Priority setting for adult malnutrition and nutritional screening in healthcare: a James Lind Alliance.

BACKGROUND: Malnutrition is one the greatest global health challenges of our generation, leading to the increased utilisation of healthcare resources, as well as morbidity and mortality. Research has primarily been driven by industry, academia and clinical working groups and has had little involvement from patients and carers. The project described in the present study aimed to establish a priority setting partnership allowing patients, carers and healthcare professionals an opportunity to influence the research agenda. METHODS: A national survey was conducted to gather malnutrition uncertainties and identify key issues (i.e. areas within scope where an evidence-base is lacking) from those with experience of malnutrition. Uncertainties were analysed according to themes. Similar questions were grouped and summary questions were developed. A second survey was conducted and respondents were asked to choose their 10 most important summary questions. A workshop was conducted to finalise the top 10 research priorities from the most frequently indicated uncertainties on the interim survey. RESULTS: Overall, 1128 uncertainty questions were submitted from 268 people. The interim survey had 71 responses and a list of the top 26 questions was generated for the workshop. There were 26 questions discussed, ranked and agreed by healthcare professionals, carers and patients at the workshop. The top 10 research priorities were then chosen. These included questions on oral nutritional supplements, vulnerable groups, screening, community care, use of body mass index and technology. CONCLUSIONS: The top 10 research priorities in malnutrition and nutritional screening have been identified from a robust process involving patients, carers and healthcare professionals.

The novel NADPH oxidase 4 inhibitor GLX351322 counteracts glucose intolerance in high-fat diet-treated C57BL/6 mice.

In type 2 diabetes, it has been proposed that pancreatic beta-cell dysfunction is promoted by oxidative stress caused by NADPH oxidase (NOX) overactivity. Five different NOX enzymes (NOX1-5) have been characterized, among which NOX1 and NOX2 have been proposed to negatively affect beta-cells, but the putative role of NOX4 in type 2 diabetes-associated beta-cell dysfunction and glucose intolerance is largely unknown. Therefore, we presently investigated the importance of NOX4 for high-fat diet or HFD-induced glucose intolerance using male C57BL/6 mice using the new NOX4 inhibitor GLX351322, which has relative NOX4 selectivity over NOX2. In HFD-treated male C57BL/6 mice a two-week treatment with GLX351322 counteracted non-fasting hyperglycemia and impaired glucose tolerance. This effect occurred without any change in peripheral insulin sensitivity. To ascertain that NOX4 also plays a role for the function of human beta-cells, we observed that glucose- and sodium palmitate-induced insulin release from human islets in vitro was increased in response to NOX4 inhibitors. In long-term experiments (1-3 days), high-glucose-induced human islet cell reactive oxygen species (ROS) production and death were prevented by GLX351322. We propose that while short-term NOX4-generated ROS production is a physiological requirement for beta-cell function, persistent NOX4 activity, for example, during conditions of high-fat feeding, promotes ROS-mediated beta-cell dysfunction. Thus, selective NOX inhibition may be a therapeutic strategy in type 2 diabetes.

Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5'-phosphatase interaction with c-Abl.

AIMS/HYPOTHESIS: It is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway. METHODS: Human EndoC-ßH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-ßH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-ßH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining. RESULTS: Imatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total ß-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability. CONCLUSIONS/INTERPRETATION: Imatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

C/EBP homologous protein contributes to cytokine-induced pro-inflammatory responses and apoptosis in ß-cells.

Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic ß-cells. Pro-inflammatory cytokines are early mediators of ß-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in ß-cells, but the role for CHOP overexpression in cytokine-induced ß-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-κB (NF-κB) is a crucial transcription factor regulating ß-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-κB activity and expression of key NF-κB target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased IκB degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting ß-cell death: (1) CHOP directly contributes to cytokine-induced ß-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-κB activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis.

A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans.

AIMS/HYPOTHESIS: Successful gene transfer to pancreatic islets might be a powerful tool for dissecting the biological pathways involved in the functional impairment and destruction of beta cells in type 1 diabetes. In the long run, such an approach may also prove useful for promoting islet graft survival after transplantation in diabetic patients. However, efficient genetic modification of primary insulin-producing cells is limited by the specific compact structure of the pancreatic islet. We present here a whole-pancreas perfusion-based transduction procedure for genetic modification of intact pancreatic islets. MATERIALS AND METHODS: We used flow cytometry analysis and confocal microscopy to evaluate the efficiency of in vitro and perfusion-based transduction protocols that use adenoviral and lentiviral vectors expressing green fluorescent protein. Islet cell viability was assessed by fluorescence microscopy and beta cell function was determined via glucose-stimulated insulin secretion. RESULTS: In intact rat and human pancreatic islets, adenoviral and lentiviral vectors mediated gene transfer to about 30% of cells, but they did not reach the inner cellular mass within the islet core. Using the whole-pancreas perfusion protocol, we demonstrate that at least in rodent models the centrally located insulin-producing cells can be transduced with high efficiency, while preserving the structural integrity of the islet. Moreover, islet cell viability and function are not impaired by this procedure. CONCLUSIONS/INTERPRETATION: These results support the view that perfusion-based transduction protocols may significantly improve the yield of successfully engineered primary insulin-producing cells for diabetes research.

Effective and accurate screening for diabetic retinopathy using a 60 degree mydriatic fundus camera.

UNLABELLED: OBJECTIVES. To establish whether an experienced endocrinologist could screen accurately for diabetic retinopathy using mydriatic 60 degree fundus photographs compared with a reference standard, viz. the combined highest scores of two experienced ophthalmologists. DESIGN: Retrospective review of 60 degree colour transparency photographs taken over a 6-year period. Retinopathy was graded in a standardised way. SETTING: Patients attending the diabetic clinic at Johannesburg Hospital, South Africa. SUBJECTS: Fifteen hundred and seventeen patients (2446 eyes) formed the basis for the study. Patients were included if there was more than 50% readability of the fundus photographs. OUTCOME MEASURES: Outcome measures were prevalence of any retinopathy and presence of referable (severe) retinopathy. Interobserver agreement was measured using the kappa statistic, and sensitivity and specificity of the screener were evaluated. RESULTS: The prevalence of retinopathy at the clinic was approximately 30%, but only about 12% was severe enough to warrant referral to the ophthalmology outpatient department. The endocrinologist was very accurate in determining cases requiring referral; there was 97% agreement with the reference standard, viz. the combined highest score of two experienced ophthalmologists (gold standard). Correlation on the determination of any retinopathy was less accurate (80% agreement), mostly owing to the endocrinologist reporting more isolated microaneurysms than the ophthalmologists. The screening method used gave a sensitivity of 83% and specificity of 99% which are within recommended standards. CONCLUSIONS: The screening strategy using a mydriatic fundus camera at the diabetic clinic was found to be effective and accurate and greatly reduced the number of possible referrals to the ophthalmology outpatient department.

Protection of rat pancreatic islets by potassium channel openers against alloxan, sodium nitroprusside and interleukin-1beta mediated suppression--possible involvement of the mitochondrial membrane potential.

AIMS/HYPOTHESIS: We aimed to study the effects of two K(ATP) channel openers (KCO), diazoxide and the more potent compound NNC 55-0118, on beta-cell suppression and/or toxicity induced by alloxan, sodium nitroprusside and IL-1beta. METHODS: Islets from rats were exposed to 0.3 mmol/l diazoxide or NNC 55-0118 for 30 min and either alloxan (0.5 mmol/l), sodium nitroprusside (0.5 mmol/l) or IL-1beta (12.5 or 25 U/ml) were added and the incubation continued for 30 min. Islets were then washed and incubated for 24 h before examination. RESULTS: After exposure to alloxan, islets showed reduced glucose oxidation rate and impaired glucose-stimulated insulin release. NNC 55-0118 counteracted the effects of alloxan, while diazoxide was less effective. After treatment with sodium nitroprusside, islet glucose oxidation rates were reduced and this was prevented by pretreatment with NNC 55-0118. In short-term experiments the potassium channel openers (KCOs) did not influence the IL-1beta effect on insulin secretion. However, long-term addition (24 h) of NNC 55-0118 counteracted IL-1beta induced inhibition of the glucose oxidation rate. It was shown, using the fluorescent probe JC-1, that the mitochondrial membrane potential was reduced by the potassium channel openers (KCOs), most strongly by NNC 55-0118. Nevertheless culture with KCOs for 72 h did not cause irreversible damage to the islets. CONCLUSION/INTERPRETATION: Potassium channel openers (KCOs), in particular NNC 55-0118, prevented the toxic effects of alloxan and sodium nitroprusside. IL-1beta mediated suppression was reduced by NNC 55-0118 provided the long-term addition of the potassium channel opener (KCO). The protective mechanism of potassium channel openers (KCOs) might involve a decrease of the mitochondrial membrane potential.

Pharmacological characterization of cholecystokinin receptors mediating contraction of human gallbladder and ascending colon.

Cholecystokinin (CCK) produces contractions of gallbladder and colon in a number of different species. Although the effects of CCK on the human gallbladder are relatively well documented, the CCK receptors in the human colon have not been clearly characterised. Therefore, in this study, the CCK receptors in the human gallbladder and colon were compared using pharmacological techniques. Contraction of specimens of the human tissue was measured using in vitro organ bath bioassay. The effect of selective concentrations of CCK(1) and CCK(2) receptor antagonists (L-364,718 and JB93182, respectively) was determined on agonist concentration-effect (E/[A]) curves obtained by cumulative dosing with sulphated CCK. The CCK(1) antagonist L-364,718 produced a rightward shift of the CCK-8S [E/[A] curve in the human gallbladder (pA(2)=9.15 +/- 0.26) and ascending colon (pA(2)=9.20 +/- .33). In both tissues, the CCK(2) receptor antagonist, JB93182, had no effect on the CCK E/[A] curves. In addition, in the colon, pentagastrin responses were inhibited by L-364,718 but unaffected by JB93182. These data indicate that the CCK-induced contraction of the human colon and gallbladder smooth muscle is mediated solely through the CCK(1) receptor subtype, and the antagonist affinity estimates are consistent with those previously obtained in experiments on animal tissue.

Absence of a significant linkage between Na(+),K(+)-ATPase subunit (ATP1A3 and ATP1B3) genotypes and bipolar affective disorder in the old-order Amish.

Previous studies provide evidence for a genetic component for susceptibility to bipolar affective disorder (BPAD) in the old-order Amish population. El-Mallakh and Wyatt [1995: Biol Psychiatry 37:235-244] have suggested that the Na(+),K(+)-ATPase may be a candidate gene for BPAD. This study examines the relationship between BPAD in the old-order Amish cohort and the Na(+),K(+)-ATPase alpha1 and beta3 subunit genes (ATP1A3, ATP1B3). A total of 166 sibling pairs were analyzed for linkage via nonparametric methods. Suggestive levels of statistical significance were not reached in any stratification model for affective illness. Overall, the results do not support linkage of bipolar disorder to the Na(+),K(+)-ATPase alpha subunit gene (ATP1A3) and beta subunit gene (ATP1B3) in these old-order Amish families and they show that these Na(+),K(+)-ATPase subunit genes are not major effect genes (>or=fourfold increased genetic risk of disease) for BPAD in the old-order Amish pedigrees. We cannot exclude other genetic variants of the Na(+),K(+)-ATPase hypothesis for BPAD, whereby other loci may modifying Na(+),K(+)-ATPase activity.

Role of p38 mitogen-activated protein kinase (p38 MAPK) in cytokine-induced rat islet cell apoptosis.

The signaling pathways mediating nitric oxide production and apoptosis in pancreatic beta-cells are not fully understood. We investigated cytokine-induced protein phosphorylation events in insulin-producing cells and evaluated their role in inducible nitric oxide synthase (iNOS) induction and cell death. Interleukin-1beta (IL-1beta), but not interferon-gamma (IFN-gamma), induced phosphorylation of p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, and mitogen- and stress-activated protein kinase 1 (MSK1) in rat insulin-producing RINm5F cells. This was paralleled by an increased phosphorylation of the transcription factors activating transcription factor-2 (ATF-2) and cAMP-responsive element-binding protein (CREB). The p38 inhibitor SB203580 prevented cytokine-induced phosphorylation of CREB and MSK1, but not of ATF-2. IFN-gamma induced the phosphorylation of signal transducer and activator of transcription 1. The combination of IL-1beta and IFN-gamma increased both apoptosis and necrosis in rat islet cells. SB203580, but not the extracellular signal-regulated kinase inhibitor PD98059, partially prevented cytokine-induced apoptosis, an effect that was not associated with reduced nitrite production or lowered iNOS expression. In conclusion, cytokine-induced p38 activation participates in beta-cell apoptosis, possibly by a nitric oxide-independent mechanism or by enhancing the sensitivity to nitric oxide.

Inhibition of gastric acid secretion by nimesulide: a possible factor in its gastric tolerability.

OBJECTIVE: To study the effect of nimesulide on acid secretion in mouse isolated stomach. METHODS: Isolated lumen-perfused mouse stomachs were monitored by pH-electrodes (1). Gastric acid secretion was stimulated with histamine or 5-methylfurmethide (5-MeF, a stable acetylcholine derivative), and the effect of nimesulide and indomethacin were studied alone and in combination with famotidine. RESULTS: The concentration-dependent stimulation of gastric acid output by histamine (Hill equation fitting parameters: log[A]50 5.44 +/- 0.15; p, 1.01 +/- 0.11; alpha, 0.64 +/- 0.05) was inhibited by famotidine, and also by nimesulide (log r = 0.79 +/- 0.10 at 30 microM). However, nimesulide also reduced the maximum acid output. The shift produced by nimesulide and famotidine in combination indicated a greater than additive effect, suggesting that nimesulide was not acting at histamine H2-receptors (Shankley et al., 1988) (2). Indomethacin reduced acid secretion only at the highest concentration (100 microM). Furthermore, the histamine-receptor-independent stimulation of gastric acid output by 5-MeF was greatly inhibited by nimesulide, which also suggests that nimesulide was acting on the parietal cell signaling pathway at a non-histamine-receptor site. CONCLUSION: The relatively low risk of gastric mucosal damage with nimesulide is thought to involve its weak inhibition of gastric prostaglandin synthesis and its weak acidity, but another factor might be the ability to reduce gastric acid production. However, the effect of nimesulide on human gastric acid secretion remains to be investigated.

Cardiovascular disease risk stratification and comparison in a California population.

This study was designed to identify the need for primary prevention of cardiovascular disease in an HMO population and to develop appropriate interventions for individuals in different risk groups, based on risk stratification and comparison. The analysis is based on a cross-sectional survey of the HMO members of a large employer group. Respondents (n=17,878) were stratified based on the Framingham model; 34% of respondents without cardiovascular disease were classified as moderate to high attributable risk for the disease, and 66% were classified as low attributable risk. Results of logistic regression analyses suggest that, compared with respondents with pre-existing cardiovascular disease, moderate- to high-risk respondents are more likely to smoke, have unhealthy diets, and be overweight, hypertensive, and hypercholesterolemic. More low-risk respondents had unhealthy diets than did those with pre-existing cardiovascular disease. There were no differences between these groups for physical activity and stress. Respondents had fewer modifiable risk factors and healthier lifestyles than did those who were at risk. These findings suggest that primary prevention should be enhanced, especially among those with significantly increased risk for the disease. Moreover, the approaches of this project-population-based risk assessment, stratification, and comparison-were instrumental in identifying the target population and designing appropriate interventions. (c) 2001 by CHF, Inc.

Novel experimental strategies to prevent the development of type 1 diabetes mellitus.

Type 1 diabetes is an autoimmune disease leading to extensive destruction of the pancreatic beta-cells. Our research focusses on the role of beta-cells during the course of the disease, aiming at finding novel strategies to enhance beta-cell resistance against the cytotoxic damage inflicted by the immune system. Special attention has been paid to the possibility that cytokines released by the immune cells infiltrating the pancreatic islets can directly suppress and kill beta-cells. Certain cytokines (interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma) either alone or in combination, are able to activate signal transduction pathways in beta-cells leading to transcription factor activation and de novo gene expression. In this context, it has been found that induction of inducible nitric oxide synthase mediates an elevated production of nitric oxide, which impairs mitochondrial function and causes DNA damage eventually leading to apoptosis and necrosis. However, other induced proteins SUCH AS heat shock protein 70 and superoxide dismutase may reflect a defense reaction elicited in the beta-cells by the cytokines. Our strategy is to further seek for proteins involved in both destruction and protection of beta-cells. Based on this knowledge, we plan to apply gene therapeutic approaches to increase expression of protective genes in beta-cells. If this is feasible we will then evaluate the function and survival of such modified beta-cells in animal models of type 1 diabetes such as the NOD mouse. The long-term goal for this research line is to find novel approaches to influence beta-cell resistance in humans at risk of developing type 1 diabetes.

Gastric acid secretion in cyclooxygenase-1 deficient mice.

BACKGROUND: Constitutive cyclooxygenase-1 enzyme synthesizes prostaglandins which are thought to play an important role in the functional integrity of the stomach gastric mucosa. Recently, it was shown that cyclooxygenase-1 deficient mutant mice did not develop spontaneous gastric pathology and appear less sensitive to indomethacin-induced gastric damage. AIM: To investigate gastric acid secretion in cyclooxygenase-1 deficient mutant mice. METHODS: The basal and histamine or isobutyl methylxanthine-stimulated acid secretion in stomachs of cyclooxygenase-1 deficient homozygous mice and the effect of indomethacin was compared with that of heterozygous and wild-type mice using isolated lumen perfused mouse stomachs, in organ baths, monitored by pH-electrodes. RESULTS: There was no significant difference in the basal or histamine stimulated gastric acid secretion between wild-type or heterozygous or homozygous mice. However, isobutyl methylxanthine was more potent in the cyclooxygenase-1 deficient and heterozygous mice than in wild-type mice. Indomethacin, at concentrations below 1 mM, had no effect on either basal or histamine stimulated acid secretion in any of the mice populations. CONCLUSION: Gastric acid secretion is maintained without prostaglandin involvement in cyclooxygenase-1 deficient mice. The finding that basal and histamine-stimulated gastric acid secretion was similar in the cyclooxygenase-1 deficient, compared to wild-type mice is consistent with the lack of spontaneous gastric pathology in the cyclooxygenase-1 deficient mice.

Interleukin-1beta regulates phospholipase D-1 expression in rat pancreatic beta-cells.

The cytokine interleukin (IL)-1beta induces a biphasic effect in rat pancreatic islets, with an early and transitory stimulation of insulin release followed by progressive functional suppression. To clarify the mechanisms involved in these effects, we have recently performed a differential display of messenger RNA (mRNA) by RT-PCR (DDRT-PCR) on rat beta-cells exposed for 6 or 24 h to IL-1beta. Among the different IL-1beta-induced genes, there was an early and transient increase in phospholipase D-1 (PLD1) expression. PLD1 can induce phosphatidic acid formation and subsequent activation of protein kinase C, a process which stimulates insulin release. In the present study, we characterized the regulation of PLD isoforms by IL-1beta in pancreatic beta-cells. By using different combinations of primers and RT-PCR, we observed that IL-1beta induces an early increase (2 and 6 h) in the expression of both alternatively spliced isoforms of PLD1 (PLD1alpha and 1b). Prolonged exposure to IL-1beta (12 and 24 h) caused a decrease of PLD1a mRNA expression compared with control beta-cells, and lead to a return of PLD1b mRNA to basal level. NG-methyl-L-arginine (LMA), a blocker of the inducible form of nitric oxide synthase (iNOS), prevented this late inhibitory effect of IL-1beta, suggesting that IL-1beta-induced decrease in PLD1a expression is NO-mediated. IL-1beta induced an early (2-6 h) and sustained (16-24 h) increase in PLD1a mRNA expression in insulin-producing RINm5F cells. This was paralleled by a cytokine-induced increase in PLD1 protein expression and enzyme activity. RINm5F cells, but not primary beta-cells, expressed PLD2, and the expression of this gene was not affected by IL-1beta. In conclusion, we have shown that the cytokine IL-1beta regulates PLD1 expression in primary and clonal beta-cells. The early induction of PLD1 probably contributes to the early stimulatory effects of IL-1beta on islet insulin release.

Liposome-mediated transfer of IL-1 receptor antagonist gene to dispersed islet cells does not prevent recurrence of disease in syngeneically transplanted NOD mice.

IL-1beta is cytotoxic to pancreatic beta-cells in vitro but its role in the vicinity of beta-cells in vivo is unknown. We explored whether liposome-mediated transfer of the interleukin 1 receptor antagonist (IL-1ra) gene to islet cells might prevent recurrence of disease in syngeneically transplanted non-obese diabetic (NOD) mice. NOD mouse islet cells were transfected using liposome-mediated gene transfer with a human IL-1ra cDNA construct and transplanted two days later to prediabetic NOD mice. Graft infiltration and destruction were monitored three, five and eight days posttransplantation by histology and determination of insulin and cytokine content. IL-1ra gene transfer resulted in transient expression of IL-1ra protein in islet cells in vitro as assessed by ELISA and of IL-1ra mRNA in transplanted islets as revealed by RT-PCR. However, both control and IL-1ra transfected NOD grafts exhibited massive infiltration and loss of insulin-positive cells, paralleled by a decreased insulin content. Increased IL-1ra expression did not clearly affect other cytokine profiles (IL-1beta, IFN-gamma, IL-2), except for an increase of IL-10 on day eight. In conclusion, liposome-mediated IL-1ra gene transfer to mouse islet cells results in transient expression of IL-1ra which is, however, insufficient to confer resistance to destruction of grafted insulin-producing cells in the NOD mouse.

An improved in vitro bioassay for the study of 5-HT(4) receptors in the human isolated large intestinal circular muscle.

Recently, it was demonstrated that 5-HT induces relaxation of human colon circular muscle through activation of 5-HT(4) receptors and 5-HT(7) receptors. The aim of the current study was to develop a new in vitro bioassay of human colon that would facilitate the pharmacological analysis of 5-HT responses mediated solely by 5-HT(4) receptors. Contracting circular muscle strips with KCl (80 mM) yielded a stable contractile tension and, in contrast to muscarinic cholinoceptor agonists and histamine, a profound reduction of spontaneous contractility. This allowed the establishment of reproducible, fully-defined, agonist concentration-response curves by cumulative dosing. Under these conditions, 5-HT induced a concentration-dependent relaxation (pEC(50) 7.31, Hill slope 0.91). Neither methysergide (10 microM) nor granisetron (1 microM) affected the 5-HT-induced relaxation, suggesting that 5-HT(1), 5-HT(2), 5-HT(3), 5-ht(5), 5-HT(6) or 5-HT(7) receptors are not involved. The lack of effect of tetrodotoxin (0.3 microM) indicated a direct effect of 5-HT on the smooth muscle. The selective 5-HT(4) receptor antagonists GR 113808, GR 125487 and RS 39604 competitively antagonized the 5-HT-induced relaxation (pK(B) 9.43, 10.12 and 8.53, respectively). SB 204070 (1 nM) produced a rightward shift (pA(2) 10.34) and depression of the 5-HT curve. These affinity estimates are similar to those previously reported for 5-HT(4) receptors. The selective 5-HT(4) receptor agonists, prucalopride and R076186, induced relaxations (pEC(50) 7.50 and 7.57, respectively), that were blocked by GR 113808 (3 nM), yielding pA(2) estimates of 9.31 and 9.21, respectively. To summarise, in KCl (80 mM)-contracted muscle strips, 5-HT induces relaxation through activation of a homogeneous smooth muscle 5-HT(4) receptor population. This new bioassay allows the focused, pharmacological characterization of human colonic 5-HT(4) receptors in vitro.