M.B.M. de Smet

Effects of experimental psychological stress on distribution and function of peripheral blood cells

&NA; Fifty male subjects (aged 24 to 55 years) were subjected to a mild and potentially uncontrollable interpersonal stress situation. They were asked to solve a difficult puzzle. Subsequently they were requested to explain their solution to “another subject,” actually a confederate to the researchers. The confederate frustrated the subjects explanation efforts. Care was taken that neither solving nor explaining of the puzzle was successful. The experimental situation induced mild psychological strain as documented by mood changes in the experimental group when compared with a control group of 36 male subjects. Peripheral blood was drawn by an indwelling catheter just before, directly after, 15 minutes after, and 30 minutes after the stress situation. Numbers of leukocytes, lymphocytes, monocytes, T‐cell subsets, natural killer (NK) cells, and B‐cells were determined. As functional assays we used in vitro proliferative responses of T‐ and B‐cells to mitogenic stimulation (PHA and PWM) and to an antigen cocktail. The potential influences of health‐ and biobehavioral variables were taken into account in the analyses, as well as incidental differences in initial mood or immunological baseline. The results replicated and expanded on previous research. In contrast to controls, experimental subjects showed a significant increase in numbers of NK cells after the stress‐period, returning to baseline values after 15 minutes of rest. A similar effect was shown on T‐suppressor/cytotoxic cells and, inversely, on T‐helper/suppressor ratio, but these effects could be attributed to changes in the numbers of CD8+CD57+ cells. No effects were observed on proliferation. From the results we conclude that the effects of a short lasting mild psychological stressor are mainly restricted to cells of the NK cell population.

Relationships Between Cardiovascular and Immunological Changes in an Experimental Stress Model

To investigate the relationships between cardiovascular variables (SBP, DBP, and HR) and circulating natural killer (NK) cell numbers, 70 male volunteers were subjected to a rest condition (N = 30) or a stressful laboratory task (N = 40). At baseline, no significant relationships could be demonstrated between the number of NK cells and the cardiovascular variables. Analysis of covariance showed that the stressor induced increases in the number of NK cells, SBP, DBP, and HR. Changes in Nk cell numbers were highly correlated to changes in cardiovascular variables in both the task and the no-task group. These results indicate that there is no relationship between the number of circulating NK cells and cardiovascular levels per se, but that changes in these variables, either stress-induced or under rest conditions, are regulated by a common mechanism.

Small intestinal glucose absorption and duodenal motility in type 1 diabetes mellitus

OBJECTIVE:Small intestinal glucose absorption is increased in animal models of diabetes mellitus, but little data are available in humans. Small intestinal motility is reported to be frequently abnormal in patients with diabetes and could potentially affect glucose absorption. Our aim was to evaluate small intestinal glucose absorption and duodenal motor responses to intraduodenal nutrients, in patients with type 1 diabetes and controls.METHODS:Eight type 1 patients (two with autonomic neuropathy) and nine controls were studied during euglycemia. A manometric catheter was positioned across the pylorus, and nutrient infused intraduodenally (90 kcal over 30 min), followed by a bolus of 3-O-methylglucose (3-OMG). Blood was sampled to measure glucose and 3-OMG concentrations.RESULTS:During nutrient infusion, the number of duodenal waves did not differ between patients and controls. After the infusion, patients with diabetes had more propagated duodenal wave sequences (p < 0.05). The area under the plasma 3-OMG curve did not differ between the groups but correlated with both the blood glucose concentration at the time of 3-OMG administration (r = 0.64, p < 0.005) and the number of duodenal waves (r = 0.52, p < 0.05) and antegrade propagated duodenal sequences (r = 0.51, p < 0.05) preceding the 3-OMG bolus.CONCLUSIONS:During euglycemia, duodenal motor responses to small intestinal nutrient are comparable in patients with relatively uncomplicated type 1 diabetes and healthy subjects, but duodenal motility after nutrient infusion is increased in patients. Small intestinal glucose absorption is similar in patients and controls, but may be dependent on the blood glucose concentration and duodenal motor activity.

Indomethacin enhances bile salt detergent activity : Relevance for NSAIDs-induced gastrointestinal mucosal injury

Gastroduodenal toxicity of nonsteroidal anti-inflammatory drugs (NSAIDs) is partly independent from cyclooxygenase inhibition, possibly related to increased intermixed micellar–vesicular (nonphospholipid–associated) bile salt concentrations thought to be responsible for bile salt cytotoxicity. We evaluated the effects of indomethacin on bile salt cytotoxicity with complementary in vitro and ex vivo systems. In the erythrocyte model, indomethacin alone did not induce hemolysis. In contrast, indomethacin enhanced and phospholipids decreased hemolysis induced by hydrophobic taurodeoxycholate (TDC). Hydrophilic tauroursodeoxycholate (TUDC) enhanced rather than decreased TDC-induced hemolysis in the presence of indomethacin. Indomethacin did not affect intermixed micellar–vesicular bile salt concentrations or compositions. Indomethacin also increased TDC-induced lactate dehydrogenase release in CaCo-2 cells and bile salt-induced rat colonic mucosal injury, and prevented potential protective effects of TUDC in these systems. Our data show that indomethacin enhances bile salt–induced cytotoxicity without affecting intermixed micellar–vesicular bile salt concentrations or compositions. These findings may be relevant for gastroduodenal injury during NSAID therapy.

Intestinal barrier function in patients undergoing colectomy.

Aimu2002 The aim of this pilot study was to determine whether the type of approach (open or laparoscopic) and the order of devascularization during laparoscopic colectomy affect intestinal barrier function, local inflammatory response and clinical outcome.

FRI0009 Il4-10 synerkine: a novel anti-inflammatory drug to inhibit immunopathology in rheumatic diseases

Background A considerable percentage of patients shows a limited response to biologics targeting one specific inflammatory mediator, largely because of redundancy of these mediators. An attractive alternative is inhibition of multiple proinflammatory mediators and induction of immunoregulatory activity by regulatory cytokines such as IL4 and IL10. Many studies demonstrated the strong capacity of IL4 and IL10 as stand alone drugs to inhibit inflammation and tissue-destructive responses in animal and human in vitro models. Clinical results of these cytokines, however, have been disappointing, possibly because of poor bioavailability that is mainly due to their low molecular weight and rapid renal clearance. We have executed a feasibility study to develop IL4 and IL10 as one biologic, preserving distinct characteristics of each molecule plus improving bioavailablity by increasing the molecular size. Objectives Development and testing of anti-inflammatory properties of an IL4/IL10 fusion protein; IL4-10 synerkine. Methods IL4-10 synerkine was expressed in human HEK293 cells. Biochemical properties of IL4-10 synerkine were determined by western blot, size exclusion chromatography, and ELISA. Functional properties were studied by measuring the capacity of IL4-10 synerkine to regulate production of pro-inflammatory cytokines and their inhibitors, as well as proinflammatory and regulatory T cell activity, and expression Fcγ and Fcε receptors (FcεRI, IIa, IIb, III and FcεR). In addition, blocking IL4 receptor and IL10 receptor strategies were performed to confirm the specific activities of IL4 and IL10. Results IL4-10 synerkine appeared as a glycosylated dimeric protein with a molecular size of ~70kDa. ELISA as well as immunoblotting confirmed that the IL4-10 synerkine indeed consisted of IL10 and IL4 subunits. In whole blood assays IL4-10 synerkine dose-dependently inhibited multiple pro-inflammatory cytokines, which was almost complete at 20 ng/ml (IL1β, TNFα, IL6 and IL8, all p<0.001). This effect was dependent on interaction with IL10R and IL4R. Oppositely, the synerkine significantly induced production of IL1RA and preserved sTNFR levels. In addition, IL4-10 synerkine strongly inhibited Th1 and Th17 cytokine secretion, while maintaining FoxP3 expression and FoxP3-expressing CD4 T cells. Finally, while IL4 up regulated FcεR expression and IL10 up regulated expression of activating FcγRs on monocytes (all p<0.001), both Fcε and FcγR expression were largely down regulated to control levels in the presence of IL4-10 synerkine (all at least p<0.01). Conclusions The IL4-10 synerkine is a novel anti-inflammatory drug that shifts multiple proinflammatory pathways towards immunoregulation. The increased molecular mass predicts better bioavailabilty in humans than the wild-type molecules, which potentially enhances its clinical efficacy. The strong and improved inhibitory activities of IL4-10 synerkine (as compared to IL4 or IL-10 monotherapy and combination therapy) that were observed in several animal models for inflammatory pain underscores the potential for treatment of inflammatory and possibly degenerative rheumatic diseases. Disclosure of Interest None Declared

IL4-10 fusion protein:a novel immunoregulatory drug combining activities of IL-4 & IL-10

The objective of this study was to test the capacity of a newly developed fusion protein of interleukin 4 (IL‐4) and IL‐10 [IL4‐10 fusion protein (FP)] to shift multiple pro‐inflammatory pathways towards immune regulation, and to inhibit pro‐inflammatory activity in arthritis models. The effects of IL4‐10 FP in comparison with IL‐4, IL‐10 and IL‐4 plus IL‐10 on pro‐ and anti‐inflammatory mediators, T cells and immunoglobulin (Ig) receptors in favour of immunoregulatory activity were studied. In addition, the capacity of IL4‐10 FP to inhibit pro‐inflammatory activity in ex‐vivo and in‐vivo arthritis models was investigated. IL4‐10 FP robustly inhibited pro‐inflammatory cytokine [IL‐1β, tumour necrosis factor (TNF)‐α, IL‐6 and IL‐8] production in whole blood cultures, mediated by both the IL‐10 and the IL‐4 moiety. IL4‐10 fusion protein induced IL‐1 receptor antagonist (IL‐1RA) production and preserved soluble TNF receptor (sTNFR) levels, strongly increasing IL‐1RA/IL‐1β and sTNFR/TNF‐α ratios. In addition, IL4‐10 FP strongly inhibited T helper (Th) type 1 and 17 cytokine secretion, while maintaining FoxP3 expression and up‐regulating Th2 activity. In addition, while largely leaving expression of activating Fc gamma receptor (FcγR)I, III and Fc epsilon receptor (FcεR) unaffected, it significantly shifted the FcγRIIa/FcγRIIb ratio in favour of the inhibitory FcγRIIb. Moreover, IL4–10 FP robustly inhibited secretion of pro‐inflammatory cytokines by rheumatoid arthritis synovial tissue and suppressed experimental arthritis in mice, without inducing B cell hyperactivity. IL4‐10 fusion protein is a novel drug, signalling cells to induce immunoregulatory activity that overcomes limitations of IL‐4 and IL‐10 stand‐alone therapy, and therefore has therapeutic potential for inflammatory diseases such as rheumatoid arthritis.

A8.4 IL4–10 synerkine: a novel anti-inflammatory drug to inhibit immunopathology in inflammatory rheumatic diseases

Background A considerable percentage of patients shows a limited response to biologics targeting one specific inflammatory mediator, largely because of redundancy of these mediators. An attractive alternative is inhibition of multiple proinflammatory mediators and induction of immunoregulatory activity by regulatory cytokines such as IL4 and IL10. Many studies demonstrated the strong capacity of IL4 and IL10 as stand-alone drugs to inhibit inflammation and tissue-destructive responses in animal and human in vitro models. Clinical results of these cytokines, however, have been modest, possibly because of poor bioavailability that is mainly due to low molecular weight and rapid renal clearance. Objectives To execute a feasibility study to develop IL4 and IL10 as one biologic (called IL4–10 synerkine), preserving distinct characteristics of each molecule plus improving bioavailablity by increasing the molecular size. Materials and methods Biochemical properties of IL4–10 synerkine were determined by western blot, size exclusion chromatography, and ELISA. Functional properties were studied by measuring the capacity of IL4–10 synerkine to regulate production of pro-inflammatory cytokines and their inhibitors, as well as proinflammatory and regulatory T-cell activity, and expression of Fc receptors. In addition, blockade of IL4 and IL10 receptor was performed to confirm the specific activities of IL4 and IL10. Results IL4–10 synerkine appeared as a glycosylated dimeric protein with a molecular size of ˜70 kDa, consisted of intact IL10 and IL4 subunits. In whole blood assays IL4–10 synerkine robustly and dose-dependently inhibited multiple pro-inflammatory cytokines, which was almost complete at 20 ng/ml (IL1β, TNFα, IL6 and IL8, all p < 0.001). This effect was dependent on interaction with IL10R and IL4R. Oppositely, the synerkine significantly induced production of IL1RA and preserved sTNFR levels. IL4–10 synerkine strongly inhibited Th1 and Th17 cytokine secretion (P < 0.01), while maintaining FoxP3 expression. Finally, while IL4 upregulated FcεR expression and IL10 upregulated expression of activating FcγRs on monocytes (all p < 0.001), both Fcε and FcγR expression were largely preserved at control levels in the presence of IL4–10 synerkine (all at least p < 0.01). Conclusions IL4–10 synerkine is a novel anti-inflammatory drug that shifts multiple proinflammatory pathways towards immunoregulation. The increased molecular mass predicts better bioavailabilty in humans than the wild-type molecules, which potentially enhances its clinical efficacy. The strong and improved inhibitory activities of IL4–10 synerkine (as compared to IL4 and IL-10 monotherapy) that we observed in several animal models for inflammatory pain underscores the potential for treatment of inflammatory and possibly degenerative rheumatic diseases.