Hemodynamic and metabolic variables predict porcine ex vivo liver function.

Early recognition of hepatic function during initial graft reperfusion is important in beginning hepatic support perfusions as well as in liver transplantation. We hypothesized that both hemodynamic and metabolic perfusion variables obtained immediately after reperfusion predict eventual function during liver support or transplantation. Specific hemodynamic variables, i.e., portal vein pressure and hepatic vascular resistance, as well as metabolic variables, i.e., O(2) consumption and P(CO(2)) gradients, were compared with indices of hepatic function and damage, i.e., aqueous bile production, bile lipid outputs, lactate dehydrogenase levels, and histopathology, during an ex vivo support perfusion. O(2) consumption during early reperfusion correlated directly with unstimulated bile flows (P < 0.02) and histopathology scores (P < 0.05). Hepatic venous P(CO(2)) gradients correlated inversely with unstimulated bile flows (P < 0.05). Hemodynamic variables, i.e., portal vein pressure and hepatic vascular resistance, were inversely related with taurocholate-stimulated bile flows (P < 0.05). Hemodynamic and metabolic variables of early reperfusion are useful parameters in predicting eventual effectiveness of the harvested liver for ex vivo hepatic support perfusions.

Pancreatic cancer cell proliferation is phosphatidylinositol 3-kinase dependent.

BACKGROUND: Genetic mutations found in pancreatic cancer (K-ras, p16, p53) lead to inappropriate cellular proliferation. Mitogens stimulate proliferation via the phosphatidylinositol 3-kinase (PI3K)- and/or the p44/42-mitogen-activated protein kinase [p44/42-MAPK or extracellular signal-regulated kinase (ERK)] signaling pathways. We examined whether inhibition of either PI3K or ERK could limit proliferation in human pancreatic cancer. METHODS: Proliferation was stimulated in quiescent human pancreatic cancer cell lines (BxPC3 and Panc-1) by 10% fetal calf serum (FCS). In certain samples, PD98059 (an ERK inhibitor) or LY294002 (a PI3K inhibitor) was also added. AKT phosphorylation (indicating PI3K activity) and ERK phosphorylation (ERK activation) were determined by Western blot. Cell viability was determined by MTT assay. Cell cycle progression and apoptosis were determined by flow cytometry. A two-tailed t test was used for statistical analysis of the data (significance P < 0.05). RESULTS: LY294002 inhibited the PI3K pathway without affecting ERK activation in response to serum. PD98059 inhibited the ERK pathway specifically. In both BxPC-3 and Panc-1 cell lines, LY294002 inhibited serum-induced proliferation. This was associated with G(1) cell cycle arrest and with an increase in the rate of apoptosis. PD98059 inhibited proliferation only in BxPC3 cells, and to a lesser degree than did LY294002. CONCLUSIONS: PI3K signaling appears to be necessary for G(1)-to-S phase progression and proliferation in pancreatic cancer cells. ERK plays a lesser role in mitogen-induced proliferation. Pharmacological inhibition of PI3K may decrease proliferation, increase apoptosis, and potentially confer therapeutic benefit in pancreatic cancer.

Sodium salicylate inhibits proliferation and induces G1 cell cycle arrest in human pancreatic cancer cell lines.

The mutations most common in pancreatic cancer decrease the ability to control G1 to S cell cycle progression and cellular proliferation. In colorectal cancer cells, nonsteroidal anti-inflammatory drugs inhibit proliferation and induce cell cycle arrest. We examined whether sodium salicylate, an aspirin metabolite, could inhibit proliferation in human pancreatic cancer cell lines (BxPC3 and Panc-1). Quiescent cells were treated with medium containing 10% fetal calf serum, with or without salicylate. Cellular proliferation was measured by MTT assay and bromodeoxyuridine incorporation. The fractions of cells in G0/G1, S, and G2/M phases of the cell cycle were quantitated by fluorescence-activated cell sorting. Results were compared between groups by two-tailed t test. Cyclin D1 expression was determined by Western blot analysis and prostaglandin E2 expression by enzyme-linked immunosorbent assay. Serum-starved cells failed to proliferate, with most arrested in the G1 phase. Salicylate significantly inhibited serum-induced progression from G1 to S phase, cellular proliferation, and the expression of cyclin D1. The concentrations at which 50% of serum-induced proliferation was inhibited were 1.2 mmol/L (Panc-1) and 1.7 mmol/L (BxPC3). The antiproliferative effect of sodium salicylate was not explained by inhibition of prostaglandin E2 production. This study provides further evidence in a noncolorectal cancer model for the antineoplastic effects of nonsteroidal anti-inflammatory drugs.

Laparoscopic surgery and Kupffer cell activation.

BACKGROUND: Evidence tends to support a relative preservation of the systemic immune response with laparoscopy as compared with laparotomy. However, the role of hepatic macrophages, or Kupffer cells, in modulating this immune advantage is unknown. This study investigated the functions of Kupffer cells after either laparoscopy or laparotomy in a rat model. METHODS: Rats underwent laparoscopy, laparotomy, or control operations. Kupffer cells were harvested, cultured, and stimulated with lipopolysaccharide. Culture supernatants were analyzed for tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6). Cytoplasmic lysates were analyzed for activation of two mitogen-activated protein kinases (MAPKs). RESULTS: Production of TNF-alpha and IL-6 was similar in laparoscopy, laparotomy, and control groups. Both laparotomy and laparoscopy showed increased activation of p38 MAPK as compared with controls. Activation of ERK1/2 was decreased during laparotomy as compared with laparoscopy. CONCLUSIONS: Although cytokine production was similar in the laparoscopy and laparotomy groups, changes in MAPK activation suggest that intracellular pathways are more affected during laparotomy than during laparoscopy.

Biliary secretion of extracorporeal porcine livers with single and dual vessel perfusion.

BACKGROUND: Hepatic support systems that provide detoxification without biliary secretion (i.e., isolated hepatocyte systems) are sufficient to improve encephalopathy and bridge patients to transplantation. However, biliary secretion may be critical when hepatic support attempts to restore function and regeneration of the host liver. The purpose of these studies was to optimize the support liver secretory response to bile acid by either single-vessel (portal vein; PV) or dual-vessel (hepatic artery [HA] + PV) perfusions during extracorporeal porcine liver perfusion. METHODS: Extracorporeal porcine liver perfusion of anesthetized pigs was developed using support porcine livers perfused through the PV (n=4) alone and through the HA + PV (n=4) via a venovenous circuit. Support livers were provoked with taurocholate (TC) to enhance bile aqueous and hydrophobic outputs. RESULTS: After cold preservation and reperfusion, both PV and HA + PV livers had initial 1-hr bile aqueous outputs < 15% of in vivo flow, with cholesterol (C) and phospholipid (PC) outputs <25% of in vivo flow. Bile flow was significantly greater for recovered HA + PV livers (3.0+/-0.01 ml/15 min) than PV livers (1.9+/-0.01 ml/15 min). Despite this, PC output was significantly greater for PV than HA + PV livers. The C/PC ratio of PV livers was twice that of HA + PV livers. TC infusion (48 micromol/kg/15 min) of HA + PV livers demonstrated significantly greater increments in bile flow, PC output, and C output than PV livers. CONCLUSION: In the unstimulated state, porcine support livers with dual-vessel perfusion generated greater aqueous and C outputs despite diminished PC output than in those with single-vessel perfusion. TC stimulation increased bile flow, PC output, and C output in dual-perfused livers more than in PV livers. HA + PV perfusion of support livers is the preferred technique for removing hydrophobic compounds that require PC transport for excretion or exist in the aqueous phase.

Ubiquitin-proteasome inhibition enhances apoptosis of human pancreatic cancer cells.

BACKGROUND: Tumor necrosis factor (TNF-a)-induced apoptosis is limited by coactivation of nuclear factor kappa B (NF-kb)-dependent antiapoptotic genes. Nuclear translocation of NF-kB requires degradation of ubiquitinated phospho-IkB-a by the 26S proteasome. We examined whether inhibition of the ubiquitin-proteasome pathway enhances TNF-a-induced apoptosis in BxPC-3 human pancreatic cancer cells. METHODS: Serum-starved BxPC-3 cells (12 hours) were pretreated or not for 50 minutes with PSI (30 m mol/L), a peptide aldehyde known to inhibit specifically the chymotrypsin-like activity of the 26S proteasome. Cells were subsequently stimulated with recombinant human TNF-a (400 units/mL). Western blots were performed using antibodies to IkB-a and phospho-IkB-a. Level of apoptosis was determined by two methods: enzyme-linked immunosorbent assay detection of interhistone DNA fragments and flow cytometry with propidium iodide staining. RESULTS: TNF-a-induced degradation of IkB-a was inhibited by PSI. Phospho-IkB-a accumulation was observed 20 minutes after TNF-a stimulation. Apoptosis relative to constitutive levels was significantly increased after PSI pretreatment, as measured by DNA fragmentation (P < or = .05 by Student t test). Percent apoptosis by flow cytometry confirmed marked increases in apoptotic cell fractions from 5.9% (untreated) to 6.8% (TNF-a alone), 16.4% (PSI alone), and 18.9% (PSI and TNF-a). CONCLUSIONS: PSI enhances both constitutive and TNF-a-induced apoptosis through inhibition of IkB-a degradation in BxPC-3 human pancreatic cancer cells.

Sodium salicylate inhibits macrophage TNF-alpha production and alters MAPK activation.

INTRODUCTION: Transcriptional activation of the TNF-alpha gene in LPS-stimulated macrophages is dependent upon nuclear factor kappa-B (NF-kappaB) activity. Salicylates may interfere with NF-kappaB activity through a MAPK (mitogen-activated protein kinase)-dependent process. These studies investigate the effects of sodium salicylate (NaSal) on TNF-alpha production and MAPK activation in macrophages. METHODS: Rat peritoneal macrophages were pretreated or not with sodium salicylate or ibuprofen for 1 h and then stimulated with 100 ng/ml LPS. Six hours following stimulation, cell viability was assessed by MTT assay. At specified time intervals after LPS stimulation, supernatant TNF-alpha was measured by ELISA. Western blots of cell lysates were performed for analysis of total and activated (phosphorylated) MAPKs. RESULTS: Salicylate and LPS, alone or combined, did not significantly alter macrophage viability. Salicylate, but not ibuprofen, significantly reduced TNF-alpha production in LPS-stimulated macrophages. LPS-stimulated activation of ERK and SAPK/JNK was inhibited by NaSal pretreatment. NaSal treatment of macrophages activated p38 MAPK independent of LPS stimulation. Pretreatment of samples with the specific p38 MAPK inhibitor, SB203580, did not significantly alter TNF-alpha production in either LPS or NaSal and LPS-treated samples. CONCLUSIONS: Salicylates alter MAPK signaling and suppress TNF-alpha production in LPS-stimulated macrophages. Salicylate-induced control of inflammatory mediator production in macrophages may, in part, underlie the clinically significant anti-inflammatory effects of these compounds.

Salicylates inhibit NF-kappaB activation and enhance TNF-alpha-induced apoptosis in human pancreatic cancer cells.

INTRODUCTION: Tumor necrosis factor (TNF-alpha)-induced apoptosis is limited by its coactivation of nuclear factor kappa B (NF-kappaB)-dependent anti-apoptotic genes. Sodium salicylate (NaSal) inhibits NF-kappaB activation by limiting phosphorylation and degradation of its bound inhibitor protein, IkappaB-alpha. We examined whether NaSal enhances TNF-alpha-induced apoptosis in cultured human pancreatic cancer cell lines. METHODS: Two cultured human pancreatic cancer cell lines were studied. PANC-1 and BxPC-3 cells were serum-starved for 12 h, pretreated or not for 1 h with NaSal (5-20 mM), and then stimulated with recombinant human TNF-alpha (400 units/ml). Western blots of cytoplasmic lysates were performed to demonstrate IkappaB-alpha phosphorylation and degradation. Western blots of nuclear extracts were performed to assess nuclear translocation of NF-kappaB. In separate cultures, apoptosis was measured 4.5 h after TNF-alpha stimulation by both ELISA detection of interhistone DNA fragments and flow cytometry with propidium iodide staining. RESULTS: TNF-alpha induced IkappaB-alpha phosphorylation and degradation, which was inhibited by NaSal in both cell lines. TNF-alpha-induced apoptosis (DNA fragmentation) increased significantly when BxPC-3 cells were pretreated with NaSal. Flow cytometry confirmed this, demonstrating increases in apoptotic cell fractions: 8.5% (untreated), 9.3% (TNF-alpha alone), 14.9% (15 mM NaSal), and 22.9% (NaSal and TNF-alpha). In contrast, no increases in apoptosis were measured in the PANC-1 cell line among the various treatment groups. CONCLUSIONS: NaSal enhances TNF-alpha-induced apoptosis while inhibiting IkappaB-alpha phosphorylation and degradation in BxPC-3 human pancreatic cancer cells.

Endotoxin fails to stimulate inositol triphosphate production in macrophages.

UNLABELLED: Inositol Triphosphate (IP3) production is an early cell signaling event which leads to mobilization of intracellular calcium (Ca++). We examined whether bacterial endotoxin (lipopolysaccharide, LPS) stimulates IP3 production in macrophages pretreated with LPS (tolerant) or not. METHODS: RAW 264.7 macrophages were cultured at 5 x 10(6) cells in RPMI supplemented with 10% FCS. LPS tolerance was induced by pretreating macrophages (Tol) for 19 h with 10 ng/ml of LPS. Non-tolerant (Non-Tol) macrophages received no LPS pretreatment. Macrophages were next washed, repleted with fresh media, and stimulated with 100 ng/ml LPS. Paired cultures were stimulated with 1 microM platelet activating factor (PAF), a known stimulant of IP3 production. Following 1, 10, and 15-min stimulation intervals, IP3 was extracted with trichloroacetic acid and measured by receptor displacement assay. RESULTS: LPS did not stimulate IP3 production in either Non-Tol or Tol macrophages. In contrast, PAF stimulated significant increases in IP3 levels within 1 min in both Non-Tol (9.5 +/- 3.0 pmol/ml) and Tol (9.5 +/- 2.4 pmol/ml) macrophages. Non-Tol IP3 levels returned to baseline by 10 min, while Tol IP3 levels remained significantly elevated (8.2 +/- 1.7 pmol/ml). CONCLUSIONS: Unlike PAF, bacterial LPS fails to stimulate IP3 production in macrophages. Furthermore, IP3 production could not be elicited in cultured macrophages repetitively stimulated with LPS.

The molecular and cellular biology of pancreatic cancer.

Adenocarcinoma of the pancreas carries a grave prognosis for affected patients. Certain oncogenes (K-ras and HER-2/neu) are mutated in a large proportion of these aggressive tumors. Adenocarcinoma of the pancreas has also been associated with loss of tumor suppressor genes (p53, DPC4, p16/MTS), either by deletion or by mutation and loss of function. Growth factors (EGF, TGF-alpha, HGF) and growth factor receptors (EGF-R, c-met, CCK) are expressed at levels not found in the normal pancreas. Finally, factors important for angiogenesis (FGF, integrins, selectins) are likely to play an important role in the growth and metastasis of clinically relevant tumors. This review attempts to summarize and assimilate current research into the molecular and cellular biology of pancreatic cancer.

Laparoscopic surgery and the systemic immune response.

OBJECTIVE: The authors review studies relating to the immune responses evoked by laparoscopic surgery. SUMMARY BACKGROUND DATA: Laparoscopic surgery has gained rapid acceptance based on clinical grounds. Patients benefit from faster recovery, decreased pain, and quicker return to normal activities. Only more recently have attempts been made to identify the metabolic and immune responses that may underlie this clinical success. The immune responses to laparoscopy are now being evaluated in relation to the present knowledge of immune responses to traditional laparotomy and surgery in general. METHODS: A review of the published literature of the immune and metabolic responses to laparoscopy was performed. Laparoscopic surgery is compared with the traditional laparotomy on the basis of local and systemic immune responses and patterns of tumor growth. The impact of pneumoperitoneum and insufflation gases on the immune response is also reviewed. CONCLUSIONS: The systemic immune responses for surgery in general may not apply to laparoscopic surgery. The body's response to laparoscopy is one of lesser immune activation as opposed to immunosuppression.

Fluorescent and biotin probes for dopamine receptors: D1 and D2 receptor affinity and selectivity.

Fluorophor and biotin derivatives of dopamine agonist and antagonist drugs were synthesized and evaluated for binding affinity and selectivity at D1 and D2 dopamine receptors in membranes prepared from monkey (Macaca fascicularis) caudate putamen. Binding was measured using [3H]SCH 23390 to label D1 receptors and [3H]spiperone to label D2 receptors. The selective D1 antagonist SKF 83566, whether coupled to 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD), to fluorescein, or to biotin retained high affinity for D1 dopamine receptors (Ki, 5.3 16 and 3.5 nM, respectively) and high D1/D2 receptor selectivity (130-, 300, and 600-fold, respectively). The selective D2 antagonist derivative N-(p-aminophenethyl)spiperone, (NAPS) coupled either to biotin or to NBD via the N-aminoethylphenyl group, likewise retained high D2 receptor affinity (Ki, 0.58 and 0.66 nM, respectively) and high D2/D1 selectivity (190- and 150-fold, respectively). The affinity of the NBD-coupled derivative of (S)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin hydrochloride [(S)-PPHT], a selective D2 agonist, was actually higher than that of the parent compound (Ki, 0.30 versus 2.1 nM), whereas the affinity of fluorescein-coupled (S)-PPHT was lower (Ki, 4.8 nM). Sensitivity to GTP, a characteristic of agonist binding at dopamine receptors, was demonstrated for NBD-coupled (S)-PPHT, because D2 receptor affinity was somewhat reduced in the presence of GTP. PPHT-fluorescein fluorescence labeling rimmed cells in monkey and rat anterior pituitary and outlined cells in the striatum. Fluorescent and biotin probes based on selective high affinity ligands for dopamine receptors may expedite studies of receptor localization and mobility at the cellular level.

Intracardiac extension of intravenous leiomyomatosis.

A 42-year-old woman was found to have intravenous leiomyomatosis of the uterus with extension into the inferior vena cava and right atrium. Intravenous leiomyomatosis is a rare neoplastic disease characterized by invasion of venous channels by a benign smooth muscle tumor arising either from the wall of a vessel or from a uterine myoma. Intracardiac extension is often initially misdiagnosed as a right atrial myxoma and may cause death by mechanical obstruction. The diagnosis of intravenous leiomyomatosis should be considered in young women with cardiac symptoms associated with a right atrial mass who also have a pelvic mass or who have previously undergone hysterectomy because of leiomyoma uteri.