Recombinant Human HPS Protects Mice and Nonhuman Primates from Acute Liver Injury.

Acute liver injury shares a common feature of hepatocytes death, immune system disorders, and cellular stress. Hepassocin (HPS) is a hepatokine that has ability to promote hepatocytes proliferation and to protect rats from D-galactose (D-Gal)- or carbon tetrachloride (CCl4)-induced liver injury by stimulating hepatocytes proliferation and preventing the high mortality rate, hepatocyte death, and hepatic inflammation. In this paper, we generated a pharmaceutical-grade recombinant human HPS using mammalian cells expression system and evaluated the effects of HPS administration on the pathogenesis of acute liver injury in monkey and mice. In the model mice of D-galactosamine (D-GalN) plus lipopolysaccharide (LPS)-induced liver injury, HPS treatment significantly reduced hepatocyte death and inflammation response, and consequently attenuated the development of acute liver failure. In the model monkey of D-GalN-induced liver injury, HPS administration promoted hepatocytes proliferation, prevented hepatocyte apoptosis and oxidation stress, and resulted in amelioration of liver injury. Furthermore, the primary pharmacokinetic study showed natural HPS possesses favorable pharmacokinetics; the acute toxicity study indicated no significant changes in behavioral, clinical, or histopathological parameters of HPS-treated mice, implying the clinical potential of HPS. Our results suggest that exogenous HPS has protective effects on acute liver injury in both mice and monkeys. HPS or HPS analogues and mimetics may provide novel drugs for the treatment of acute liver injury.

[Assessment of protein synthesizing function of the liver in experimental hepatitis].

Liver protein synthesis was estimated comparing the levels of prothrombin and its inactive form PIVKA-prothrombin. The latter indicates liver dysfunction. These diagnostic tests allow monitoring the effectiveness of the commonly applied preparation "Essentiale Forte" and that of the liposomal form of the biologically active additive (BAA) FLP-MD based on phospholipids of various origin.

Cancer-induced cachexia: a guide for the oncologist.

Cancer-induced cachexia (CIC) is a paraneoplastic syndrome that may account for up to 20% of deaths in cancer patients. Cachexia includes distinct metabolic changes that are the result of an acute-phase response (APR) mounted by the host as a reaction to tumor cells. These changes include increased muscle proteolysis, increased fat lipolysis, and increased hepatic production of acute-phase proteins such as C-reactive protein and fibrinogen. This APR pathogenesis is an important consideration in trying to treat cachectic patients as most therapies do not target the APR and its subsequent metabolic effects. Although there is currently no cure for CIC, the oncologist frequently encounters cachectic patients in practice, and evidence-based management is needed. We review the current data for assessment of starvation and cachexia, providing guidelines for management that include serum markers and functional assessment. In addition, a review of current therapies is provided, including hypercaloric feeding and nutritional intervention to address starvation, as well as data on appetite stimulants such as corticosteroids and megestrol acetate. Experimental therapies are also discussed, including nonsteroidal antiinflammatory drugs, tumor necrosis factor alpha antagonists, tetrahydrocannabinol, growth hormone, ghrelin, oxandrolone, and omega-3 fatty acids.

Effects of infliximab and parenteral nutrition on albumin and fibrinogen synthesis rates in pediatric Crohn disease.

OBJECTIVES: Tumor necrosis factor-alpha (TNF-alpha) may play a significant role in growth disturbance in pediatric Crohn disease. The aim of this study was to determine the effects of anti-TNF-alpha therapy on albumin and fibrinogen synthesis during both fasting and parenteral nutrition infusion in pediatric patients with active Crohn disease. PATIENTS AND METHODS: Children with active Crohn disease scheduled for their initial dose of infliximab underwent assessment immediately before and 2 weeks following infliximab infusion. Using the stable isotope [d5] phenylalanine, rates of fractional and absolute albumin and fibrinogen synthesis were calculated. Measurements were made in both the fasting and parenterally fed states. RESULTS: Fifteen children (mean age 14.9 +/- 0.3) completed the study. The mean serum albumin changed from 3.59 +/- 0.08 to 3.66 +/- 0.04 g/dL, and the mean fibrinogen level decreased from 230 +/- 17 to 187 +/- 8 mg/dL (P < 0.05) following infliximab therapy. During fasting, there were no changes in albumin and fibrinogen synthesis rates following infliximab. During parenteral nutrition infusion, the fractional albumin synthesis rate changed from 11.8% to 15.1%/day (P = 0.06), and the absolute albumin synthesis rate increased from 192 to 248 mg x kg(-1) x day(-1) (P < 0.05), whereas no changes in fibrinogen synthesis rates were observed. Synthesis rates of albumin and fibrinogen were increased during parenteral nutrition infusion compared with the fasting state. CONCLUSIONS: Following infliximab therapy, during parenteral nutrition infusion, albumin synthesis increased significantly. Conversely, serum fibrinogen levels decreased following infliximab therapy in the absence of significant change in synthesis rates.

Protein catabolism in advanced renal disease: role of cytokines.

BACKGROUND: Protein energy wasting is a maladaptive metabolic state often associated with inflammation, which is common in patients with chronic kidney disease (CKD). METHODS: A literature search was performed using MEDLINE and the reference lists of relevant review articles. The following key words were used in the MEDLINE search: "cytokines", "inflammation", "protein metabolism", "acute-phase protein", "cachexia", "chronic kidney disease", "end-stage renal disease" and "hemodialysis". The search was limited to English-language articles. RESULTS: While experimental models have shown that uremic animals are more prone for proteolysis, the results from the human studies are controversial. Intradialytic loss of amino acids and activation of proinflammatory cytokines lead to protein catabolism during hemodialysis (HD). At the whole-body level, intradialytic parenteral nutrition (IDPN) increases protein synthesis and decreases proteolysis. Amino acid infusion during HD increases muscle protein synthesis, but does not decrease protein catabolism. Activation of interleukin-6 during HD induces protein catabolism, impairs amino acid utilization for protein synthesis and increases acute-phase protein synthesis. CONCLUSION: The changes in albumin, fibrinogen and muscle protein kinetics during HD could be due to competing and complementary effects of availability of amino acids and activation of proinflammatory cytokines.

Inhibition of proteasome by bortezomib causes intracellular aggregation of hepatic serpins and increases the latent circulating form of antithrombin.

Conformational diseases include heterogeneous disorders sharing a similar pathological mechanism, leading to intracellular aggregation of proteins with toxic effects. Serpins are commonly involved in these diseases. These are structurally sensitive molecules that modify their folding under even minor genetic or environmental variations. Indeed, under normal conditions, the rate of misfolding of serpins is high and unfolded serpins must be degraded by the proteasome system. Our aim was to study the effects of bortezomib, a proteasome inhibitor, on conformationally sensitive serpins. The effects of bortezomib were analysed in patients with multiple myeloma, HepG2 cells, and Swiss mice, as well as in vitro. Levels, anti-FXa activity, heparin affinity, and conformational features of antithrombin, a relevant anticoagulant serpin, were analysed. Histological, ultrastructural features and immunohistological distribution of antithrombin and alpha1-antitrypsin (another hepatic serpin) were evaluated. We also studied the intracellular accumulation of conformationally sensitive (fibrinogen) or non-sensitive (prothrombin) hepatic proteins. The inhibition of the proteasome caused intracellular accumulation and aggregation of serpins within the endoplasmic reticulum that was associated with confronting cisternae and Mallory body formation. These effects were accompanied by a heat stress response. Bortezomib also increased the levels of intracellular fibrinogen, but has no significant effect on prothrombin. Finally, bortezomib had only minor effects on the mature circulating antithrombin, with increased amounts of latent antithrombin in plasma. These results suggest that the impairment of proteasomal activities leads to an intracellular accumulation of conformationally sensitive proteins and might facilitate the release of misfolded serpins into circulation where they adopt more stable conformations.

Interferon-gamma exacerbates liver damage, the hepatic progenitor cell response and fibrosis in a mouse model of chronic liver injury.

BACKGROUND/AIMS: Several previous studies have suggested that interferon gamma (IFNgamma) may play a key role during hepatic progenitor cell (HPC) mediated liver regeneration. However to date, no studies have directly tested the ability of IFNgamma to mediate the HPC response in an in vivo model. METHODS/RESULTS: Administration of IFNgamma to mice receiving a choline deficient, ethionine (CDE) supplemented diet to induce chronic injury resulted in an augmented HPC response. This was accompanied by increased inflammation, altered cytokine expression and hepatic fibrosis. Serum alanine aminotransferase activity, hepatocyte apoptosis and Bak staining were significantly increased in IFNgamma-treated, CDE-fed mice, demonstrating that liver damage was exacerbated in these animals. Administration of IFNgamma to control diet fed mice did not induce liver damage, however it did stimulate hepatic inflammation. CONCLUSIONS: Our results suggest that IFNgamma increases the HPC response to injury by stimulating hepatic inflammation and aggravating liver damage. This is accompanied by an increase in hepatic fibrogenesis, supporting previous reports which suggest that the HPC response may drive fibrogenesis during chronic liver injury.

Platelet function testing to assess effectiveness of platelet transfusion therapy.

BACKGROUND: Posttransfusion corrected count increments (CCI) following administration of platelets is the standard method for assessing effectiveness of platelet transfusion therapy. However, improvement in platelet count following transfusion may not necessarily indicate improvement in platelet function or restoration of primary hemostatic capacity. To address this possibility, we investigated the effectiveness of platelet transfusion based on results of the Platelet Function Analyzer (PFA-100) and post-transfusion CCI. INVESTIGATION DESIGN AND METHODS: Platelet transfusion requests with different indications received at the blood bank were evaluated for inclusion in the investigation. Pre-transfusion, the following laboratory tests were performed: (1) PFA-100 assays (blood collected in 3.2% buffered sodium citrate) performed with CEPI and CADP test cartridges; (2) complete blood count (in EDTA) and platelet count; and (3) routine coagulation profile including PT, PTT, fibrinogen and D-Dimer. Only patients with normal coagulation profiles were included. The same set of tests were performed on a new blood sample collected 10-60 min post-transfusion. Chart review and clinical evaluation for response to platelet therapy were performed on each occasion of transfusion. RESULTS: Thirty-one patients, five of whom were transfused on more than one occasion were evaluated. Thirty-five transfusion incidents were included. Posttransfusion outcomes were divided into two groups--those that resulted in shortening (>40 s) or normalization of the closure time (Group A) and those that had no change or greater prolongation of the closure time (Group B) when compared to the pre-transfusion value. Seventeen and eighteen transfusion episodes were categorized as Groups A and B, respectively. In Group A with improved PFA testing, nine patients had bleeding as indication for transfusion and six of these had concomitant improvement in their clinical picture as confirmed by control of hemorrhage. In contrast in Group B with no improvement in PFA testing, seven patients had bleeding as indication for transfusion and none showed cessation of hemorrhagic symptoms. These findings were statistically significant (p=0.0114). Similar evaluation using the post-transfusion CCI showed no correlation to bleeding symptoms in these patients (p-=0.500). CONCLUSIONS: In this evaluation, platelet function testing using the PFA-100 provided a better indication of transfusion outcome than did the post-transfusion CCI. Using this approach, PFA-100 may be an effective aid for supporting platelet transfusion decisions and may further aid in improving management of the hospital blood bank platelet inventory.

Modulation of the liver export protein synthetic response to feeding by an n-3 fatty-acid-enriched nutritional supplement is associated with anabolism in cachectic cancer patients.

The acute-phase protein response is associated with accelerated weight loss and shortened survival in cancer. This may be due to hepatic protein synthesis increasing demand for amino acids. An n -3 fatty-acid-enriched nutritional supplement will moderate aspects of cachexia in cancer patients. The present study examined the effect of such a supplement on hepatic synthesis of albumin and fibrinogen. Albumin and fibrinogen synthesis were measured in the fed and fasting state in eight weight-losing patients with pancreatic cancer by an intravenous flooding dose technique. Tracer incorporation into proteins was measured by GC/MS. Patients were restudied after 3 weeks of oral supplement enriched with fish oil (providing 2510 kJ/day and 2 g of eicosapentaenoic acid/day). At baseline, all patients were losing weight (median, 2.4 kg/month). After 3 weeks of consumption of the fish-oil-enriched nutritional supplement, patients' weight stabilized (median change, +1 kg; P = 0.01). At baseline, albumin and fibrinogen synthesis rates were stimulated in the fed compared with the fasting state [14.2 compared with 11.3 g/day (29% rise; P = 0.01) and 4.5 compared with 3.3 g/day (38% rise; P = 0.01) respectively]. After 3 weeks of the supplement, this stimulation in the fed state was no longer observed for albumin and was reduced for fibrinogen [11.2 compared with 10.5 g/day (3% rise; P = 0.21) and 3.7 compared with 2.9 g/day (17% rise; P = 0.01) respectively]. After 3 weeks, the combined albumin plus fibrinogen synthetic rate tended to fall in the fasting state (14.7 compared with 12.3 g/day; P = 0.09) and was significantly reduced in the fed state (18.7 compared with 14.6 g/day; P = 0.01). Modulation of hepatic export protein synthesis with feeding may have contributed to the net whole-body anabolism observed with administration of the n -3 fatty-acid-enriched oral supplement.

Antigen-specific IgE and IgA antibodies in bronchoalveolar lavage fluid are associated with stronger antigen-induced late phase reactions.

The mechanism(s) leading to the development of late phase allergic reactions is (are) unknown. Previous studies have indicated that a relationship between serum IgE and the late phase exists. To explore the relationships between allergen-specific immunoglobulins in bronchoalveolar lavage (BAL) fluids and the magnitude of airflow limitation during the late phase response to inhaled allergen. Ragweed-specific IgE, IgA, secretory IgA (sIgA) and IgG were measured in BAL fluid and in the serum 1-5 weeks before whole lung antigen challenge with ragweed extract, in 16 ragweed allergic asthmatics. In addition, BAL and serum eosinophil cationic protein (ECP) and BAL fibrinogen levels were determined and BAL cells counted and differentiated. The latter procedures were repeated in a second BAL performed 24 h after the end of the ragweed challenge. After the challenge, lung function was monitored hourly for 8 h, to record the magnitude of airflow limitation. Ragweed-specific immunoglobulins were detected in 25% to 37.5% of BAL samples. Compared to the subjects with undetectable BAL fluid ragweed-specific IgE levels at baseline, those with detectable antibodies had stronger late phase reactions as determined by the nadir of FEV1 between hours 4 and 8 after the ragweed inhalation challenge (P = 0.0007). Allergen-induced changes in BAL ECP and fibrinogen levels were also higher in those subjects with detectable ragweed-specific IgE in baseline fluids (P = 0.03 and P = 0.005, respectively). Significant relationships between BAL antigen-specific IgA, serum ragweed-specific IgE and IgA and the late phase reaction were also found. The results of this study point towards the possibility that allergen-specific IgE and IgA may be independently involved in the pathogenesis of the late phase reaction. This notion merits further exploration.

Single administration of thrombopoietin to lethally irradiated mice prevents infectious and thrombotic events leading to mortality.

A sufficiently high dose of thrombopoietin to overcome initial c-mpl-mediated clearance stimulates hematopoietic reconstitution following myelosuppressive treatment. We studied the efficacy of thrombopoietin on survival after supralethal total body irradiation (9 Gy) of C57BL6/J mice and the occurrence of infectious and thrombotic complications in comparison with a bone marrow graft or prophylactic antibiotic treatment. Administration of 0.3 microg thrombopoietin, 2 hours after irradiation, protected 62% of the mice as opposed to no survival in placebo controls. A graft with a supraoptimal number of syngeneic bone marrow cells (10(6) cells) fully prevented mortality, whereas antibiotic treatment was ineffective. Blood cell recovery was observed in the thrombopoietin-treated mice but not in the placebo or antibiotic-treated group. Bone marrow and spleen cellularity as well as colony-forming unit granulocyte-macrophage and burst-forming unit erythroid were considerably increased in thrombopoietin-treated mice relative to controls. Histologic examination at day 11 revealed numerous petechiae and vascular obstructions within the brain microvasculature of placebo-treated mice, which was correlated with hypercoagulation and hypofibrinolysis. Thrombopoietin treatment prevented coagulation/fibrinolysis disorder and vascular thrombosis. High fibrinogen levels were related to bacterial infections in 67% of placebo-treated mice and predicted mortality, whereas the majority of the thrombopoietin-treated mice did not show high fibrinogen levels and endotoxin was not detectable in plasma. We conclude that thrombopoietin administration prevents mortality in mice subjected to 9-Gy total body irradiation both by interfering in the cascade leading to thrombotic complications and by amelioration of neutrophil and platelet recovery and thus protects against infections and hemorrhages.

The effect of nutritional and hormonal supplementation on protein synthesis immediately after liver transplantation.

We have previously shown that immediately after liver transplantation (LT) the porcine recipient exhibits elevated plasma glucagon, increased fractional synthetic rate (FSR) of fibrinogen, and decreased FSR of fixed or structural liver proteins. The purpose of this study was to evaluate the effect of nutritional and hormonal supplementation on these observations 24 h after LT. Two groups of nine pigs were studied 1 day after LT using radioisotopic and arteriovenous difference techniques. A control group underwent LT with saline infusion and a supplemented group underwent LT with infusion of glucose, amino acids (6 and 1.06 mg/kg. min, respectively), and intraportal insulin (0.6 mU/kg. min) and glucagon (1.3 ng/kg. min). Primed constant infusions of [3H]leucine were used to determine leucine flux, an estimate of whole body protein breakdown, and fractional synthetic rates (FSR). The following changes were noted with supplementation: elevated plasma insulin (6 +/- 1 versus 29 +/- 4 microU/ml, control versus supplemented, respectively, P < 0.05), decreased glucagon to normal levels (323 +/- 65 versus 102 +/- 12 pg/ml, P < 0.05), decreased fibrinogen FSR (108 +/- 15 versus 70 +/- 6%/day, P < 0.025), and increased fixed liver protein FSR (8 +/- 1 versus 13 +/- 2%/day, P < 0.05, respectively). Albumin FSR was unaltered by supplementation (8 +/- 2 versus 6 +/- 1%/day, respectively). Nutritional and hormonal supplementation immediately after LT restored the measured protein synthesis in the allograft to near normal levels 1 day after transplantation.

Glucocorticoid-induced attenuation of mucosal exudation of fibrinogen and bradykinins in seasonal allergic rhinitis.

The mucosal plasma exudate with its proteins, enzymes, derived peptides, and matrix molecules is an important factor in inflammatory airway diseases. This study investigated whether topical glucocorticosteroid treatment influences mucosal exudation of bulk plasma (fibrinogen) and the generation of plasma-derived mediators (bradykinins) in seasonal allergic rhinitis. Twenty-two patients with birch-pollen-induced allergic rhinitis participated in a double-blind, randomized, placebo-controlled study during the birch pollen season in 1989. After a 2-week run-in period, the participants received treatment with budesonide (200 micrograms per nasal cavity and day) or placebo. The patients kept a diary to record their daily nasal symptoms (itching, sneezing, nasal blockage, and secretion). The amount of birch pollen in the air was determined with the aid of a Burkhard pollen trap. A nasal lavage was performed once a week, and the levels of bradykinins and fibrinogen were determined in the lavage fluid samples. The birch pollen season was very mild, resulting in only minor nasal symptoms. In spite of the low pollen exposure, treatment with budesonide reduced the lavage fluid levels of both bradykinins and fibrinogen. The present results show that topical glucocorticosteroid treatment attenuates plasma exudation and the generation of plasma-derived mediators in seasonal allergic rhinitis. This action may not result from simple vascular antipermeability effects of the drug but may rather reflect the anti-inflammatory efficacy of topical glucocorticoids in the airway mucosa.

Harry M. Vars Research Award. Arginine supplementation improves histone and acute-phase protein synthesis during gram-negative sepsis in the rat.

Mechanisms of nutrient alteration of hepatic protein synthesis during sepsis are unclear. In vitro, arginine downregulates endotoxin-stimulated hepatocyte protein synthesis but in vivo effects are unknown. This study evaluated the effects of supplemental arginine or glycine on fibrinogen (acute-phase protein), histone, albumin, and liver protein synthesis after Gram-negative sepsis in the rat. Adult rats (225 g, n = 36) were randomized to receive isonitrogenous isocaloric total parenteral nutrition supplemented with 264 mg of N per kilogram per day as either arginine or glycine. On day 5, each group was further randomized to control or sepsis. Sepsis was induced by injection of 8 x 10(7) Escherichia coli per 100 g body weight, and then a continuous infusion of [1-14C]leucine was started. The rats were sacrificed 4 hours later. The fractional protein synthesis rates (percent per day) of histone, fibrinogen, albumin, and liver were determined. Supplemental arginine led to significantly increased histone (p less than 0.05, analysis of variance) and fibrinogen (p less than 0.01, analysis of variance) synthesis in the septic rats compared with all other groups. Histone and albumin synthesis were also significantly increased (p less than 0.05) in the arginine-supplemented control group compared with the glycine-supplemented control group. Arginine supplementation during sepsis significantly increased (p less than 0.05) albumin and liver protein synthesis compared with controls. Histones which are involved in DNA synthesis and are rich in arginine may play a role in the host response to stress and sepsis. These in vivo results appear to contradict hepatocyte-Kupffer cell coculture studies perhaps because of the hormonal and cytokine responses to nutrient substrate and acute septicemia.

Arginine supplementation improves histone and acute-phase protein synthesis during gram-negative sepsis in the rat.

Mechanisms of nutrient alteration of hepatic protein synthesis during sepsis are unclear. In vitro, arginine downregulates endotoxin-stimulated hepatocyte protein synthesis but in vivo effects are unknown. This study evaluated the effects of supplemental arginine or glycine on fibrinogen (acute-phase protein), histone, albumin, and liver protein synthesis after Gram-negative sepsis in the rat. Adult rats (225 g, n=36) were randomized to receive isonitrogenous isocaloric total parenteral nutrition supplemented with 264 mg of N per kilogram per day as either arginine or glycine. On day 5, each group was further randomized to control or sepsis. Sepsis was induced by injection of 8 x 10(7) Escherichia coli per 100 g body weight, and then a continuous infusion of [1-14C] leucine was started. The rats were sacrificed 4 hours later. The fractional protein synthesis rates (percent per day) of histone, fibrinogen, albumin, and liver were determined. Supplemental arginine led to significantly increased histone (p < 0.05, analysis of variance) and fibrinogen (p < 0.01, analysis of variance) synthesis in the septic rats compared with all other groups. Histone and albumin synthesis were also significantly increased (p < 0.05) in the arginine-supplemented control group compared with the glycine-supplemented control group. Arginine supplementation during sepsis significantly increased (p < 0.05) albumin and liver protein synthesis compared with controls. Histones which are involved in DNA synthesis and are rich in arginine may play a role in the host response to stress and sepsis. These in vivo results appear to contradict hepatocyte-Kupffer cell coculture studies perhaps because of the hormonal and cytokine responses to nutrient substrate and acute septicemia.

Oral contraceptives and plasma protein metabolism.

PIP: Plasma concentrations of various physiologically important proteins, including transferrin, ceruloplasmin, haptoglobin, transcortin, sex hormone-binding globulin, thyroxin-binding globulin, renin-substrate, fibrinogen, coagulation factors VII and VIII, antithrombin-III, plasminogen, prealbumin, albumin, retinol-binding protein, and lipoprotein fractions, were measured before treatment with oral contraceptives (OCs) and then again after 6 cycles of treatment to measure changes in the daily synthesis rate of 2 proteins, albumin and fibrinogen, under the influence of various OC formulations. Results are presented for 38 women using high-dose (50 mcg estrogen) preparations, 38 using low-dose (30 mcg estrogen preparations), and 20 using a continuous-dose progestagen-only minipill (30 mcg levonorgestrel). Most of the proteins measured showed significant alterations in women using the high-dose OCs. Changes with the lower dose product were less marked, and most proteins were unchanged in women using the minipill. (For example, synthesis rates of fibrinogen, mg/kg/day, for controls, high-, low-, and mini-dose subjects were: 24, 43, 28, and 25 respectively). Data from isotope studies indicated that synthetic estrogens act on liver synthesis and secretion rates for many plasma proteins; hence the clinical associations seen with OC use.^ieng