Pegylated interferons Lambda-1a and alfa-2a display different gene induction and cytokine and chemokine release profiles in whole blood, human hepatocytes and peripheral blood mononuclear cells.

Pegylated interferon-lambda-1a (Lambda), a type III interferon (IFN) in clinical development for the treatment of chronic HCV infection, has shown comparable efficacy and an improved safety profile to a regimen based on pegylated IFN alfa-2a (alfa). To establish a mechanistic context for this improved profile, we investigated the ex vivo effects of Lambda and alfa on cytokine and chemokine release, and on expression of IFN-stimulated genes (ISGs) in primary human hepatocytes and peripheral blood mononuclear cells (PBMCs) from healthy subjects. Our findings were further compared with changes observed in blood analysed from HCV-infected patients treated with Lambda or alfa in clinical studies. mRNA transcript and protein expression of the IFN-λ-limiting receptor subunit was lower compared with IFN-α receptor subunits in all cell types. Upon stimulation, alfa and Lambda induced ISG expression in hepatocytes and PBMCs, although in PBMCs Lambda-induced ISG expression was modest. Furthermore, alfa and Lambda induced release of cytokines and chemokines from hepatocytes and PBMCs, although differences in their kinetics of induction were observed. In HCV-infected patients, alfa treatment induced ISG expression in whole blood after single and repeat dosing. Lambda treatment induced modest ISG expression after single dosing and showed no induction after repeat dosing. Alfa and Lambda treatment increased IP-10, iTAC, IL-6, MCP-1 and MIP-1ß levels in serum, with alfa inducing higher levels of all mediators compared with Lambda. Overall, ex vivo and in vivo induction profiles reported in this analysis strongly correlate with clinical observations of fewer related adverse events for Lambda vs those typically associated with alfa.

Transgenic overexpression of hepatocyte growth factor in the beta-cell markedly improves islet function and islet transplant outcomes in mice.

Recent advances in human islet transplantation have highlighted the need for expanding the pool of beta-cells available for transplantation. We have developed three transgenic models in which growth factors (hepatocyte growth factor [HGF], placental lactogen, or parathyroid hormone-related protein) have been targeted to the beta-cell using rat insulin promoter (RIP). Each displays an increase in islet size and islet number, and each displays insulin-mediated hypoglycemia. Of these three models, the RIP-HGF mouse displays the least impressive phenotype under basal conditions. In this study, we show that this mild basal phenotype is misleading and that RIP-HGF mice have a unique and salutary phenotype. Compared with normal islets, RIP-HGF islets contain more insulin per beta-cell (50 +/- 5 vs. 78 +/- 9 ng/islet equivalent [IE] in normal vs. RIP-HGF islets, P < 0.025), secrete more insulin in response to glucose in vivo (0.66 +/- 0.06 vs. 0.91 +/- 0.10 ng/ml in normal vs. RIP-HGF mice, P < 0.05) and in vitro (at 22.2 mmol/l glucose: 640 +/- 120.1 vs. 1,615 +/- 196.9 pg. microg protein(-1). 30 min(-1) in normal vs. RIP-HGF islets, P < 0.01), have two- to threefold higher GLUT2 and glucokinase steady-state mRNA levels, take up and metabolize glucose more effectively, and most importantly, function at least twice as effectively after transplantation. These findings indicate that HGF has surprisingly positive effects on beta-cell mitogenesis, glucose sensing, beta-cell markers of differentiation, and transplant survival. It appears to have a unique and unanticipated effective profile as an islet mass- and function-enhancing agent in vivo.

The processing and utilization of hepatocyte growth factor/scatter factor following partial hepatectomy in the rat.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pluripotent growth factor capable of acting as a motogen, a morphogen, and a mitogen. Originally, HGF/SF was found as a blood-borne mitogen for hepatocytes and has since been determined to be very important in liver repair. Previous studies have established that HGF/SF must be proteolytically cleaved to elicit its effects. After liver injury by toxins such as carbon tetrachloride or after surgical resection, partial hepatectomy (PHX), HGF/SF concentrations increase in the blood. The aims of this study were to examine (1) which form of HGF/SF is present in the normal liver, (2) which form is present in the regenerating liver after PHX, and (3) if the HGF/SF used after PHX is derived from existing liver reservoirs. Both single-chain HGF/SF and active two-chain HGF/SF are present in normal liver, with the former being the dominant form. After PHX, the liver can be described as having two phases with regard to the use of endogenous HGF/SF. The first phase from 0 to 3 hours is the consumptive phase and is characterized by a decrease in both single-chain HGF/SF and active two-chain HGF/SF. The second phase is the productive phase. It is characterized by a pronounced reappearance of both single-chain HGF/SF as well as two-chain HGF/SF. The activation index shows a 5-fold increase over sham operations during the productive phase. The use of radiolabeled HGF/SF showed that during the first 3 hours, HGF/SF is used in part from hepatic stores. Furthermore, during the first 3 hours after PHX, only active two-chain HGF/SF is seen in the plasma.

The C-terminal region of PTHrP, in addition to the nuclear localization signal, is essential for the intracrine stimulation of proliferation in vascular smooth muscle cells.

PTHrP is secreted by most cell types. In addition to a paracrine/autocrine role, PTHrP has "intracrine" actions, entering the nuclear compartment under the direction of a classic bipartite nuclear localization signal. In vascular smooth muscle cells, nuclear entry stimulates mitogenesis. In the current study, we sought to more precisely define the regions of PTHrP required for the activation of mitogenesis in vascular smooth muscle cells. PTHrP deletion mutants missing large regions [i.e. the signal peptide, N terminus (1--36), mid region (38--86), nuclear localization signal, C terminus (108--139), or combinations of the above] were expressed in A-10 vascular smooth muscle cells. The consequences on nuclear localization and proliferation were examined. Deletion of the nuclear localization signal prevented nuclear entry and slowed proliferation. Deletion of the highly conserved N terminus or mid region had no impact on nuclear localization or on proliferation. Deletion of the C terminus had no deleterious effect on nuclear localization but dramatically reduced proliferation. Thus, the nuclear localization signal is both necessary and sufficient for nuclear localization of PTHrP. In contrast, activation of proliferation in vascular smooth muscle cells requires both an intact nuclear localization signal and an intact C terminus. Whereas the nuclear localization signal is required for nuclear entry, the C terminus may serve a trans-activating function to stimulate mitogenesis once inside the nucleus of vascular smooth muscle cells.

Altered content and modulation of soluble guanylate cyclase in the cerebellum of rats with portacaval anastomosis.

It is shown that the glutamate-NO-cGMP pathway is impaired in cerebellum of rats with portacaval anastomosis in vivo as assessed by in vivo brain microdialysis in freely moving rats. NMDA-induced increase in extracellular cGMP in the cerebellum was significantly reduced (by 27%) in rats with portacaval anastomosis. Activation of soluble guanylate cyclase by the NO-generating agent S-nitroso-N-acetyl-penicillamine and by the NO-independent activator YC-1 was also significantly reduced (by 35-40%), indicating that portacaval anastomosis leads to remarkable alterations in the modulation of guanylate cyclase in cerebellum. Moreover, the content of soluble guanylate cyclase was increased ca. two-fold in the cerebellum of rats with portacaval anastomosis. Activation of soluble guanylate cyclase by NO was higher in lymphocytes isolated from rats with portacaval anastomosis (3.3-fold) than in lymphocytes from control rats (2.1-fold). The results reported show that the content and modulation of soluble guanylate cyclase are altered in brain of rats with hepatic failure, resulting in altered function of the glutamate-NO-cGMP pathway in the rat in vivo. This may lead to alterations in cerebral processes such as intercellular communication, circadian rhythms, including the sleep-waking cycle, long-term potentiation, and some forms of learning and memory.

Using beta-cell growth factors to enhance human pancreatic Islet transplantation.

This is a particularly exciting time in the field of pancreatic islet growth, development, and survival. The recent publication of a study demonstrating that human pancreatic islet transplantation is both technically and immunologically feasible has highlighted the need for large supplies of pancreatic islets or pancreatic beta cells for larger-scale islet transplantation in patients with diabetes. This, together with a rapid expansion in the past several years of the understanding of mechanisms of islet growth, development, and survival, has accelerated and invigorated efforts to therapeutically harness the cellular mechanisms responsible for pancreatic beta-cell proliferation, survival, and development and to take advantage of this new knowledge to enhance the availability, survival, and function of pancreatic beta cells in human islet transplantation for diabetes mellitus. Here, we briefly review the confluence of events that have provided optimism and energy to the islet transplant field, and we focus on peptide growth factors that eventually may be deployed in the effort to augment islet mass and function in patients with diabetes.

Outcome and hepatic hemodynamics in liver transplant patients with portal vein arterialization.

UNLABELLED: Few cases of successful portal vein arterialization in orthotopic and auxiliary liver transplantation have been reported. AIM: To evaluate the effect of portal vein arterialization on hepatic hemodynamics and long-term clinical outcome in three patients undergoing liver transplantation. METHODS: Two patients with extensive splanchnic venous thrombosis received an orthotopic liver transplant and one with fulminant hepatic failure received an auxiliary heterotopic graft. Portal vein arterialization was performed in all cases. RESULTS: One patient died 4 months after transplant and two are still alive. Auxiliary liver graft was removed 3 months post-transplant when complete native liver regeneration was achieved. Immediate post-transplant liver function was excellent in all cases. Only one patient developed encephalopathy and variceal bleeding owing to prehepatic portal hypertension secondary to arterioportal fistula 14 months after transplant. He was successfully treated by embolization of the hepatic artery. Hepatic hemodynamic measurements demonstrated a normal pressure gradient between wedged and free hepatic venous pressures in all cases. Liver biopsy showed acceptable graft architecture in two cases and microsteatosis in one. CONCLUSIONS: Liver transplantation with portal vein arterialization is an acceptable salvage alternative when insufficient portal venous flow to the graft is present. The double arterial supply does not imply changes in hepatic hemodynamics, at least in the early months post-transplant.

Somatostatin plus isosorbide 5-mononitrate versus somatostatin in the control of acute gastro-oesophageal variceal bleeding: a double blind, randomised, placebo controlled clinical trial.

BACKGROUND: Variceal bleeding is a severe complication of portal hypertension. Somatostatin reduces portal pressure by decreasing splanchnic blood flow, and nitrates by diminishing intrahepatic resistance. Experimental studies have shown that the combination of somatostatin and nitrates has an additive effect in decreasing portal pressure. AIM: To compare the therapeutic efficacy of either intravenous infusion of somatostatin plus oral isosorbide 5-mononitrate or somatostatin alone in gastro-oesophageal variceal bleeding associated with liver cirrhosis. METHODS: A unicentre, double blind, placebo controlled, clinical trial was conducted. Sixty patients bleeding from oesophageal or gastric varices were randomised to receive intravenous infusion of somatostatin (250 microg/hour) plus oral isosorbide 5-mononitrate (40 mg/12 hours) (group I) or somatostatin infusion plus placebo (group II) for 72 hours. RESULTS: The two groups of patients had similar clinical, endoscopic, and haematological characteristics. Control of bleeding was achieved in 18 out of 30 patients (60%) in group I and 26 out of 30 patients (87%) in group II (p<0.05). There was no significant difference in mean transfusion requirements between the two groups: 2.6 (2.2) v 1.8 (1.6) respectively; means (SD). Mortality and side effects were similar in the two groups, but development of ascites was higher in group I (30%) than in group II (7%) (p<0.05). CONCLUSION: In cirrhotic patients with acute gastro-oesophageal variceal bleeding, addition of isosorbide 5-mononitrate to somatostatin does not improve therapeutic efficacy, induces more adverse effects, and should not be used.

Cleavage of the HER2 ectodomain is a pervanadate-activable process that is inhibited by the tissue inhibitor of metalloproteases-1 in breast cancer cells.

HER2/neu, a Mr 185,000 tyrosine kinase receptor that is overexpressed in breast cancer, undergoes proteolytic cleavage of its extracellular domain (ECD). In contrast with other membrane-bound proteins, including growth factor receptors, that are cleaved by a common machinery system, we show that HER2 cleavage is a slow process and is not activated by protein kinase C. Pervanadate, a general inhibitor of protein-tyrosine phosphatases, induces a rapid and potent shedding of HER2 ECD. The shedding of HER2 ECD is inhibited by the broad-spectrum metalloprotease inhibitors EDTA, TAPI-2, and batimastat. The tissue inhibitor of metalloproteases-1; an inhibitor of matrix metalloproteases that does not inhibit cleavage by the general protein kinase C-dependent shedding machinery, also inhibited HER2 ECD shedding, whereas tissue inhibitor of metalloproteases-2 did not. These data suggest that HER2 cleavage is a process regulated by an as-yet-unidentified distinct protease.

Interleukin-6, nitric oxide, and the clinical and hemodynamic alterations of patients with liver cirrhosis.

OBJECTIVE: Nitric oxide has been proposed as a mediator of hyperdynamic circulation in cirrhosis. Endotoxin and cytokines induce the synthesis of nitric oxide. The aim of this study was to investigate the relationship between endotoxemia, cytokines, and nitric oxide in patients with cirrhosis, and to correlate these findings with clinical, biochemical, and hemodynamic parameters. METHODS: Clinical, biochemical, and hemodynamic parameters were assessed in 66 patients with cirrhosis and 15 controls. Levels of antidiuretic hormone, plasma renin activity, aldosterone, interferon gamma, interleukin-1, interleukin-6, tumor necrosis factor alpha, endotoxin, and nitrates-nitrites were determined. RESULTS: Mean arterial pressure was lower and interleukin-6, tumor necrosis factor alpha, nitrites-nitrates levels, and endotoxin positivity rates were higher in cirrhotics than in controls (p < 0.005). Mean arterial pressure decreased and interleukin-6 levels increased with worsening of Child score (p < 0.005). Patients with ascites had higher levels of interleukin-6, tumor necrosis factor alpha, and nitrates-nitrites than patients without ascites (p < 0.01). Elevated levels of interleukin-6 were found in patients with encephalopathy grade I, compared with patients without (p < 0.001); this association was independent of the severity of liver disease. In patients with low mean arterial pressure, interleukin-6 levels were higher than in patients with high mean arterial pressure (p = 0.001), whereas tumor necrosis factor alpha and nitrates-nitrites levels were not different. By multivariate analysis, high interleukin-6 levels showed independent associations with the presence of ascites, encephalopathy, and low mean arterial pressure. Only interleukin-6 levels had significant correlations with Child score, plasma renin activity, serum and urinary sodium, and mean arterial pressure (r > or = 0.4, p < 0.005). CONCLUSIONS: Although the activity of the nitric oxide pathway is increased in patients with cirrhosis and might contribute to the hemodynamic alteration, other factors are involved. Interleukin-6, possibly through nitric oxide-independent mechanisms, also might play a role in the vasodilatation of cirrhosis and the pathogenesis of hepatic encephalopathy.

Tyrosine kinase inhibition ameliorates the hyperdynamic state and decreases nitric oxide production in cirrhotic rats with portal hypertension and ascites.

Tumor necrosis factor-alpha (TNF) causes vasodilatation and a hyperdynamic state by activating nitric oxide (NO) synthesis. Tyrphostins, specific inhibitors of protein tyrosine kinase (PTK), block the signaling events induced by TNF and NO production. A hyperdynamic circulatory syndrome (HCS) is often observed in portal hypertension (PHT). TNF and NO seem to mediate these hemodynamic changes. The aim of this work was to study the effect of PTK inhibition on the systemic and portal hemodynamics, TNF and NO production, in cirrhotic rats with portal hypertension. Rats with liver cirrhosis induced by chronic inhalation of carbon tetrachloride were used. Animals were treated daily with tyrphostin AG 126 (alpha-cyano-(3-hydroxy-4-nitro) cinnamonitrile) or placebo for 5 d. Mean arterial pressure (MAP), heart rate (HR), and portal pressure (PP) were measured by indwelling catheters. Cardiac output (CI) and stroke volume (SV) were estimated by thermodilution, systemic vascular resistance (SVR) was calculated (MAP/CI), and portal systemic shunting (PSS) was quantitated using radioactive microspheres. Serum and mesenteric lymph node (MLN) TNF levels were measured using an immunoassay kit, and serum NOx was determined photometrically by its oxidation products. The AG 126-treated group showed a statistically significant increase in MAP and SVR, and decreases in CI, SV, MLN TNF, and serum NO oxidation products nitrite and nitrate (NOx) in comparison with the placebo-treated rats. No significant differences were noticed in HR, PP, PSS, or serum TNF. Significant correlations were observed between MAP and NOx, MAP and MLN TNF, PSS and NOx, and serum TNF and serum NOx. The HCS observed in PHT seems to be mediated, at least in part, by TNF and NO by the activation of PTKs and their signaling pathways. PTK activity inhibition ameliorates the hyperdynamic abnormalities that characterize animals with cirrhosis and PHT.

Thalidomide inhibits tumor necrosis factor alpha, decreases nitric oxide synthesis, and ameliorates the hyperdynamic circulatory syndrome in portal-hypertensive rats.

A hyperdynamic circulatory state frequently is observed in portal hypertension with liver failure or extensive portal-systemic shunting. Tumor necrosis factor alpha (TNF) causes marked hypotension in mammals by inducing nitric oxide synthesis and has been shown to play a role in the development of the hemodynamic changes observed in portal hypertension. Thalidomide selectively inhibits TNF production by enhancing messenger RNA degradation. We investigated the systemic and portal hemodynamic effects of thalidomide in a prehepatic model of portal hypertension and evaluated whether suppressing TNF synthesis decreases NO production. Portal hypertension was induced by partial ligation of the portal vein (PVL). Animals received thalidomide (T) (50 mg/kg/d) + water or water alone (W), orally, daily for 2 days before and 13 days after PVL operation, at which time hemodynamic studies were performed and TNF plasma levels were obtained. Sham-operated animals were studied identically. In an additional group of PVL animals, 24-hour urinary excretion of NO2- and NO3- was measured during treatment. PVL animals receiving T presented with a significantly higher mean arterial pressure and systemic vascular resistance and significantly lower portal pressure, TNF plasma levels, and 24-hour urinary excretion of NO2- and NO3-, in comparison with rats receiving W. A significant correlation (r = -0.61) was observed between TNF plasma levels and mean arterial pressure among PVL animals. Thalidomide did not have any significant effects on sham rats. Thalidomide inhibits TNF synthesis and reduces NO production, blunts the development of the hyperdynamic circulation, and decreases portal pressure in PVL-operated rats.

Epstein-Barr virus infection associated with interstitial nephritis and chronic fatigue.

Severe renal disease in the setting of Epstein-Barr virus (EBV) infection is exceedingly rare. We report here the case of a 22-year-old man with acute EBV infection associated with severe interstitial nephritis. The patient developed chronic fatigue and chronic renal failure with a serological profile typical of primary EBV infection. Clinical improvement with anti-EBNA seroconversion occurred after acyclovir therapy. Our patient illustrates that chronic fatigue with major organ dysfunction and a serological profile of primary infection can be seen in chronic EBV infection. In such a case, acyclovir may prove beneficial.

Tumor necrosis factor alpha: a major contributor to the hyperdynamic circulation in prehepatic portal-hypertensive rats.

BACKGROUND/AIMS: Portal hypertension is often accompanied by a hyperdynamic circulatory syndrome. Tumor necrosis factor (TNF) alpha causes vasodilatation and a hyperdynamic state in mammals by activating nitric oxide synthesis. The aim of this study was to investigate whether TNF-alpha plays a role in developing the hyperdynamic syndrome in portal hypertension. METHODS: Portal-hypertensive rats, induced by partial ligation of the portal vein (PVL), were used. In experiment 1, rats that underwent PVL were treated with polyclonal anti-mouse TNF-alpha or placebo intravenously the same day of the PVL operation and 24 hours before hemodynamic studies. Hemodynamic studies were performed 5 days after PVL. In experiment 2, rats that underwent PVL received anti-TNF-alpha or placebo intravenously 3 days and 24 hours before hemodynamics as in experiment 1. Hemodynamics were performed 14 days after the PVL operation. TNF-alpha blood levels were measured using a bioassay. RESULTS: Anti-TNF-alpha treatment induced a significant increase in mean arterial pressure, heart rate, and systemic vascular resistance and a significant decrease in cardiac index, portal pressure, and TNF-alpha levels in comparison with placebo animals. No significant effects were observed in sham rats. CONCLUSIONS: Anti-TNF-alpha treatment in rats that underwent PVL significantly blunts the development of the hyperdynamic circulation and reduces portal pressure. TNF-alpha may play a role in the hemodynamic abnormalities of portal hypertension.

Noninvasive measurement of femoral blood flow and portal pressure response to propranolol in patients with cirrhosis.

This study investigated the correlation between changes in hepatic and systemic hemodynamics and femoral blood flow (FBF), measured by dual-beam pulsed wave Doppler, in 58 portal hypertensive patients receiving propranolol (0.15 mg/Kg intravenously; n = 44) or placebo (n = 14) under double-blind conditions. Placebo administration had no effects. Propranolol caused significant reductions (P < .0001) in hepatic venous pressure gradient (HVPG; from 19.1 +/- 4.1 to 16.2 +/- 4.2 mm Hg), azygos blood flow (from 563 +/- 204 to 387 +/- 176 mL/min), cardiac index (CI; from 4.4 +/- 1.0 to 3.3 +/- 0.8 L/m2/min), and FBF (from 237 +/- 79 to 176 +/- 58 mL/m2/min). In 17 patients HVPG decreased below 12 mm Hg and/or more than 20% of the baseline value (good response; mean change, -26 +/- 8%); in the remaining 27 patients (poor response) the mean change in HVPG was less: -9 +/- 6%. Patients with a good response had bled less often from varices, had significantly higher FBF (272 +/- 73 vs. 215 +/- 76 mL/m2/min) and lower baseline HVPG (16.8 +/- 3.9 vs. 20.6 +/- 3.6 mm Hg) than those with poor response in HVPG. The good response was also associated with greater decreases in FBF (-33 +/- 12 vs. -19 +/- 13% in poor responders), CI (-30 +/- 9 vs. -19 +/- 12%), and heart rate (-19 +/- 5 vs. -16 +/- 6%). A decrease in FBF of > 20% predicted a good response in 16 of 28 patients (positive predictive value, 57%).(ABSTRACT TRUNCATED AT 250 WORDS)