcinogenesis, severe alterations of insulin signaling were induced by the pathological local action of islet hormones in the livers and may substantially contribute to the carcinogenic process.

Animals↗

PubMed2004

Aberrant acinar cell CA 19-9 expression and peri-insular acinar cell alterations in an adult human pancreas.

We herein report on a 23-year-old female patient who suffered from Crohn's disease and anorexia nervosa and died after long-term malnutrition and a perforated colitis. At autopsy, her pancreas displayed two peculiar findings. First, there was a constant and aberrant expression of CA 19-9 in the acinar cells. The expression of the carbohydrate antigen CA 19-9 is normally confined to duct-like epithelia, including centroacinar cells, while islet and acinar cells have repeatedly been reported to be immunohistochemically negative. Thus, to the best of our knowledge, our case is the first to show aberrant acinar CA 19-9 expression, and its potential meaning is discussed. Second, the pancreas showed a heterogeneity in acinar morphology, with peri-insular acini being considerably larger than tele-insular acini. The existence of enlarged peri-insular acini, mainly in animals, has occasionally been reported. Its origin, however, is still unclear. Some authors have proposed an influence of high insulin concentrations, exerted via the insulo-acinar capillary axis. We agree with the concept of an islet-derived mechanism. However, as we have observed a similar heterogeneity in streptozotocin- and autoimmune-diabetic rats, we presume that other islet hormones, in particular glucagon, might be more important for this phenomenon in the animals, as well as in the present case.

Adult↗

PubMed2004

Insulin-like growth factor (IGF)-binding protein-1 is highly induced during acute carbon tetrachloride liver injury and potentiates the IGF-I-stimulated activation of rat hepatic stellate cells.

Hepatic stellate cells (HSC) play a pivotal role in hepatic tissue repair and fibrogenesis. IGF-I has been considered a mitogenic signal for activation and proliferation of HSC in vitro. In the present study IGF-I and IGF-binding protein (IGFBP) gene expression was studied in a model of acute liver injury induced by a single intragastric dose of carbon tetrachloride (CCl(4)) in adult rats. Northern blot analysis revealed a marked increase in IGFBP-1 mRNA levels, with a maximum between 3 and 9 h after CCl(4) application, whereas steady state mRNA levels of IGF-I were only moderately altered. In situ hybridization experiments demonstrated that this increase in IGFBP-1 mRNA was due to a strong expression of IGFBP-1 in the perivenous region 6-12 h after CCl(4) application, extending to the midzonal region of the acinus within 24-48 h. Consequently, a prominent immunostaining for IGFBP-1 was observed in perivenous areas, with a maximum 24-48 h after intoxication. Preincubation of early cultured HSC with a nonphosphorylated IGFBP-1 from human amniotic fluid resulted in a 3.4-fold increase in IGF-I-induced DNA synthesis. The mitogenic effect of IGF-I was also potentiated when HSC were cocultivated with IGFBP-1-overexpressing BHK-21 cells compared with nontransfected cells. These data suggest that IGFBP-1 released during the early steps of liver tissue damage and repair may interact with HSC and potentiate the sensitivity of IGF-I to mitogenic signals.

Acute Disease↗

PubMed2003

L-carnitine ameliorates abnormal vulnerability of steatotic rat livers to cold ischemic preservation.

BACKGROUND: Up to 30% of all livers retrieved for organ transplantation exhibit steatotic transformations. Chronic organ-donor shortage has led to the acceptance of these organs for transplantation, although a higher risk of graft nonfunction is associated with the preservation of steatotic livers. METHODS: A dietary steatosis was induced in Wistar rats by fasting them for 2 days and feeding them with a fat-free diet. Fatty livers (n=14) were retrieved and flushed with 60 mL of histidine, tryptophane, alpha-ketoglutarate (HTK) solution. In half of the experiments, L-carnitine (5 mM) was added to the HTK. Functional integrity of the livers was evaluated by isolated reperfusion with KHB in a recirculating system at 37 degrees C for 45 minutes. RESULTS: Addition of L-carnitine to the HTK promoted a significant reduction of the enzyme leakage from the livers upon reperfusion. Release of alanine-aminotransferase was reduced to one third (127+/-22 vs. 423+/-61 U/L), and the loss of glutamate dehydrogenase in the perfusate could be reduced significantly (42+/-7 vs. 542+/-134 U/L) when compared with livers stored without additional medication. Morphologic corroboration of these data was obtained by electron microscopy. Although normal appearance of liver mitochondria was preserved at the end of the cold ischemic storage, reperfusion of cold-stored fatty livers entailed massive alterations and frequent destruction of hepatic mitochondria. However, these morphologic impairments were remarkably mitigated in the carnitine-treated group. CONCLUSIONS: L-carnitine represents a feasible metabolic adjunct for a safe and more successful preservation of ischemia-reperfusion-sensitive steatotic livers.

Alanine Transaminase↗

PubMed2003

Involvement of integrins in osmosensing and signaling toward autophagic proteolysis in rat liver.

Inhibition of autophagic proteolysis by hypoosmotic or amino acid-induced hepatocyte swelling requires osmosignaling toward p38MAPK; however, the upstream osmosensing and signaling events are unknown. These were studied in the intact perfused rat liver with a preserved in situ environment of hepatocytes. It was found that hypoosmotic hepatocyte swelling led to an activation of Src (but not FAK), Erks, and p38MAPK, which was prevented by the integrin inhibitory hexapeptide GRGDSP, but not its inactive analogue GRGESP. Src inhibition by PP-2 prevented hypoosmotic MAP kinase activation, indicating that the integrin/Src system is located upstream in the osmosignaling toward p38MAPK and Erks. Inhibition of the integrin/Src system by the RGD motif-containing peptide or PP-2 also prevented the inhibition of proteolysis and the decrease in autophagic vacuole volume, which is otherwise observed in response to hypoosmotic or glutamine/glycine-induced hepatocyte swelling. These inhibitors, however, did not affect swelling-independent proteolysis inhibition by phenylalanine. In line with a role of p38MAPK in triggering the volume regulatory decrease (RVD), PP-2 and the RGD peptide blunted RVD in response to hypoosmotic cell swelling. The data identify integrins and Src as upstream events in the osmosignaling toward MAP kinases, proteolysis, and RVD. They further point to a role of integrins as osmo- and mechanosensors in the intact liver, which may provide a link between cell volume and cell function.

Animals↗

PubMed2003

Apolipoprotein A-IV mRNA overexpression in early preneoplastic hepatic foci induced by low-number pancreatic islet transplants in streptozotocin-diabetic rats.

After low-number transplantation of islets of Langerhans into the liver of streptozotocin-diabetic rats, the hepatocytes in the acini, draining the blood from the islets, are exposed to a local hyperinsulinemia, whereas the remaining tissue is affected by hypoinsulinemia. In this model, insulin induces alterations that resemble preneoplastic foci of altered hepatocytes (FAH) and develop into hepatocellular tumors in later stages of carcinogenesis. In rodents, apolipoprotein A-IV (A-IV) is synthesized in the small intestine and the liver. Whereas intestinal production is mainly influenced by lipid intake and chylomicrone formation, little is known about mechanisms regulating hepatic A-IV synthesis. As it is known that insulin modulates lipoprotein metabolism in different ways, we investigated the effect of insulin on hepatocytic A-IV mRNA expression in this model. After Laser microdissection of FAH and quantitative RT-PCR (LightCycler), we found a 3.2 to 7.4-fold increase of A-IV mRNA in the FAH. To the best of our knowledge, these results represent the first data of insulin-stimulated A-IV mRNA overexpression in rat hepatocytes in vivo, and are in line with previously reported results of experiments with cultured hepatocytes. It remains to be elucidated whether A-IV mRNA overexpression is only an epiphenomenon of insulin action or is relevant for hepatocarcinogenesis in this model.

Animals↗

PubMed2002

Quantitative analysis of gap-junctional intercellular communication in precision-cut mouse liver slices.

Direct intercellular communication through gap junction channels is involved in the maintenance of tissue homeostasis and suppression of carcinogenesis. Gap-junctional communication is often altered in tumor cells but it can also be modulated in response to tumor promotors or inflammatory signals. In order to evaluate the effect of nongenotoxic compounds, suggested to be involved in tumor promotion, on gap junctional intercellular communication in the liver, we have developed a direct dye transfer method. The fluorescent dye Alexa Fluor 488 was iontophoretically injected into hepatocytes of freshly prepared, precision-cut mouse liver slices (250 microm). The area of dye spreading was monitored and quantified by microscopy. Comparison of dye spreading in connexin-32-deficient versus wild-type liver revealed a 96% decrease in connexin-32-deficient tissue. Induction of an acute phase response in connexin-32-deficient mice by intraperitoneal injection of lipopolysaccharide increased dye coupling by 33%, probably due to upregulation of connexin-26-containing gap junction channels.

Animals↗

PubMed2002

Liver preservation with HTK: salutary effect of hypothermic aerobiosis by either gaseous oxygen or machine perfusion.

The aim of the present study was to improve the viability of marginal livers from non-heart beating donors upon cold preservation using two different techniques for the provision of tissue aerobiosis. Livers from male Wistar rats (250-300 g bw) were harvested after 60 min of cardiac arrest, flushed via the portal vein with 20 mL of heparinized Ringer's solution and 60 mL of histidine-tryptophan-ketoglutarate (HTK) preservation solution. Control livers were then stored submerged in HTK for 24 h at 4 degrees C while other organs were subjected to aerobic conditions by either insufflation of gaseous oxygen via the venous vascular system of the cold stored organ (VSOP) or pulsatile machine perfusion (MP) with oxygenated HTK at 5 mL/min at 4 degrees C. Superoxide dismutase (SOD) (7500 IU) was added to the last 10 mL of HTK in order to prevent adverse effects of high oxygen tensions at hypothermia. Viability of the livers was assessed upon isolated perfusion in vitro with oxygenated Krebs-Henseleit buffer at constant flow. VSOP or MP, both significantly improved vascular conductivity upon reperfusion as evaluated by portal venous pressure, reduced hepatic enzyme release and led to a rise in hepatic bile production upon reperfusion. Induction of apoptosis was also looked for in tissue homogenates by Western analysis for cleavage of poly(ADP-ribose)polymerase (PARP). Expression of cleaved PARP fragment could be found in reperfused control livers but also, though to a lesser extend, after VSOP or MP. In conclusion, provision of oxygen during cold preservation significantly contributes to improve organ viability upon reperfusion and must be regarded as a useful adjunct for marginal or pre-damaged livers. HTK has been shown for the first time to be also suitable for long-term MP preservation of the liver, but, as inferred from these data, simple insufflation of gaseous O2 may be considered a feasible alternative.

Aerobiosis↗

PubMed2002

DNA vaccination with AFP-encoding plasmid DNA prevents growth of subcutaneous AFP-expressing tumors and does not interfere with liver regeneration in mice.

The oncofetal alpha-fetoprotein (AFP) is reexpressed in the majority of hepatocellular carcinomas and may be used as a target molecule for an immunotherapy or prophylaxis against this tumor. We investigated the potential of DNA vaccination with AFP-expressing plasmid DNA to induce an immune response against AFP-expressing tumor cells in DBA/2 mice. 62.5% of mice vaccinated with AFP-expressing plasmid DNA, rejected subcutaneous syngeneic AFP-expressing P815 tumors, whereas only 16.7% of mice vaccinated with control plasmid rejected these tumor cells (P=.03). Mean survival of mice after challenge with subcutaneous AFP-expressing tumor cells was prolonged for 8 days in mice vaccinated with AFP-expressing DNA (35 days) compared to mice vaccinated with control plasmid (27 days). To rule out possible autoimmune reactions against regenerating liver, which also reexpresses AFP, we evaluated the influence of AFP-specific DNA vaccination on liver regeneration in DBA/2 mice. Histologic quantification of proliferating hepatocytes and of the amount of necrotic liver tissue in carbon tetrachloride-damaged liver did not reveal statistically significant differences in mice vaccinated with AFP-expressing plasmid compared to control mice. These data suggest that AFP-specific DNA vaccination represents a useful tool to inhibit growth of AFP-expressing tumors in mice that does not affect liver regeneration.

Animals↗

PubMed2002

Atypical manifestation of a cytoplasmic antineutrophil cytoplasmic antibody (PR3-ANCA)-associated vasculitis with involvement of aortic intima and parietal endocardium.

The traditional classification of vasculitis, based on the size of affected vessels, has meanwhile been extended by using antineutrophil cytoplasmic antibodies (ANCAs) as seromarkers in the differential diagnosis of different types of vasculitis. We report an autopsy case of fulminant generalized vasculitis positive for C-ANCA (1:320) and anti-proteinase 3 (PR3) antibodies (>100 U/mL) in a 63-year-old man. The unusually broad histologic spectrum included periarteritis nodosa-like lesions in medium-sized vessels and leucocytoclastic vasculitis in small vessels, as well as capillaritis. In addition, the left atrial and ventricular endocardium and the intima of the aorta thoracalis were patchily involved in the inflammatory process. Glomerulonephritis and/or immune complexes were not detectable by electron microscopy or immunohistochemistry. To the best of our knowledge, involvement of the aortic intima ("intimitis") and the parietal endocardium has not been described in PR3-ANCA-positive vasculitis to date.

Antibodies, Antineutrophil Cytoplasmic↗

PubMed2002

Morphology and morphometric investigation of hepatocellular preneoplastic lesions and neoplasms in connexin32-deficient mice.

Gap junctions are composed of protein subunits, called connexins, and provide a pathway for the exchange of ions and small molecules between contacting cells. This transfer of molecules is thought to be an important pathway for direct cell communication, and is involved in tissue homeostasis, growth control and embryonic development. Impairment of gap junctional intercellular communication (GJIC) via different mechanisms may therefore contribute to dysregulated cellular proliferation and subsequent tumor development. We investigated the effect of Connexin32-deficiency on liver histology and the formation of preneoplastic foci and hepatocellular neoplasms in transgenic knockout mice, as Connexin32 (Cx32) is the major gap junction protein in the liver. Loss of Cx32 does not alter the morphology of extrafocal liver tissue. However, after administration of a single dose of diethylnitrosamine (DEN), given 2 weeks after birth, the number and volume fraction of preneoplastic foci showed a 3.3-fold to 12.8-fold increase in the Cx32-deficient mice as compared with the corresponding wildtype groups, regardless of sex and age of the animals. Number and volume fraction of hepatocellular adenomas and carcinomas also increased significantly in these animals. The experimental groups did not differ in the morphology of the different types of preneoplastic foci and neoplasms. On the other hand, Cx32-deficiency without DEN treatment did not lead to an increase in the spontaneous development of any type of preneoplastic hepatic foci or hepatocellular neoplasms in up to 18-month-old Cx32-deficient mice as compared with wildtype controls. In conclusion, our results indicate that impairment of GJIC in mouse liver due to deletion of the Cx32 coding DNA clearly promotes the carcinogenic effect of DEN administration and results in a higher susceptibility to hepatocellular neoplasms, but does not appear to initiate hepatic tumor development.

Animals↗