bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-term administration of isosorbide-5-mononitrate does not impair renal function in cirrhotic patients
Authors: Salerno F , Borroni G , Lorenzano E , Solenghi D , Cazzaniga M , Bissoli F , Ceriani R , deFranchis R
Published: November 2004

Isosorbide-5-mononitrate (Is-5-Mn), alone or combined with β- blockers, has been proposed for prophylaxis of variceal bleeding in cirrhosis. However, renal insufficiency, might be an important undesirable effect of this therapy, especially in patients with ascites. We assessed the changes in renal function induced in 26 cirrhotic patients by acute or chronic administration of Is-5-Mn. The acute administration of 20 mg of Is-5-Mn to 21 patients reduced mean blood pressure (83.4 ± 2.4 vs. 92.8 ± 3.4 mm Hg, P < .001), urine volume (5.5 ± 0.8 vs. 8.7 ± 1.1 mL/min, P < .05), urine sodium excretion (114 ± 19 vs. 244 ± 41 muEq/min, P < .001), urine potassium excretion (41 ± 3.4 vs. 67 ± 8.5 muEq/min, P < .001), and atrial natriuretic factor (74 ± 10 vs. 98 ± 12 pg/mL, P < .005). The glomerular filtration rate was decreased in the 11 patients with ascites (57 ± 9 vs. 68 ± 12 mL/min, P < .05), and plasma renin activity was increased in 4 ascitics. Twenty-one patients (16 from the acute study + 5 other patients) were given Is-5-Mn for 3 months at the dose of 80 mg/d. This did not affect blood pressure and renal function in patients without ascites, but reduced mean blood pressure (91.9 ± 3.4 vs. 89.6 ± 3 mm Hg, P < .05), urine volume (5.8 ± 1.1 vs. 3.4 ± 0.9 mL/min, P < .05), and urine sodium excretion (205 ± 38 vs. 99 ± 16 muEq/min, P < .01) in those with ascites. There were no changes in glomerular filtration rate and renal plasma flow, while plasma renin activity increased in only 3 patients with ascites and 1 without. Systemic hemodynamics and renal function of cirrhotic patients, especially those with ascites, are affected adversely by acute administration of Is-5-Mn. Long-term administration of the drug is well tolerated by compensated patients and does not affect renal plasma flow nor glomerular filtration rate, but can induce hypotension and sodium retention in patients with ascites.
Is the Mayo model for predicting survival useful after the introduction of ursodeoxycholic acid treatment for primary biliary cirrhosis?
Authors: Kilmurry M R , Heathcote E J , Cauch-Dudek K , O’Rourke K , Bailey R J , Blendis L M , Ghent C N , Minuk G Y , Pappas S C , Scully L J , Steinbrecher U P , Sutherland L R , Williams C N , Worobetz L J
Published: November 2004

Treatment of patients with primary biliary cirrhosis (PBC) using ursodeoxycholic acid (UDCA) leads to a reduction in serum bilirubin. The first objective of this study was to assess the performance of certain prognostic indicators for PBC after the introduction of treatment with UDCA. Serum bilirubin is an important prognostic indicator for PBC and an important component of the Mayo model for grading patients into risk categories. In an analysis of patients enrolled in the Canadian multicenter trial, the Mayo score was calculated before and after treatment with UDCA. After treatment, the Mayo score continued to divide patients with PBC into groups with varying risk. In addition, the serum bilirubin alone was shown to do the same even after the introduction of treatment with UDCA. A second objective was to establish whether UDCA had an effect on long-term (2- to 6-year) survival in patients with PBC.
Modulation of rat hepatocyte proliferation by bile salts: In vitro and In vivo studies
Authors: Barone M , Francavilla A , Polimeno L , Ierardi E , Romanelli D , Berloco P , Di Leo A , Panella C
Published: November 2004

In this study, the stimulatory effect of bile salts (BS) was evaluated both In vitro, using hepatocyte primary cultures, and In vivo, in normal and 40% partially hepatectomized rats previously fed on BS- enriched diets for 4 weeks. In vitro results show that conjugated cholate (CA) and chenodeoxycholate (CDCA) augmented proliferative activity in rat hepatocytes cultured in absence of mitogens, whereas conjugated deoxycholate (DCA), and ursodeoxycholate (UDCA) did not have any significant effect. None of these BSs increased significantly the replicative response induced by submaximal concentrations of epidermal growth factor (EGF). In vivo, at the end of dietary treatment all animals fed on CA or DCA but not those fed on either CDCA, or UDCA, or tauroursodeoxycholate (TUDCA) developed cholestatic hepatitis and a burst of damage-induced hepatocyte proliferation. After 40% partial hepatectomy (PH), CA- and DCA-treated groups underwent a deterioration of cholestatic hepatitis. On the other hand, in CDCA-, and UDCA-, and TUDCA-treated groups liver histology, serum glutamic pyruvic transaminase (SGPT) and cholestasis indices did not change significantly compared with controls. As far as the proliferative activity, a significant increase was observed not only in CA and DCA but also in UDCA- and TUDCA-fed groups compared with controls, whereas a slight decrease was observed in CDCA-treated animals. In conclusion, our data indicate that conjugated BSs had only a modest stimulatory effect on hepatocyte proliferation In vitro. However, In vivo, in PH rats, UDCA or TUDCA treatment determined a further increase of hepatocellular proliferation not attributable to hepatotoxic effects. Our result suggest that modifications of bile acid pool could modulate hepatocellular proliferation.
Inhibitory actions of cyclic adenosine monophosphate and pertussis toxin define two distinct epidermal growth factor-regulated pathways leading to activation of mitogen-activated protein kinase in rat hepatocytes
Authors: Gines P , Li X , Brown S E , Nakamura T , Guzelian P S , Heasley L E , Schrier R W , Nemenoff R A
Published: November 2004

Increased intracellular cyclic adenosine monophosphate (cAMP) levels have been shown in some reports to inhibit and in other studies to stimulate growth factor-mediated activation of the mitogen-activated protein kinase (MAP kinase) pathway, depending on the cell type examined. The relationship between cAMP and MAP kinase in hepatocytes has not been examined. In the current study, stimulation of primary cultures of rat hepatocytes with hepatocyte growth factor (HGF) or epidermal growth factor (EGF) increased Ras, Raf, and MAP kinase activity. Incubation of hepatocytes with cAMP-increasing agents blocked activation of Raf by both HGF and EGF, whereas activation of Ras was unaffected. MAP kinase activation by HGF was completely inhibited, whereas EGF-stimulated MAP kinase activity was only slightly reduced. Incubation of hepatocytes with pertussis toxin slightly blunted MAP kinase activation by EGF but not HGF. Increasing cAMP in hepatocytes preincubated with pertussis toxin completely inhibited the activation of MAP kinase by EGF. In conclusion, HGF activates MAP kinase in hepatocytes exclusively through an Raf-dependent pathway and this activation may be completely blocked by increasing cAMP. In contrast, EGF activates MAP kinase in hepatocytes through both Raf-dependent and Raf-independent pathways: the latter pathway probably involves a pertussis toxin-sensitive G protein.
Bacterial lipopolysaccharide antagonizes transforming growth factor β1-induced apoptosis in primary cultures of hepatocytes
Authors: Martin-Sanz P , Diaz-Guerra M J , Casado M , Bosca L
Published: November 2004

Incubation of primary cultures of fetal hepatocytes with lipopolysaccharide (LPS) elicited the expression of nitric oxide (NO) synthetase as well as antagonized the apoptotic cell death evoked by treating the cells with transforming growth factor β1 (TGF-β1). In addition to LPS, exposure of the cells to chemical NO donors also protected against apoptotic cell death when assayed at concentrations in the low micromolar range. Treatment of hepatocytes with large concentrations of NO donors promoted both apoptotic and necrotic cell death. These results suggest that NO synthesis by hepatocytes might be involved in the protection against apoptotic death.
Long-term octreotide treatment prevents vascular hyporeactivity in portal-hypertensive rats
Authors: Sieber C C , Lee F , Groszmann R J
Published: November 2004

Chronically portal-hypertensive rats show in vitro vascular hyporeactivity in large part mediated by the endothelium-derived vasodilator nitric oxide. We tested whether long-term octreotide treatment (15 micrograms/kg subcutaneously in 5% D/W, 8-hourly) corrects the in vitro vascular hyporeactivity. Increases in perfusion pressures (delta mm Hg) to potassium chloride (30-300 mmol/L) of in vitro perfused superior mesenteric arterial vascular beds of partial portal vein-ligated (PVL) rats were significantly (P < .05) higher in octreotide (n = 9) compared with placebo (n = 10, 5% D/W) treated animals. Octreotide significantly (P < .05) increased mean arterial pressure compared with placebo, the values being 129 ± 3 and 117 ± 4 mm Hg, respectively. Furthermore, a significant (P < .001) correlation was observed between in vitro vascular reactivity and mean arterial pressure. Incubation of separate vascular beds (n = 7 for both PVL and sham-operated rats) with octreotide (10-6 mol/L) did not enhance pressure responses to 125 mmol/L potassium chloride, and failed to increase perfusion pressures in preconstricted vessel preparations (n = 6), excluding a direct inhibitory effect on NO. In summary, long-term octreotide treatment prevents in vitro vascular hyporeactivity in prehepatic portal-hypertensive rats, and octreotide does not exert its action through direct effects on endothelium-derived NO.
Role of Kupffer cells in arresting circulating tumor cells and controlling metastatic growth in the liver
Authors: Bayon L G , Izquierdo M A , Sirovich I , van rooijen N , Beelen R H , Meijer S
Published: November 2004

Metastasis to the liver is a common event in clinical oncology. Blood-borne tumor cells (TCs) arriving to the liver sinusoids run into a special vascular bed. The lining of liver sinusoids is shared by Kupffer cells (KCs) and endothelial cells. KCs, liver-fixed macrophages, are responsible for detection and removal of “non-self” particles. To investigate their role in arresting blood-borne TCs and controlling tumor growth, we injected a syngeneic colon carcinoma cell line into a mesenteric vein of two groups of rats; one group was without Kupffer cells and the other normal controls. We removed the liver of these animals at different time intervals and performed immunohistochemical analysis with monoclonal antibodies (MoAbs) against our tumor cell line, three macrophage subpopulations, natural killer cells, and B and T lymphocytes. Additionally, we showed in vitro spontaneous cytotoxicity of KCs against our tumor cell line. Results suggest that KCs play a relevant role in arresting circulating TCs at the liver sinusoid, although it is limited to a small number of malignant cells. They also seem to play a major role in clearing neoplastic cells from the liver parenchyma, in controlling tumor growth in the very early stages of metastatic development, and in modulating the host immune response to cancer cells.
Expression of a rat liver phosphatidylcholine translocator in Xenopus laevis oocytes
Authors: Kullak-Ublick G , Gerloff T , Hagenbuch B , Berr F , Meier P J , Stieger B
Published: November 2004

A phospholipid translocating protein from rat liver has been expressed in Xenopus laevis oocytes. Injection of oocytes with total rat liver messenger RNA (mRNA) resulted in the function expression of saturable uptake of the water soluble phophatidylcholine derivative L-α- dibutyroylglycero-3-phophatidylcholine (diC4PC), Kinetic studies revealed an apparent Km value of approximately 10 mmol/L, which is similar to the value previously obtained in isolated rat liver canalicular plasma membrane vesicles for an adenosine triphosphate (ATP)-independent phosphatidylcholine translocator. Size fractionation of total rat liver mRNA yielded an active mRNA species between 1.8 and 2.6 kb, that stimulated the expressed phophatidylcholine uptake activity approximately fivefold as compared with differently sized mRNA subfractions. This active mRNA size class is too small to code for the mdr2 P-glycoprotein, which has been suggested to function as an ATP- dependent canalicular phosphatidylcholine translocator. Hence, the data indicate that there are at least two separate polypeptides involved in phospholipid translocation from hepatocytes into bile.
Changes in Serum Fibrogenesis Markers During Interferon Therapy for Chronic Hepatitis Type C
Authors: Ishibashi K , Kashiwagi T , Ito A , Tanaka Y , Nagasawa M , Toyama T , Ozaki S , Naito M , Azuma M
Published: November 2004

We serially measured the levels of serum N–terminal peptide of type III procollagen (PIIINP), the 7S domain of type IV collagen (IV–7S), and hyaluronate (HA) before (0 month), at the end (6 months), and 24 weeks after the end of interferon therapy (12 months) in patients with chronic hepatitis type C to estimate the effects of interferon alfa (IFN–alpha) on serum levels of hepatic fibrogenesis markers. One hundred twenty–one patients with chronic hepatitis type C received intramuscular injection of 6 million U of natural IFN–alpha for 24 weeks. Patients were divided into three groups: sustained complete response (CR–S), complete response with rebound (CR–R), and nonresponse (NR). Serum PIIINP, IV–7S, and HA levels were significantly decreased and reached normal levels at 12 months in CR–S; only IV–7S levels were significantly decreased at 12 months in CR–R, whereas these levels remained abnormally high in the NR. These results suggest that IFN– alpha therapy could lower the levels of serum hepatic fibrogenesis markers along with improvements in hepatic inflammation.
Variable Gene Expression Within Human Tyrosinemia Type 1 Liver May Reflect Region–Specific Dysplasia
Authors: Haber B A , Chuang E , Lee W , Taub R
Published: November 2004

Patients with hereditary tyrosinemia type 1 have a deficiency of fumarylacetoacetate hydrolase (FAH) and develop progressive hepatocellular dysfunction with a high risk of malignant transformation. Serum alpha–fetoprotein levels are frequently elevated in these patients; therefore, this commonly used marker of tumorigenesis is inadequate. To date, no literature exists describing the hepatic gene alterations in patients with this disease. We analyzed the expression of a panel of proliferation associated and liver–specific genes in the liver of a 33 month–old girl at the time of orthotopic liver transplantation. This study provides information that may be useful in developing markers for malignancy and understanding the pathogenesis of this disease. Gene expression patterns of two regenerating nodules and total liver from the patient with FAH deficiency were compared with control donor liver. Liver–specific and growth–induced genes with altered expression in the tyrosinemic liver included several functional classes: structural proteins (

actin, thrombospondin

), transcription factors (

c–fos, egr–1, C/EBPalpha

), liver–specific enzymes

glucose–6–phosphatase

[G6Phase], and secreted factors

insulin–like growth factor binding protein 1 [GFBP–1]

. Isolated macronodules demonstrated varied patterns of expression, suggesting that they do not form a homogeneous cellular environment. In the tyrosinemic liver,

IGFBP–1

messenger RNA expression was high and

G6Phase

messenger RNA was not detectable. Although G6Phase and IGFBP–1 are coexpressed in regenerating liver, immunohistochemistry in the tyrosinemic liver demonstrated a mutually exclusive distribution for the two proteins in a tissue section with features of dysplasia. We propose that cells in these areas may have an aberrant transcription factor and growth factor “milieu” that leads to altered gene and protein expression. These molecular alterations are reflected in dysplastic histologic changes and may ultimately predispose to the development of malignancy.
Use of A Designer Triple Expression Hybrid Clone for Three Different Lipoyl Domain for the Detection of Antimitochondrial Autoantibodies
Authors: Moteki S , Leung P S , Coppel R L , Dickson E R , Kaplan M M , Munoz S , Gershwin M E
Published: November 2004

The detection of antimitochondrial autoantibodies (AMAs) is critical in the diagnosis of primary biliary cirrhosis (PBC). However, conventional laboratory assays to detect AMA are dependent on the time–consuming method of immunofluorescence microscopy, a method often plagued by problems of nonspecificity. AMAs react against mitochondrial autoantigens including the E2 components of the pyruvate dehydrogenase complex (PDC–E2), the branched–chain 2–oxo–acid dehydrogenase complex (BCOADC–E2), and the 2–oxo–glutarate dehydrogenase complex (OGDC–E2). Interestingly, the immunodominant epitopes of PDC–E2, BCOADC–E2, and OGDC–E2 are all conformational lipoate binding sites, but antibodies against them do not cross–react. Although 80% to 90% of sera from patients with PBC react to PDC–E2, approximately 10% patients with PBC react only to BCOADC–E2 and/or OGDC–E2. We have taken advantage of our epitope–mapping studies of the E2 components of PDC, BCOADC, and OGDC, and constructed a “designer” hybrid clone, designated as pML–MIT3, that coexpresses the immunodominant epitopes within the three distinct lipoyl domains. We examined a total of 321 sera, including 186 sera from patients with PBC, to test the immunoreactivity of pMIT3. Of 186 sera from patients with PBC, 152 sera (81.7%) reacted with recombinant fusion protein of PDC–E2, whereas 171 sera (91.9%) showed positive reactivities when probed by immunoblotting against the recombinant fusion protein expressed from the pML–MIT3 clone. Of 34 PBC sera that did not react with recombinant PDC–E2, 18 sera contained BCOADC–E2–specific AMA and 1 serum possessed only OGDC–E2–specific AMA. We also developed an enzyme–linked immunosorbent assay (ELISA), using affinity–purified recombinant fusion protein of pML–MIT3 clone as the antigen source, to quantify specific AMAs in patients with PBC. None of the 135 control sera from patients with primary sclerosing cholangitis (PSC), chronic autoimmune hepatitis (CAH), systemic lupus erythematosus (SLE), or healthy volunteers showed significant reactivity against pML–MIT3 recombinant fusion protein in the ELISA assay. Our results indicate that an ELISA using recombinant, cloned autoantigen of pML–MIT3 is a powerful and very specific method for the detection of AMA.
The Metabolic Organization of the Adult Human Liver: A Comparative Study of Normal, Fibrotic, and Cirrhotic Liver Tissue
Authors: Racine-Samson L , Scoazec J , D'Errico A , Fiorentino M , Christa L , Moreau A , Roda C , Grigioni W F , Feldmann G
Published: November 2004

Little is known about the alterations of metabolic organization of the human liver tissue in chronic liver diseases. We therefore compared the distribution of the following zonal metabolic markers in 10 samples of normal liver tissue, 10 samples of fibrotic tissue, and 22 samples of cirrhotic tissue: (a) the enzymatic activities of glucose–6–phosphatase (G6P), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), nicotinamide–adenine–dinucleotide–phosphate [NAPH] dehydrogenase (ND), beta–hydroxybutyrate dehydrogenase (HBDH), and glutamate dehydrogenase (GDH); (b) the protein glutamine synthetase (GLS); and (c) albumin messenger RNA (mRNA). The normal human hepatic lobule was characterized by the periportal predominance of G6P and SDH enzymatic activities and albumin mRNAs, the perivenous predominance of ND and GDH, the restriction of GLS to a small perivenous compartment, and the predominanc of beta–HBDH at the contact of both portal tracts and centrilobular veins. In fibrosis, the overall metabolic organization of the normal liver tissue was retained. The expression of periportal markers predominated around enlarged portal tracts and that of perivenous markers around residual centrilobular veins. GLS was constantly detected at the contact of centrilobular veins. In cirrhotic nodules, no zonation was observed for most enzymatic activities or for albumin. Only G6P usually predominated at the periphery of the nodules. GLS was constantly undetectable. No difference accordingly to the etiology of the underlying disease was observed. In conclusion, the normal human hepatic lobule presents a marked metabolic zonation, preserved in fibrotic lesions, but lost in cirrhotic nodules. The alterations of the metabolic organization observed in cirrhosis might contribute to the pathogenesis of some of the metabolic disorders associated with advanced liver disease.
Role in Nitric Oxide in Kupffer Cell-Mediated Hepatoma Cell Cytotoxicity In Vitro And Ex Vivo
Authors: Fukumura D , Yonei Y , Kurose I , Saito H , Ohishi T , Higuchi H , Miura S , Kato S , Kimura H , Ebinuma H , Ishi H
Published: November 2004

The metabolic change in tumor cells (AH70, a rat hepatoma cell line) cocultured with isolated rat Kupffer cells were visualized and analyzed by a laser scanning confocal imaging system. When AH70 cells were cocultured with Kupffer cells, fluorescence intensity of rhodamine 123 (Rh123) decreased, indicating the reduction of mitochondrial function. The reduction in Rh123 was eliminated by NG–monomethyl–L–arginine (L– NMMA), an analogue of L–arginine, suggesting the involvement of nitric oxide (NO). Two hour after the cells were cocultured, membrane compromised AH70 cells which were observed as propidium from 2.8% to 25%. This increase was also attenuated by L–NMMA, suggesting that Kupffer cell–mediated injury of tumor cells largely depends on NO. The concentrations of NO–2 + NO–3 in the culture medium markedly increased after coculture of AH70 cells with Kupffer cells. Moreover, NO synthase (NOS) activity in Kupffer cells significantly increased after coculture. These in vitro results suggest that NO mediates Kupffer cell– induced tumor cell damage characterized by reduced mitochondrial function and diminished barrier function. In the ex vivo study of the perfused liver to which AH70 cells were injected via the catheter inserted into the portal vein, some AH70 cells were arrested in the upper stream of sinusoid and the fluorescence intensity of Rh123 in adherent AH70 cells decreased in a time–dependent manner within 2 hours. The number of PI–positive AH70 cells also increased 2 hours after the injection of AH70 cells. These changes were inhibited by either administration of N omega–L–nitroarginine–methylester (L–NAME) to perfusate or pretreatment of the rat liver with GdCl3, which is known to deplete Kupffer cell function. Thus, the present study suggests that NO from Kupffer cells induces mitochondrial dysfunction in tumor cells followed by membrane barrier dysfunction in the liver sinusoid.
Liver Cell Uptake and Degradation of Soluble Immunoglobulin G Immune Complexes In Vivo And In Vitro In Rats
Authors: Johansson A G , Lövdal T , Magnusson K , Berg T , Skogh T
Published: November 2004

Immune complexes were formed between dinitrophenylated human serum albumin (DNP–HSA) and polyclonal rabbit immunoglobulin G (IgG) anti–DNP antibodies at antibody excess. The antigen was labelled with isotope (125I–tyramine–cellobiose) or fluorochrome, (6–[fluorescein–5–(and–6)–carboxamido] hexanoic–acid, succinimidyl ester). The radiolabelled antigen, native or antibody complexed, was given intravenously to rats. Radioactivity was measured in various organs at 1 hour following injection. The liver was the main site for removal of the antigen as well as of the immune complexes. Within the liver, immune complexes were mainly associated with nonparenchymal liver cells, the total recovery from Kupffer cells being about 10 times greater than from the liver endothelial cells. The uncomplexed radiolabelled antigen was readily degraded by both cells types. After IgG complexing, the degradation decreased, both in Kupffer cells and in liver endothelial cells.

In vitro

experiments with isolated liver cells, showed that IgG complexing increased antigen uptake to about the same extent in Kupffer cells and in liver endothelial cells. The degradation of both antigen and immune complexes was less efficient

in vitro

than

in vivo

. Immune complex uptake

in vitro

was shown also by confocal fluorescence microscopy in Kupffer cells and in liver endothelial cells. Also

in vitro

, only minor uptake was found in the hepatocytes. We conclude that both liver endothelial cells and Kupffer cells are involved in the hepatic handling of soluble IgG immune complexes, but we found no evidence for substantial uptake by hepatocytes.
Role of Kupffer Cells and the Spleen in Modulation of Endotoxin–Induced Liver Injury After Partial Hepatectomy
Authors: Suzuki S , Nakamura S , Serizawa A , Sakaguchi T , Konno H , Muro H , Kosugi I , Baba S
Published: November 2004

The hypothesis that both activated Kupffer cells and the spleen may be responsible for endotoxin–induced liver injury following partial hepatectomy was investigated. Male rats were divided into a sham group receiving laparotomy alone and three groups receiving a two–thirds hepatectomy; one group was given normal saline (NS) solution as a vehicle control, one group received intravenous gadolinium chloride (GC group) (7 mg/kg body weight) for 2 days before intravenous injection of endotoxin to inhibit Kupffer cell phagocytosis, and the third group simultaneously underwent splenectomy and partial hepatectomy (SH group). As endotoxin, lipopolysaccharide (LPS) (1 mg/kg body weight) was administered intravenously 2 days after surgery. In the GC and SH groups, phagocytic activity was reduced to approximately 40% of that in the sham group. The highest plasma tumor necrosis factor alpha (TNF–alpha) level (8,544 ± 1,223 pg/mL) was observed in the NS group at 1 hour after LPS administration, and the level was significantly reduced by GdCl3 or splenectomy (

P

< 0.05). Inhibition of Kupffer cell function and splenectomy attenuated functional and structural liver damage associated with the decreased hepatic infiltration of polymorphonuclear leukocytes (PMNs) and reduced priming of circulating PMNs in the early stage of endotoxemia following partial hepatectomy. Consequently, the 24–hour survival rate of the SH and GC groups was significantly improved to 50% and 80%, respectively (

P

< .05), while that of the NS group was 12.5%. These findings indicate that the modification of inflammatory mediator generation by splenectomy or inhibition of Kupffer cell function may be beneficial for the prevention of endotoxin–induced liver injury after partial hepatectomy.
Altered Lipid Metabolism in Apolipoprotein E-Deficient Mice Does Not Affect Cholesterol Balance Across the Liver
Authors: Kuipers F , van Ree J M , Hofker M H , Wolters H , Veld G I , Havinga R , Vonk R J , Princen H M , Havekes L M
Published: November 2004

Adaptation of cholesterol and bile acid synthesis and of biliary cholesterol secretion represent key metabolic responses to maintain cholesterol homeostasis and have been suggested to be influenced by apolipoprotein E (apoE) phenotype in humans. We have investigated hepatic metabolism and secretion of cholesterol into bile in homozygous apoE–deficient (apoE –/–) mice fed normal lab chow. Plasma cholesterol levels were 10 times higher in apoE (–/–) mice than in controls (+/+); triacylglycerol levels were only minimally affected. Hepatic cholesterol (+56%) and triacylglycerol (+232%) contents were significantly increased in apoE (–/–) mice, whereas those of cholesteryl ester and of phospholipids were similar in both groups. Lipid accumulated predominantly in periportal areas of apoE (–/–) livers. Hepatic 3–hydroxy–3–methylglutaryl–coenzyme A reductase (HMG CoA reductase) messenger RNA (mRNA) level and activity were reduced by 45% and 50%, respectively, in apoE (–/–) mice. In contrast, plasma lathosterol/cholesterol ratios, indicative for whole–body cholesterol synthesis, were fourfold increased in these mice. Acyl–coenzyme A: cholesterol acyltransferase (ACAT) activity was similar in livers of both groups. Despite the marked changes in hepatic cholesterol metabolism, neither hepatic bile acid synthesis, bile acid pool size and composition, nor hepatic cholesterol 7alpha–hydroxylase and sterol 27–hydroxylase mRNA levels differed between apoE (–/–) and (+/+) mice. In addition, biliary cholesterol secretion was unaffected in the knock– out mice. Our results show that lack of apoE leads to marked changes in hepatic cholesterol metabolism without altering cholesterol balance across the liver. The data are compatible with increased peripheral cholesterol biosynthesis in apoE–deficient mice.
Alterations in Glutathione Homeostasis in Mutant Eisai Hyperbilirubinemic Rats
Authors: Lu S C , Cai J , Kuhlenkamp J , Sun W , Takikawa H , Takenaka O , Horie T , Yi J , Kaplowitz N
Published: November 2004

Eisai hyperbilirubinemic rats (EHBR) are mutant Sprague–Dawley rats that exhibit impaired biliary organic anion and reduced glutathione (GSH) secretion. In addition, liver GSH levels are twice that of age– matched controls. The mechanisms for the defect in biliary GSH secretion and the increase in cell GSH are not fully understood. We previously showed that canalicular membrane–enriched vesicles isolated from EHBR livers exhibited normal GSH transport. In the present study, we examined the steady–state rat canalicular reduced glutathione transporter (RcGshT) messenger RNA (mRNA) and protein levels, as well as the mechanisms for the increase in cell GSH. Both Northern and Western blot analyses of EHBR livers showed nearly identical RcGshT mRNA and polypeptide levels, respectively, as compared with controls. Treatment with phenobarbital, which increased steady–state RcGshT mRNA by five– to sixfold, RcGshT polypeptide, and biliary GSH secretion by onefold in controls, had a smaller effect on steady–state RcGshT–mRNA level in EHBR (by 1.5–fold) and did not increase RcGshT polypeptide or biliary GSH secretion. In examining possible mechanisms for increased liver GSH, both cysteine level and gamma–glutamylcysteine synthetase (GCS) activity were significantly higher than controls, while the activity of GSH synthetase was unchanged. Northern and Western blot analyses also showed increased steady–state GCS heavy subunit (GCS–HS) mRNA and polypeptide levels, respectively. In addition to liver, GSH levels in kidney, duodenal, jejunal, and ileal mucosa of EHBR were 200% to 300% of age–matched control rats. GCS activity was also increased in kidney cytosol of EHBR. Thus, the defect in biliary GSH secretion in EHBR most likely is either at the posttranslational level of RcGshT or in the inhibition exerted by retained endogenous organic anions. In addition, there is a widespread up–regulation of GSH synthesis capacity in the tissues of EHBR.
The role of nutrition in cancer development and prevention
Authors: Michels Karin B.
Published: November 2004

Nutrition has long been suspected to play an important role in cancer etiology. The biologic properties of nutrients make them prime candidates to aid in cancer prevention. Indeed, early epidemiologic cancer studies seemed to confirm the relevance of diet. These studies, however, were plagued by recall bias and confounding and may have thus been misleading. The results from large, prospective cohorts do not support a strong relation between nutrition and cancer. Given the difficulties in precisely assessing habitual diet, modest associations may exist, which may be impossible to capture when relying on self‐reported dietary information. Energy balance, reflected in a low body weight and high level of physical activity, has been more convincingly related to lower cancer rates. The potential importance of more extreme dietary regimens and of nutrition during earlier periods of life remains to be explored. Finally, epigenetic research is likely to contribute to the understanding of nutritional regulation of gene expression. © 2004 Wiley‐Liss, Inc.
Endostatin inhibits nitric oxide and diminishes VEGF and collagen XVIII in squamous carcinoma cells
Authors: Hebert Carla , Siavash Hessam , Norris Kathleen , Nikitakis Nikolaos G. , Sauk John J.
Published: November 2004

Low pO2 values are a common finding among oral squamous cell carcinomas (SCC). Our objective was to determine the role that oxygen tension plays on the direct tumor effect of endostatin (ES). Squamous carcinoma cell lines were grown under normoxic or hypoxic conditions and treated with endostatin (ES), nitric oxide (NO) donors, NO scavengers, NO synthase inhibitors, or transduced with AdenoVec™‐hEndo or AdenoVec™Null vectors. The expression of vascular endothelial growth factor (VEGF) and collagen XVIII were determined by RT‐PCR and protein levels assessed by Western blot analyses. Our studies demonstrated that collagen XVIII and VEGF are expressed and responsive to ES in a limited number of SCC cell lines during normoxia but were most responsive when grown under hypoxic conditions. VEGF and collagen XVIII were downregulated by both ES and transduction of cells with AdenoVec™‐hEndo. The effects of ES on SCC cells were enhanced by aminoguanidine (Ag), L‐NAME, and diphenyleneiodonium chloride (DPI). Endostatin and transduced with ES vectors diminished the levels of NO whereas NO donors enhanced VEGF expression and collagen XVIII expression. In conclusion, the direct effect of endostatins on tumor cells is most effective under conditions of low oxygen tension and can be potentiated by the use of nitric oxide synthase inhibitors or NO scavengers. © 2004 Wiley‐Liss, Inc.
Upregulation of Wilms' tumor gene 1 (WT1) in desmoid tumors
Authors: Amini Nik Saeid , Hohenstein Peter , Jadidizadeh Ali , Van Dam Kim , Bastidas Adriana , Berry Rachel L. , Patek Charles E. , Van der Schueren Bernadette , Cassiman Jean‐Jacques , Tejpar Sabine
Published: November 2004

Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors in which β‐catenin/TCF3 mediated Wnt signaling is activated. More than 80% of desmoid tumors contain activating mutations in β‐catenin. It has been shown that the Wnt signaling pathway interacts with Wilms' tumor gene 1 (WT1) in normal kidney development and plays a role in the genesis of some Wilms' tumors. About 15% of Wilms' tumors contain WT1 mutations and of these, about 50% contain β‐catenin mutations. This overlap in mutation pattern of WT1 and β‐catenin in Wilms' tumor suggests that these 2 genes may collaborate in the genesis of a subset of Wilms' tumors. To investigate whether this hypothesis could be extended to other Wnt‐dependent tumor types, we searched for WT1 mutations and studied WT1 expression in β‐catenin mutant desmoid tumors. We investigated the expression of WT1 mRNA and protein in desmoid tumors. Medium to high abundant levels of WT1 mRNA were detected by TaqMan quantitative PCR in all tested desmoids cells, whereas adjacent normal fibroblasts showed less expression of WT1. Western blot analysis and immunohistochemistry confirmed this overexpression at the protein level. A mutational screen of the WT1 zinc‐finger region by sequence analysis did not identify any mutations. Finally, we investigated a possible role of β‐catenin on WT1 regulation and vice versa. Overexpression of different β‐catenin mutants in the HEK293T cell line did not modulate WT1 promoter activity and WT1 did not affect β‐catenin /TCF transcriptional activity in this cell line. These results show that the wild‐type WT1 gene is strongly overexpressed in β‐catenin mutant desmoid tumors and may play a role in tumorigenesis of desmoid tumors, similar to what has been suggested in some epithelial malignancies. © 2004 Wiley‐Liss, Inc.
Differential effect of a bone morphogenetic protein‐7 (OP‐1) on primary and revision loaded, stable implants with allograft
Authors: Søballe Kjeld , Jensen Thomas B. , Mouzin Olivier , Kidder Louis , Bechtold Joan E.
Published: November 2004

Morselized impacted bone allograft is often used to reconstruct the bone bed in the revision of failed total joint arthroplasties. We hypothesized that addition of the bone morphogenetic protein OP‐1 (BMP‐7) to bone allograft would improve early implant fixation. We inserted one loaded 6‐mm‐diameter titanium implant (surrounded by 0.75‐mm gap) in each medial condyle of 24 canines. On one side, the implant was inserted in a controlled experimental revision setting resembling the clinical revision situation. A primary implant was inserted on the contralateral side in a previously unoperated site. Three groups were studied: 1) allograft alone, 2) allograft + 0.4 mg OP‐1, and 3) allograft + 0.8 mg OP‐1. Implant fixation was evaluated at 4 weeks. Grafted implants inserted in the primary setting without OP‐1 had better fixation than the grafted revision setting with or without OP‐1 (significantly more bone coverage, more mineralized tissue in the gap, and better mechanical interface strength). However, grafted primary implants with OP‐1 had impaired fixation compared with grafted primary implants without OP‐1 (less bone coverage of the implant and less bone formation in the gap). In contrast, grafted revision implants with OP‐1 significantly increased implant fixation compared with grafted revision implants without OP‐1 (increased mechanical interface strength and fraction of mineralized tissue in the gap). We found no differences between the two doses in any of the settings. Addition of OP‐1 to bone allografted implants may show benefit at sites with impaired bone healing capacities, such as the revision setting. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 569–576, 2004
Novel heparin/alginate gel combined with basic fibroblast growth factor promotes nerve regeneration in rat sciatic nerve
Authors: Ohta Masayoshi , Suzuki Yoshihisa , Chou Hirotomi , Ishikawa Namiko , Suzuki Shigehiko , Tanihara Masao , Suzuki Yasuo , Mizushima Yutaka , Dezawa Mari , Ide Chizuka
Published: November 2004

We have developed an alginate gel crosslinked with covalent bonds for regeneration of dermis, nerve, and bone. Recently, a novel matrix (H/A gel) which consists of heparin and alginate covalently crosslinked with ethylenediamine, was designed. It can stabilize and release biologically active basic fibroblast growth factor (bFGF) for 1 month, which is one of the heparin‐binding growth factors. In the present report, we examined the effect of this novel H/A gel on nerve regeneration in the rat sciatic nerve. In this study, regenerated axons in H/A gel with bFGF grew faster than in ordinary alginate gel with bFGF in the early stage. Myelinated fibers showed a tendency to increase in diameter toward the normal size in the later stage. Nerve bundles in the implantation exhibited minimal fibrosis and good vascularization. H/A gel with bFGF exhibited better‐developed vascularization than ordinary alginate gel with bFGF. These findings suggested that H/A gel with bFGF could serve not only as an efficient cellular scaffold, but also as a stabilizing matrix for bFGF for peripheral nerve regeneration. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 661–668, 2004
DNA vaccination against tumors
Authors: Prud'homme Gérald J.
Published: November 2004

DNA vaccines have been used to generate protective immunity against tumors in a variety of experimental models. The favorite target antigens have been those that are frequently expressed by human tumors, such as carcinoembryonic antigen (CEA), ErbB2/neu, and melanoma‐associated antigens. DNA vaccines have the advantage of being simple to construct, produce and deliver. They can activate all arms of the immune system, and allow substantial flexibility in modifying the type of immune response generated through codelivery of cytokine genes. DNA vaccines can be applied by intramuscular, dermal/epidermal, oral, respiratory and other routes, and pose relatively few safety concerns. Compared to other nucleic acid vectors, they are usually devoid of viral or bacterial antigens and can be designed to deliver only the target tumor antigen(s). This is likely to be important when priming a response against weak tumor antigens. DNA vaccines have been more effective in rodents than in larger mammals or humans. However, a large number of methods that might be applied clinically have been shown to ameliorate these vaccines. This includesin vivoelectroporation, and/or inclusion of various immunostimulatory molecules, xenoantigens (or their epitopes), antigen‐cytokine fusion genes, agents that improve antigen uptake or presentation, and molecules that activate innate immunity mechanisms. In addition, CpG motifs carried by plasmids can overcome the negative effects of regulatory T cells. There have been few studies in humans, but recent clinical trials suggest that plasmid/virus, or plasmid/antigen‐adjuvant, prime‐boost strategies generate strong immune responses, and confirm the usefulness of plasmid‐based vaccination. Copyright © 2004 John Wiley & Sons, Ltd.
The DNA minor groove binding agents Hoechst 33258 and 33342 enhance recombinant adeno‐associated virus (rAAV) transgene expression
Authors: Li Lina , Yang Linda , Kotin Robert M.
Published: November 2004

Background

Recombinant adeno‐associated viruses (rAAV) are commonly used in pre‐clinical and clinical gene transfer studies. However, the relatively slow kinetics of rAAV transgene expression complicates in vitro and in vivo experiments.

Methods

293 and COS‐1 cells were transduced with rAAV2‐EGFP, rAAV1‐EGFP, or rAAV5‐EGFP. The rAAV‐EGFP expression was analyzed in the presence of Hoechst 33 258 or 33 342 as a function of time and concentration by flow cytometry and fluorescent microscope. Effects of Hoechst on cell cycle populations were determined by flow cytometry. Enhanced green fluorescent protein (EGFP) expression plasmids with or without AAV inverted terminal repeats (ITR) were constructed and gene expression by transient transfection was compared in the presence of Hoechst.

Results

We found that Hoechst 33 258 and 33 342 increase both the level and the population of EGFP gene expressing cells, transduced by several different serotypes of rAAV‐EGFP. The augmentation of rAAV‐EGFP expression occurs in different cell types in a concentration‐dependent manner. In addition, the Hoechst 33 258 or 33 342 mediated enhancement of rAAV gene expression correlated with an increase of cells in S phase and G2/M phases of the cell cycle. Finally, gene expression from transfected ITR‐containing plasmid DNA was also enhanced by Hoechst dyes.

Conclusions

Our results revealed that two different, although related, DNA‐binding drugs, Hoechst 33 258 and 33 342, accelerate the kinetics of rAAV transgene expression. These findings may provide the basis for more sensitive assessment of rAAV biological activity and also extend the applications of rAAV for in vivo gene transfer. Published in 2004 John Wiley & Sons, Ltd.
Enantiomeric separation of chlorophenoxy acid herbicides by nano liquid chromatography‐UV detection on a vancomycin‐based chiral stationary phase
Authors: Rosales‐Conrado Noelia , León‐González Ma Eugenia , D'Orazio Giovanni , Fanali Salvatore
Published: November 2004

Enantiomeric separation of mecoprop, dichlorprop, and fenoprop herbicides in their acid form, commonly used to control the growth of broad‐leaved weeds, was carried out by nano‐liquid chromatography (nano‐LC) at a flow rate of 60 nL/min, using a packed capillary column with vancomycin‐modified silica particles of 5 μm. The length of chiral stationary phase was 21 cm, while the total and effective lengths were 43 and 33 cm, respectively. Inner diameter was 0.075 mm. Separated peaks were detected at 195 nm. Several mixtures of methanol, water, and 500 mM ammonium acetate buffer at different pH's were tested as mobile phase, and experimental parameters such as resolution (Rs), capacity factor (k), efficiency (N/m), and enantioselectivity factor (α) were measured under all the test conditions. Baseline enantiomeric separation was obtained for the three studied herbicides with α in the range 1.6–1.9, using as the mobile phase aqueous solutions containing 85% methanol, 5% of 500 mM ammonium acetate pH 4.5 buffer, and 10% water. Experimental results show that the vancomycin stationary phase presents a great enantiorecognition capability towards chlorophenoxy acid herbicides on using nano‐LC.
Extended thermodynamic approach to ion interaction chromatography. A mono‐ and bivariate strategy to model the influence of ionic strength
Authors: Cecchi Teresa , Pucciarelli Filippo , Passamonti Paolo
Published: November 2004

Recent breakthroughs in the theory of ion interaction chromatography (IIC) permit new analyses of the dependence of retention on different interdependent factors. The influence of the ionic strength I on the surface potential, the Donnan effect, and salting effects are taken into account to model the chromatographic behaviour of charged analytes in IIC. The most reliable experimental results found in the literature were used to test the retention equations that were developed following both a monovariate (I changes as the concentration of H, ion interaction reagent, changes) and a bivariate (I changes because of the simultaneous variation of H and of the background electrolyte concentrations) approaches. The present extended thermodynamic model builds on the sound intuition of the electrostatic approach and proves to provide the most successful and exhaustive quantitative explanation of experimental evidence. It is also able to rationalise the less extensive agreement between the pure electrostatic approach predictions and experimental results. The adequacy of the model is supported by physically reliable estimates of the adjustable constant (ion‐pair constants, ΔG°). Moreover statistical practice demonstrates that all the adjustable parameters (three at the most) are statistically significant. A linear, zero crossing function with unit slope is obtained when kpred is plotted against kexp. The mean percent error between kpred and kexp is 4.5% at most. In the absence of H the present retention equation reduces, as expected, to the relationship that describes the influence of I on the retention behaviour in reversed‐phase liquid chromatography.
Csaba Horváth's contribution to the theory and practice of capillary electrochromatography
Authors: Svec Frantisek
Published: November 2004

This overview has been written as a tribute to a luminary of chromatography – Csaba Gyula Horváth, who passed away earlier this year. Since the scope of his work was enormous, the following pages focus only on one single aspect of his scientific activities, capillary electrochromatography. He was a visionary, recognizing the great potential of this method and devoted a large part of his efforts to studies of problems related to CEC, covering a huge variety of issues embracing the theoretical foundation, instrumentation, and column technology. During the period of time from 1996 to 2004, Csaba Horváth published almost 30 excellent papers concerning capillary electrochromatography, which are reviewed in this article.
Two-photon Raman-type self-induced
transparency for few-cycle laser pulses
Authors: Prokopovich I P
Published: November 2004

We show the both amplitude and spectrum dynamics of
few-cycle laser pulses in two-photon Raman-type self-induced
transparency in Ar+ ions, that permit to generate half-cycle
laser pulses (FWHM) with more high amplitudes and photon energy
as well as spectral widths.