NPY family of hormones: clinical relevance and potential use in gastrointestinal disease.

The neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) family of hormones exhibit a wide variety of biological actions on the mammalian gastrointestinal tract through known G-protein coupled receptor pathways. At least four receptor subtypes, denoted as Y(1), Y(2), Y(4) an Y(5), each with specific affinities to NPY ligands, serve as regulators of mucosal function, gastrointestinal motility and secretion. Investigations to date, however, have implicated the NPY peptides as mediators in the pathogenesis of numerous gastrointestinal disorders, including malabsorption, short gut, inflammatory bowel diseases, and forms of pancreatitis. Our understanding of these diseases and the interactions of NPY peptides have been advanced by the development of receptor agonists and antagonists that can be used experimentally in animal models. Potent selective PYY agonists have been developed that exhibit clinical potential as proabsorptive agents. NPY receptor agonists and antagonists as well as mice harboring null mutations in the Y(1) and Y(4) receptors have provided novel approaches in preventing intestinal inflammation and diarrhea. The use of competitive antagonists and Y(2) receptor knockouts have also aided in understanding secretory tone and electrogenic ion transport in the colon. In the pancreas, PYY suppresses amylase and cytokine release, which would be desirable in the clinical therapy of pancreatitis. Protein/DNA array analysis has revealed that PYY reduces transcription factor binding activity and disrupts signal transduction pathways activated by TNF-alpha in acinar cells. The present review gives an overview of NPY research in gastrointestinal disease and discusses its clinical relevance and potential use as therapy.

Laparoscopic management of Chilaiditi's syndrome.

Chilaiditi's syndrome refers to the symptoms of abdominal pain, distention, vomiting, anorexia, and constipation caused by hepatodiaphragmatic interposition of the intestine. Although patients with this radiographic finding are commonly asymptomatic, presentation with symptoms is rare and accurately refers to this syndrome. There is an increased incidence of Chilaiditi's syndrome among mentally ill adults. Traditionally, Chilaiditi's syndrome is managed medically by discontinuing causative medicines. However, among the mentally ill population whose psychotropic medications precipitate the interposition of the colon, ceasing these psychotropic medications is not an appropriate option. The case presented involves a mentally ill patient with Chilaiditi's syndrome who was successfully managed with laparoscopic colopexy. At follow-up, the patient reported marked improvement of abdominal symptoms.

Vitamin E and the Y4 agonist BA-129 decrease prostate cancer growth and production of vascular endothelial growth factor.

BACKGROUND: A biologically active form of vitamin E, alpha-tocopherol succinate (ATS), has been shown to induce apoptosis of hormone-refractory prostate cancer in vitro and inhibit cell growth in vivo. The gastrointestinal hormone peptide YY (PYY) has growth inhibitory activity against multiple cancer cell lines and is synergistic with ATS against breast and pancreatic cancer growth. BA-129, a specific Y4 receptor agonist, has growth inhibitory effects on pancreatic cancer in vitro. We investigated the effects of BA-129 and ATS on prostate cancer growth and evaluated their effects on vascular endothelial growth factor (VEGF) production. METHODS: A hormone-refractory human prostate cancer cell line, PC-3, was treated with ATS alone at 10 pg/ml, PYY or BA-129 alone at doses of 75 and 500 pmol/ml, or a combination of the two agents. Cell growth was measured by MTT assay and hemocytometry using trypan blue. Quantitative measurement of VEGF was performed by ELISA. Statistical analysis was achieved by ANOVA. RESULTS: ATS exhibited significant (P < 0.05) growth inhibitory effects in prostate cancer cells. PYY also inhibited growth (P < 0.05). ATS treatment reduced VEGF production (P < 0.05). PYY treatment increased VEGF. When ATS was given in combination with BA-129, VEGF production was further reduced (P < 0.05). CONCLUSIONS: Both PYY and ATS inhibit growth in hormone-refractory prostate cancer, with augmentation when used in combination. VEGF production is inhibited by vitamin E, but increased by PYY. ATS abolishes the augmented VEGF response to PYY. Our data suggest that PYY is involved in the regulation of VEGF production and prostate cancer growth.

EGF and TGF stimulate proabsorption of glucose and electrolytes by Na+/glucose cotransporter in awake canine model.

Growth factor-stimulated intestinal absorption has recently been described, but the cellular transport mechanisms mediating this response are unknown. The purposes of this study were to examine the effect that intraluminal and systemic EGF and TGF have in intestinal absorption, elucidate a possible mechanism through which they exert their activity, and compare this response to that of a mixed meal only. Jejunal and ileal Thiry-Vella intestinal segments were constructed in six dogs. Absorption was measured by infusing the loops with a physiological electrolyte solution containing either 10 mmol or 50 mmol glucose and [14C]PEG as the impermeant marker. In vivo studies show that the addition of either EGF or TGF resulted in increased absorption of Na+, Cl-, H2O, and glucose in the intestine. This response was significantly greater than that seen when giving a mixed meal alone. Luminal phloridzin, an inhibitor of the SGLT-1 transporter, inhibited intestinal absorption observed in response to EGF and TGF. In conclusion, these results suggest that growth factors are capable of up-regulating intestinal absorption of electrolytes and nutrients and, these effects are mediated, at least in part, by SGLT-1 pathways.

Platelet activating factor antagonism reduces the systemic inflammatory response in a murine model of acute pancreatitis.

BACKGROUND: The platelet activating factor (PAF) antagonist, Lexipafant, has been used in experimental models and clinical trials to treat severe acute pancreatitis (AP). The purpose of this study was to determine whether Lexipafant reduces the local and systemic components of AP in a murine model of mild, edematous AP. MATERIALS AND METHODS: Forty-eight female Swiss-Webster mice were divided into four groups. Group 1 received 50 microl of saline ip every hour for 6 h (sham). Group 2 received saline treatment, plus Lexipafant (25 mg/kg dose ip, every 3 h starting 1 h after the first saline injection) (sham/Lex). Group 3 received cerulein (50 microg/kg dose ip, every hour for 6 h) (AP). Group 4 received AP, plus therapeutic treatment with Lexipafant (AP/Lex). Animals were sacrificed 3 h after the last injection. Serum cytokine levels were determined by ELISA. Standard assays were performed for serum amylase activity and lung myeloperoxidase activity (MPO). Histology was scored by two blinded investigators. RESULTS: Serum cytokines (TNFalpha, IL-1beta), lung MPO, and serum amylase activity were reduced by PAF antagonism. Histology showed a trend toward improvement with Lexipafant, but did not reach statistical significance. CONCLUSION: The PAF antagonism reduces the severity of systemic inflammation when given after the induction of mild AP in mice. These results suggest that Lexipafant may be useful in the treatment of mild pancreatitis after its clinical onset.

Synthetic peptide YY analog binds to a cell membrane receptor and delivers fluorescent dye to pancreatic cancer cells.

Pancreatic cancer continues to have a dismal prognosis despite multimodality treatment plans. Peptide YY (PYY) is a gut hormone that suppresses pancreatic exocrine and endocrine function. Previous experiments have shown that shortened synthetic PYY(22-36) analog decreases pancreatic cancer cell growth while also decreasing intracellular cyclic adenosine monophosphate. Our purpose was to construct an optimal synthetic PYY analog that binds to pancreatic cancer cells that may be used for imaging and therapy. Biotinylated PYY analogs with lengths ranging from PYY(1-36), PYY(9-36), PYY(14-36), PYY(22-36), and PYY(27-36) were tested with flow cytometry and receptor cross-linking studies to measure cell membrane binding. Growth inhibition studies were also performed using monotetrazolium tests to determine potency of various PYY analogs. Quantitative flow cytometry reveals the highest specific binding of PYY(14-36) to pancreatic cancer cells. Cross-linking studies reveal a receptor on the cell membrane of human pancreatic ductal adenocarcinoma cells. Growth inhibition studies reveal that PYY (14-36) has the highest potency against PANC-1 and MiaPaCa-2 cells. A novel synthetic PYY analog binds to the cell surface of pancreatic cancer cells and has the ability to deliver fluorescent dyes. The strategy of using biotinylated peptides to deliver avidin-dye complexes to cancer cells will allow imaging of pancreatic tumors and delivery of therapeutic agents.

Porcelain gallbladder is not associated with gallbladder carcinoma.

The surgical management of porcelain gallbladder is based on studies performed in 1931 and 1962, which indicated a correlation between porcelain gallbladder and carcinoma. We sought to evaluate the characteristics of patients with porcelain gallbladder and the risk for gallbladder carcinoma. The medical records of 10,741 cholecystectomies performed between 1955 and 1998 were reviewed and recorded. The pathology slides were evaluated for evidence of calcification and gallbladder carcinoma. Fifteen (0.14%) of 10,741 specimens were porcelain gallbladders. Ten patients (67%) had symptoms suggestive of biliary colic or cholecystitis. Five (33%) were asymptomatic and diagnosed incidentally. All specimens demonstrated chronic cholecystitis and partial calcification of the gallbladder wall. Nine (60%) had cholelithiasis. None had gallbladder carcinoma by recent review of pathologic material. During this same period 88 (0.82%) patients had gallbladder carcinoma, none of which showed calcification of the wall. This report represents the largest modern review of porcelain gallbladders. No carcinoma was identified among patients with porcelain gallbladder. In addition no patient with gallbladder carcinoma had calcified gallbladder. With a better understanding of the natural history of the porcelain gallbladder the current management of these patients may change.

Alpha-tocopherol succinate inhibits growth of gastric cancer cells in vitro.

BACKGROUND: Vitamin E in the form of alpha-tocopherol succinate (ATS) has been shown to inhibit growth of several cancer cell lines in vitro, including pancreas, breast, and prostate. No data exist on the effect of ATS on gastric cancer cell viability. METHODS: A gastric cancer cell line in suspension form, KATO-III, was plated in 96-well plates at 30,000 cells per well with 100 microl RPMI media. The cells were allowed to incubate for 24 h and were then treated with ATS at doses of 25, 50, or 100 microg/ml. The ATS was dissolved in 1% EtOH solution and control cells received an identical solution of EtOH without ATS. Treated cells were incubated for 24, 48, or 72 h. At the completion of the treatment period, MTT assay was performed to determine cell viability. Statistical analysis was performed using Student's t test. RESULTS: All doses of ATS resulted in inhibition of growth of the KATO-III cells. Both 100 and 50 microg/cc doses inhibited growth at all time points (P<0.005), with 48- and 72-h treatments more effective than 24-h treatment. At 24 and 48 h, 100 microg/cc was more effective at inhibition of growth than 50 microg/ml (P<0.005), but by 72 h the effects of the doses were equivalent; 25 microg/ml inhibited cell growth only at 48 and 72 h. At all time points, 50 and 100 microg/ml doses were more effective at inhibiting cell growth than 25 microg/ml. Conclusions. ATS inhibits gastric carcinoma cell growth in vitro in a dose- and time-dependent fashion. In vivo studies are indicated to further evaluate the potential benefit of this antioxidant against gastric cancer.

Structure-activity studies including a Psi(CH(2)-NH) scan of peptide YY (PYY) active site, PYY(22-36), for interaction with rat intestinal PYY receptors: development of analogues with potent in vivo activity in the intestine.

Peptide YY (PYY) is a gut hormone that inhibits secretion and promotes absorption and growth in the intestinal epithelium. We have performed structure-activity studies with the active site, N-alpha-Ac-PYY(22-36)-NH(2), for interaction with intestinal PYY receptors. Investigation of aromatic substitutions at position 27 resulted in analogues that exhibited potent in vitro antisecretory potencies with N-alpha-Ac-[Trp(27)]PYY(22-36)-NH(2) exhibiting even greater potency than intact PYY. In vivo studies in dogs revealed that this analogue also promoted intestinal absorption of water and electrolytes during continuous intravenous and intraluminal infusion. Investigations carried out to identify features that would enhance stability revealed that incorporation of Trp(30) increased affinity for PYY receptors. A "CH(2)-NH" scan revealed that incorporation of reduced bonds at position 28-29 or 35-36 imparted greater receptor affinity. In general, disubstituted analogues designed based on the results of single substitutions exhibited good receptor affinity with N-alpha-Ac-[Trp(27),CH(2)-NH(35-36)]PYY(22-36)-NH(2) having the greatest affinity (IC(50) = 0.28 nM). Conservative multiple substitutions with Nle-->Leu and Nva-->Val also imparted good affinity. An analogue designed to encompass most of the favored substitutions, N-alpha-Ac-[Nle(24,28),Trp(30),Nva(31), CH(2)-NH(35-36)]PYY(22-36)-NH(2), exhibited a proabsorptive effect in dogs comparable to, but longer lasting than, that of intact hormone. Selected analogues also exhibited good antisecretory potencies in rats with N-alpha-Ac-[Trp(30)]PYY(22-36)-NH(2) being even more potent than PYY. However, the potencies did not correlate well with the PYY receptor affinity or the proabsorptive potencies in dogs. These differences could be due to species effects and/or the involvement of multiple receptors and neuronal elements in controlling the in vivo activity of PYY compounds. PYY(22-36) analogues exhibited good affinity for neuronal Y2 receptors but poor affinity for Y1 receptors. Also, crucial analogues in this series hardly bound to Y4 and Y5 receptors. In summary, we have developed PYY(22-36) analogues which, via interacting with intestinal PYY receptors, promoted potent and long-lasting proabsorptive and antisecretory effects in in vivo models. These compounds or analogues based on them may have useful clinical application in treating malabsorptive disorders observed under a variety of conditions.

Laparoscopic port sites do not require fascial closure when nonbladed trocars are used.

The development of nonbladed obturators with integrated stability sleeves allows for creation of a muscle-splitting dilated laparoscopic port site with minimal abdominal wall defects after removal of trocar sleeves. Our objective was to determine the safety of using nonbladed obturators and not closing laparoscopic fascial port sites. Seventy patients underwent various laparoscopic procedures including the following: seven laparoscopic Roux en Y gastric bypasses, 21 laparoscopic cholecystectomies, 23 laparoscopic hernia repairs, 10 laparoscopic Nissen fundoplications, two laparoscopic appendectomies, two laparoscopic liver biopsies, one laparoscopic common bile duct exploration, one laparoscopic jejunal resection, one laparoscopic low anterior resection, one laparoscopic splenectomy, and one bedside diagnostic laparoscopy. A total of 180 laparoscopic port sites did not undergo fascial closure involving 110 10- to 12-mm ports. One hundred eighty nonbladed trocars were inserted without complication during laparoscopic surgery. In all cases the nonbladed obturator did not cause bleeding or injure viscera. Upon removal of large laparoscopic ports, the fascial defect was less than 6 to 8 mm, and the muscles of the abdominal wall covered the port site defect. The anterior fascial defect did not line up with the posterior fascial defect after removal of CO2 insufflation. No patients have developed ventral incisional hernias in the postoperative period (median follow-up of 11 months). We conclude that the use of nonbladed laparoscopic trocars is a safe technique with the ability to visualize dissection through the abdominal wall layers to create the smallest port dissection without bleeding or cutting muscle fibers. The ability to split the abdominal wall musculature allows the surgeon to forego closure of the small fascial defect.

Hypericin and photodynamic therapy decreases human pancreatic cancer in vitro and in vivo.

BACKGROUND: The treatment of pancreatic cancer has remained dismal despite advances in medical and surgical care. Recent preclinical data have revealed that hypericin, a photochemical dye, is activated by green light and generates toxic radical species in tumors. We hypothesized that interstitial hypericin and laser phototherapy would decrease pancreatic cancer growth. METHODS: MiaPaCa-2 and PANC-1 cells were grown in tissue culture. In vitro experiments were performed with addition of 10 microg of hypericin/500,000 cancer cells. Cells were incubated with hypericin for 2 h. Cells were then exposed to KTP532 green laser light for 1 min at 0.6 W using a cylindrical diffuser tip. Cell growth was measured by MTT assay 24 h after laser treatment, N = 12. MiaPaCa-2 cells were implanted subcutaneously and orthotopically in pancreas of nude mice. After 5 weeks, both tumors were injected with 100 microg of hypericin followed by insertion of a cylindrical diffuser tip into the tumor center. Mice received 200J KTP laser light at 1.0 W in two sites. Tumors were measured before and 4 weeks after laser treatment. RESULTS: Both in vitro and in vivo mice data showed a significant decrease in growth of pancreatic cancer. Pancreatic cancer cell growth was suppressed by 66.1 +/- 0.2%, n = 12, P < 0.01, ANOVA. Subcutaneous shoulder tumors were suppressed by 91.2 +/- 2.3%, n = 12, P < 0.001, and orthotopically grown pancreatic tumors were suppressed by 42.2 +/- 8.1%, n = 12, P < 0.05, compared to pretreatment sizes. Data expressed as percentage reduction vs paired controls in the MTT assay and vs pre-photodynamic therapy in mice experiments. Paired Student's t tests were performed vs pretreatment sizes. CONCLUSION: Both in vitro and in vivo results revealed a significant decrease in pancreatic cancer cell growth. Laser or dye alone had no effect, indicating that intratumor hypericin and laser therapy may prove useful in unresectable pancreatic cancer.

Improving the Surgeon's participation in research: is It a problem of training or priority?

BACKGROUND: Although numerous important contributions have originated from basic science research performed by surgeons, it seems that such dedicated work is becoming increasingly difficult to accomplish. What are the reasons for this change and what improvements can be made? This study aims to characterize the basic research training and careers of senior academic surgeons to assess and devise strategies for sustaining productive and quality surgical research. METHODS: A 25-item survey was sent to 850 senior-level members of academic societies, including the Association of Academic Surgeons, Society of University Surgeons, and American Surgical Association. It addressed each surgeon's clinical and research training and career, as well as opinions concerning surgical research. RESULTS: Three hundred seventy-seven (44%) surveys were received. Mean age was 64 years, and 73% were full professors. Seventy-two percent of respondents performed basic science research during training, and for 71% of this group, research was a significant reason for choosing a clinical specialty. Ninety-one percent performed research in the same specialty area during and after training. Of those who performed research during training, a full 99% continued to perform research on completion of training. However, 38% stopped performing basic research by age 39. Seventeen and twenty-three percent stopped basic research between 40 and 49 and between 50 and 59 years of age, respectively. The most common factors causing them to stop were increased clinical load (40%) and increased administrative duties (38%). For respondents who had stopped research prior to age 40, 73% cited increased clinical load as the primary reason. Eighty-five percent felt a dedicated research period should be included in surgery training. CONCLUSIONS: Most respondents had participated in basic research during training, and continued similar research after training. However, an overwhelming clinical practice at the junior faculty level seemed to hinder research. We conclude: (1) the majority consensus is that research training is integral to the development of academic surgeons; (2) such research training opportunities appear adequate; however, (3) faculty performing research, particularly at the junior level, need to be better protected from other academic duties, such as clinical practice and administration. The challenge to the leadership of academic surgery will be to enhance such research productivity in the context of increasing academic demands.

Peptide YY and vitamin E inhibit hormone-sensitive and -insensitive breast cancer cells.

We have shown that peptide YY, an endogenous gut hormone, and vitamin E succinate (VES) inhibit pancreatic cancer cell growth in vitro. We hypothesized that PYY and VES would inhibit breast cancer cell viability regardless of the hormone receptor status. Human breast ZR-75 ductal carcinoma (estrogen receptor negative) and MCF-7 adenocarcinoma (estrogen receptor positive) cells were cultured and exposed to VES (10 pg/ml), PYY (500 pmol), or both agents together. MTT assay was performed at 24, 48, and 72 h to evaluate cell viability. At every time interval, PYY and VES significantly inhibited cell growth compared to control. The effects of PYY were similar in magnitude to those of VES. Combining the agents resulted in a significant additive inhibition of growth with the greatest effect seen at 72 h. We have shown that PYY and vitamin E inhibit in vitro growth of breast cancer cells with variable hormone receptor status. When used in combination, the agents have a significant increase in effect. Further studies are ongoing to define the mechanism of action of these agents and to translate the experiments to an in vivo model.

Complicated enterocutaneous fistulas: failure of octreotide to improve healing.

Octreotide has been promoted as a potential aid during closure of enterocutaneous fistulas (ECFs) on the basis of clinical and experimental evidence that it can reduce gastrointestinal secretions. We retrospectively reviewed the records of patients admitted with ECF to our tertiary referral center to ascertain whether use of octreotide affected fistula duration, length of hospitalization, closure rate, and morbidity. Of 60 patients diagnosed and treated for ECF over a 4-year period, 13 underwent a therapeutic trial with octreotide. Thirteen patients from the group who did not receive octreotide were matched by cause, location, and output of the fistula, age, and primary diagnosis to the treatment group. Octreotide was administered in therapeutic dosage for a mean course of 57 +/- 29 days, resulting in a substantial acute decrease (84.7 +/- 4.8%) in fistula output. Prolonged therapy nevertheless failed to affect the outcome parameters studied, particularly fistula duration, spontaneous closure rate, and length of hospitalization. There was a significantly higher incidence of septic and thrombotic complications associated with octreotide use. In this patient population with complicated ECFs, use of octreotide showed no benefit and was associated with increased morbidity.

Role of Na+-glucose cotransport in jejunal meal-induced absorption.

In awake dogs, meal ingestion stimulates the absorption of water and electrolytes from neurovascularly intact jejunal Thiry-Vella loops, even though these loops are isolated from the remainder of the gut. This study was designed to investigate the role of Na+-glucose cotransport in mediating this event. Meal ingestion enhanced absorption when the jejunal lumen was perfused with an isotonic solution containing D-glucose, D-galactose, or 3-O-methylglucose. This response was absent when the perfusate contained mannitol or when phlorizin was added to the D-glucose solution. Mucosa from the jejunal loops was serially biopsied and assayed for brush-border Na+-glucose cotransporter (SGLT1) mRNA and protein expression. Although no changes in SGLT1 mRNA levels were observed, protein levels significantly increased within 30 min following meal ingestion. The time course of SGLT1 protein expression corresponded with that of increased Na+ and water absorption. These results suggest that meal-stimulated jejunal absorption may be mediated through an induction of mucosal SGLT1.

Interleukin-10 protects against lethality of intra-abdominal infection and sepsis.

The aim of this study was to determine whether interleukin-10 would alter locally derived and systemic proinflammatory cytokine expression and protect from the lethality of cecal ligation and puncture. Three groups of Sprague-Dawley rats were used. Group 1 underwent cecal manipulation. Groups 2 and 3 underwent cecal ligation and puncture. Group 2 received intraperitoneal saline injections beginning 1 hour after cecal ligation and puncture and every 3 hours thereafter for 24 hours. Group 3 received intraperitoneal interleukin-10 one hour after cecal ligation and puncture and every 3 hours thereafter. Animals were killed at 6 and 24 hours after cecal ligation and puncture or sham operation. Serum tumor necrosis factor-alpha (TNF-alpha) levels were determined by enzyme-linked immunosorbent assay. TNF-alpha messenger RNA expression was determined by reverse transcriptase-polymerase chain reaction using Beta-actin as the internal standard. There was a twofold increase (P <0.001) in TNF-alpha mRNA in the liver at 6 and 24 hours after cecal ligation and puncture when compared to rats treated with interleukin-10. There was a twofold increase (P <0.05) in TNF-alpha mRNA in the lung observed only at 24 hours after cecal ligation and puncture when compared to rats treated with interleukin-10. Serum levels of TNF-alpha were elevated at 6 hours in control animals, and this effect was abolished by the administration of interleukin-10. There was no difference in mortality rates at 6 hours (0% for all groups); however, at 24 hours 57% (4/7) mortality was observed in group 2 vs. 0% (0/20) in groups 1 and 3. Interleukin-10 given after the onset of cecal ligation and puncture protects against the lethality of intra-abdominal sepsis.

Peptide YY augments gross inhibition by vitamin E succinate of human pancreatic cancer cell growth.

BACKGROUND: Vitamin E succinate (VES) significantly inhibits cell growth in vitro in breast, prostate, and skin cancer cell lines. Our study demonstrated similar inhibitory effects on Mia PaCa-2 pancreatic cancer cells at the same concentration of VES (10 pg/ml). Peptide YY (PYY) also inhibits pancreatic cancer cell growth in vitro. We observed a significant additive effect on growth inhibition in Mia PaCa cells treated with both VES and PYY. METHODS: Human pancreatic ductal adenocarcinoma Mia PaCa-2 cells were cultured and treated once with either 10 pg/ml of VES or 500 pmols of PYY or with both agents together. The control group received an equivalent volume of solvents. MTT assay was performed at 24, 48, and 72 h to evaluate cell viability. RESULTS: Pancreatic cancer cell growth was reduced in all groups treated with PYY and VES. Student's t test was used to analyze the data for each treatment group. At 72 h, both PYY and vitamin E significantly inhibited cell growth compared to control. Combining the agents resulted in a dramatic additive inhibition of growth. CONCLUSION: PYY and vitamin E both inhibit growth of pancreatic cancer cells in vitro with a significant increase in effect when used in combination.

Intestinal ischemia and the gut-liver axis: an in vitro model.

BACKGROUND: Sustained intestinal ischemic injury often leads to shock and multiorgan failure, mediated in part by a cytokine cascade. Animal models have also identified a central role of Kupffer cells in amplification of cytokines following intestinal ischemia. To better understand this gut-liver axis, we developed an in vitro model. MATERIALS AND METHODS: Kupffer cells were isolated from rat livers by arabinogalactan gradient ultracentrifugation and adherence purification. Cells were grown in RPMI medium in 5% CO(2). Rat intestinal epithelial cells, IEC-6, were cultured under normoxic or anoxic (90% N(2), 10% CO(2)) conditions for 2, 12, and 24 h. Kupffer cells were then grown in the conditioned medium of the IEC-6 cultures. After 24 h, the medium was replaced with fresh medium. This final Kupffer cell supernatant was tested for tumor necrosis factor alpha and interleukin-6 production by ELISA. Trypan blue exclusion was performed to assess cell viability. RESULTS: Intestinal and Kupffer cells remained viable during the experimental time. Production of both tumor necrosis factor alpha and interleukin-6 by Kupffer cells increased with increasing ischemia time of the intestinal cells. CONCLUSIONS: Consistent with animal studies of intestinal ischemia, this study found an increase in cytokine production by Kupffer cells following hypoxia of intestinal cells. This in vitro model offers a new tool to study the expression of cytokines, proteins, and messengers involved in the cascade of events that follow intestinal ischemia.

Irradiation-induced up-regulation of Fas in esophageal squamous cell carcinoma is not accompanied by Fas ligand-mediated apoptosis.

BACKGROUND AND OBJECTIVES: Fas (APO-1) induces apoptosis after binding Fas ligand (FasL). Evidence suggests that tumors may use this interaction to evade the host immune response. Fas/FasL expression has not been reported in esophageal cancer. We hypothesized that Fas expression would render esophageal cancer cells susceptible to Fas ligation and that irradiation of the cells would increase Fas expression. METHODS: Two human esophageal squamous cell carcinoma lines, KYSE 150, which has a wild-type (wt) p53 gene, and 410 (mutated p53), were irradiated. Reverse-transcriptase polymerase chain reaction was used to detect Fas and FasL expression. Fas protein was quantitated by enzyme-linked immunosorbent assay and its presence further confirmed by Western analysis. FasL was detected by Western analysis. Cells were treated with Fas monoclonal antibody (maximum 0.05 microg/ml)+/-cycloheximide, and viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were also transduced with FasL cDNA and then quantified. RESULTS: Both lines expressed Fas and FasL, but only the KYSE 150 cell line displayed an increase in Fas following irradiation. No alteration in cell growth was detected for Fas antibody- or FasL-transduced groups versus controls. CONCLUSIONS: We have demonstrated Fas and FasL expression in esophageal tumor lines. We have also shown that Fas levels are significantly increased in response to irradiation in a wt p53 line. However, cells were resistant to treatment with Fas antibody or following transduction with FasL, suggesting that these tumor cells may use Fas/FasL expression to evade the host immune response.