IL4-10 fusion protein: a novel immunoregulatory drug combining activities of interleukin 4 and interleukin 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-1ß, 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-1ß 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.

Intestinal barrier function in patients undergoing colectomy.

AIM: 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. METHOD: Twenty-two patients undergoing elective colectomy from April 2006 to July 2008 were randomized to two sequences of vascular ligation, starting with either the inferior mesenteric artery or the ileocolic artery. Eighteen patients scheduled for open surgery served as a prospective control group. To assess the intestinal barrier function, release of intestinal fatty-acid binding protein (I-FABP; a marker of mucosal injury and ischaemia) was measured pre- and postoperatively. Mesenteric lymph nodes were harvested to assess the expression of inflammatory mediator-related genes using multiplex ligation probe amplification. The study was registered under NTR1025. RESULTS: Laparoscopic devascularization starting at the ileocolic artery resulted in a significantly increased excretion of I-FABP over time (P = 0.002). In this group, the I-FABP levels were significantly increased on postoperative days 1 and 3 compared with preoperative values (P = 0.011 and P = 0.001, respectively). There were no differences in expression of inflammatory mediator-related genes or postoperative morbidity among the groups. CONCLUSIONS: In this pilot study, devascularization commencing at the ileocolic artery during laparoscopic colectomy was associated with prolonged intestinal mucosal ischaemia.

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.

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.

Lipid solubilization in human gallbladder versus hepatic biles.

BACKGROUND/AIMS: Cholesterol crystallizes more rapidly in gallbladder than in hepatic biles, supposedly due to formation of cholesterol-supersaturated vesicles in concentrated gallbladder biles because of preferential micellization of phospholipids compared to cholesterol. We therefore aimed to compare lipid solubilization in hepatic and gallbladder biles. METHODS: Mixed micellar and vesicular phases were separated from hepatic and associated gallbladder biles of seven cholesterol gallstone patients by using state-of-the-art gel filtration with bile salts at intermixed micellar/intervesicular compositions and concentrations in the eluant. RESULTS: Vesicles were found in 6 out of 7 hepatic biles, but only in 2 of the corresponding gallbladder biles. Both percentage (7.8+/-5.1 vs. 36.3+/-7.6%; p = 0.01) and amount (0.9+/-0.2 vs. 1.7+/-0.3 mM; p = 0.06) of vesicular cholesterol were lower in gallbladder biles. Similar results were found for vesicular phospholipids (1.3+/-0.8 vs. 11.6+/-6.0%; p = 0.05; and 0.3+/-0.1 vs. 1.1+/-0.5 mM; p = 0.07). The vesicular cholesterol/ phospholipid ratio was 1.7+/-0.5 in hepatic bile but 4.3 and 1.8 in the 2 gallbladder biles which contained vesicles. Mixed micelles in gallbladder biles had a higher cholesterol saturation index than mixed micelles in hepatic biles (1.43+/-0.11 vs. 1.15+/-0.07; p = 0.02). CONCLUSIONS: Concentration of bile in the gallbladder leads to decreased vesicular lipid contents. The finding of supersaturated mixed micelles in the absence of vesicles in a significant number of patients points to the possibility of non-vesicular modes of crystallization.

A randomized trial in primary biliary cirrhosis comparing ursodeoxycholic acid in daily doses of either 10 mg/kg or 20 mg/kg. Dutch Multicentre PBC Study Group.

BACKGROUND: Ursodeoxycholic acid (UDCA) prolongs transplantation-free survival in primary biliary cirrhosis (PBC). However, the optimal therapeutic dose has not been established. AIM: To compare the effects of UDCA administered in daily doses of 10 vs. 20 mg/kg on symptoms, liver biochemistry and biliary UDCA enrichment. METHODS: A 6-month multicentre randomized open controlled trial was conducted to assess the effects of an increase in the dose of UDCA to 20 mg/kg/day vs. continuation of 10 mg/kg/day for patients who had not achieved biochemical normalization during treatment for at least 6 months with the 10 mg/kg dose. Clinical and laboratory evaluations were performed at entry and at 3-month intervals. The percentage UDCA in duodenal bile was assessed at entry and at 6 months. RESULTS: Sixty-one patients were enrolled. No side-effects of UDCA were observed. Within the 20 mg/kg/day group significant decreases were found for alkaline phosphatase (- 8%; P = 0.003), aspartate aminotransferase (- 11%; P = 0.01), alanine aminotransferase (- 17%; P < 0.001), gamma-glutamyl transferase (- 34%; P < 0.001), immunoglobulin M (- 11%; P = 0.002) and cholesterol (- 8.1%; P < 0.001). In the 10 mg/kg group none of these parameters differed significantly from baseline. No significant differences between dose groups for symptom scores or serum bilirubin were found. Biliary enrichment with UDCA increased from 37% to 46% in the 20 mg/kg group (P = 0.02) while remaining stable in the 10 mg/kg group. CONCLUSIONS: Liver biochemistry improved in PBC patients receiving UDCA 20 mg/kg/day compared to a dose of 10 mg/kg/day. Both doses were equally well tolerated. These results indicate that UDCA 10 mg/kg/ day is a suboptimal dose for treating PBC.

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.

Modulatory effects of defense and coping on stress-induced changes in endocrine and immune parameters.

We examined whether habitual defense and coping affect the response of hormones (ACTH. cortisol, prolactin. endorphins, and noradrenaline) and immune parameters (numbers of T cells. B cells. natural killer [NK] cells, and proliferative responses to mitogens or antigens) to an acute laboratory stressor (i.e., solving a 3-dimensional puzzle and explaining it to a confederate) in 86 male high school teachers. Defense and coping were assessed by Kragh's tachistoscopic Defense Mechanism Test (a measure of perceptual defense) and by 4 questionnaire-based coping styles assessing instrumental mastery-oriented coping, emotion-focused coping, cognitive defense, and defensive hostility. The laboratory stressor per se caused a relative increase in immunological (in particular NK cells) and endocrine (cortisol, prolactin) parameters. Defense and coping, however, significantly codetermined the response to the stressor. In particular, instrumental mastery-oriented coping and perceptual defense were related to stress-induced changes in numbers of B cells and in the pituitary-adrenal hormones. The results indicate that the impact of a mild psychological stressor on the immune and endocrine system depends to a considerable extent on the specific ways people deal with stressors.

Chronic stress affects immunologic but not cardiovascular responsiveness to acute psychological stress in humans.

This study deals with the effect of chronic stress on physiological responsiveness to an acute psychological stressor in male high school teachers. Chronic stress was operationalized as the self-reported number of everyday problems. Twenty-seven subjects reporting extremely low or high numbers of everyday problems were exposed to an acute psychological stressor, and changes in immunologic, endocrine, and cardiovascular parameters were monitored. The stressor included a learning process followed by a teaching session in which a confederate to the researchers was involved. Twenty subjects served as controls. The stressor had no effect on the endocrine variables measured but induced increases in heart rate and blood pressure, which were similar in both chronic stress groups. Analysis of subsets of blood lymphocytes revealed differences in natural killer (NK) and T cell responses in the low and the high stress groups. It is concluded that 1) immunologic responsiveness to an acute psychological stressor is related to problems experienced in daily life, and that 2) chronic stress differentially modifies the sensitivity of biological systems to mild acute stressors.

Adhesion of subsets of human blood mononuclear cells to endothelial cells in vitro, as quantified by flow cytometry.

Binding of leucocytes to endothelial cells (EC) is essential as an initial step in inflammatory responses. We present a rapid, non-radioactive method to measure adhesion of human lymphoid cells to EC using flow cytometry. Freshly isolated peripheral blood mononuclear cells (PBMC) were allowed to adhere to EC grown in 24-well plates. Non-adhering cells were removed, after which adhering cells and EC were dissociated using trypsin/EDTA. These samples were subsequently analysed by flow cytometry, using scatter properties to distinguish between adhering cells and EC. The ratio of the number of adhering leucocytes and EC was calculated to quantify adhesion. Results of the flow cytometric adhesion assay were comparable to those obtained with a conventional adhesion assay using chromium-labelled cells. We additionally show that by using the flow cytometric adhesion assay, adhesion of lymphocytes and monocytes present within the adhering PBMC can be quantified simultaneously. As a model, the contribution of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in adhesion of PBMC to EC was studied. It was found that adhesion of lymphocytes and monocytes is regulated differently by phorbol ester and that the relative contribution of LFA-1 and ICAM-1 differs for both cell types.

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

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.