Standardization of a human organ culture model of intestinal inflammation and its application for drug testing.

Targeting early molecular events in intestinal inflammation may represent a useful therapeutic strategy for maintaining remission in inflammatory bowel disease. Recently, we established an intestinal organ culture model (LEL model), which allows to study the initiation of an intestinal inflammatory response in human tissue. In this model, EDTA-mediated depletion of epithelial cells of colonic mucosa results in an instantaneous inflammatory response in resident lamina propria cells, which shows features of intestinal inflammation in vivo. Furthermore, activated immune cells emigrate from the lamina propria onto the luminal side of the basement membrane. Here, we standardize the LEL model and explore its suitability for drug testing. To this end, human mucosal punches of defined surface area were prepared, depleted of epithelial cells, and cultured at an optimized ratio of medium volume/punch area. The intra-assay variability of measurements of inflammatory parameters ranged from 13% for cell migration to 19% for secretion and 30% for tissue gene expression, respectively, of the inflammatory mediators IL-8 and IL-6. Importantly, known suppressive effects of dexamethasone, a drug employed for the treatment of inflammatory bowel diseases, on leucocyte migration, IL8, IL6, and TNF-α production as well as CD86 surface expression by myeloid cells were observed in this model. In conclusion, the present results suggest that the LEL model may represent a useful human experimental system not only for studying initial activation mechanisms in intestinal inflammation but also for evaluating drug compounds for the treatment of mucosal inflammation.

Immunomodulation of human intestinal T cells by the synthetic CD80 antagonist RhuDex®.

Deregulated activation of mucosal lamina propria T cells plays a central role in the pathogenesis of intestinal inflammation. One of the means to attenuate T cell activation is by blocking the CD28/CD80 co-stimulatory pathway. Here we investigate RhuDex®, a small molecule that binds to human CD80, for its effects on the activation of lamina propria T cells employing a gut-culture model of inflammation. To this end, lamina propria leukocytes (LPL) and peripheral blood lymphocytes (PBL) were stimulated either through the CD3/T-cell-receptor complex or the CD2-receptor (CD2) employing agonistic monoclonal antibodies. Co-stimulatory signals were provided by CD80/CD86 present on lamina propria myeloid cells or LPS-activated peripheral blood monocytes. Results show that RhuDex® caused a profound reduction of LPL and PBL proliferation, while Abatacept (CTLA-4-Ig) inhibited LPL proliferation to a small degree, and had no effect on PBL proliferation. Furthermore, Abatacept significantly inhibited IL-2, TNF-α, and IFN-γ release from LPL, primarily produced by CD4(+) T cells, where IL-2 blockage was surprisingly strong, suggesting a down-regulating effect on regulatory T cells. In contrast, in the presence of RhuDex®, secretion of IL-17, again mostly by CD4(+) T cells, and IFN-γ was inhibited in LPL and PBL, yet IL-2 remained unaffected. Thus, RhuDex® efficiently inhibited lamina propria and peripheral blood T-cell activation in this pre-clinical study making it a promising drug candidate for the treatment of intestinal inflammation.

Initiation of an inflammatory response in resident intestinal lamina propria cells -use of a human organ culture model.

Resident human lamina propria immune cells serve as powerful effectors in host defense. Molecular events associated with the initiation of an intestinal inflammatory response in these cells are largely unknown. Here, we aimed to characterize phenotypic and functional changes induced in these cells at the onset of intestinal inflammation using a human intestinal organ culture model. In this model, healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment. Following loss of the epithelial layer, expression of the inflammatory mediators IL1B, IL6, IL8, IL23A, TNFA, CXCL2, and the surface receptors CD14, TLR2, CD86, CD54 was rapidly induced in resident lamina propria cells in situ as determined by qRT-PCR and immunohistology. Gene microarray analysis of lamina propria cells obtained by laser-capture microdissection provided an overview of global changes in gene expression occurring during the initiation of an intestinal inflammatory response in these cells. Bioinformatic analysis gave insight into signalling pathways mediating this inflammatory response. Furthermore, comparison with published microarray datasets of inflamed mucosa in vivo (ulcerative colitis) revealed a significant overlap of differentially regulated genes underlining the in vivo relevance of the organ culture model. Furthermore, genes never been previously associated with intestinal inflammation were identified using this model. The organ culture model characterized may be useful to study molecular mechanisms underlying the initiation of an intestinal inflammatory response in normal mucosa as well as potential alterations of this response in inflammatory bowel disease.

Laparoscopic versus conventional ileoanal pouch procedure in patients undergoing elective restorative proctocolectomy (LapConPouch Trial)-a randomized controlled trial.

PURPOSE: Restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) is the standard surgical procedure for ulcerative colitis (UC) and familial adenomatous polyposis (FAP). While minimal invasive techniques have been applied increasingly, clear evidence of superiority for laparoscopic pouch procedures is not yet available. The aim of the LapConPouch Trial was to compare the effectiveness of laparoscopic (LAP) versus conventional (CON) ileoanal pouch procedure in patients undergoing elective restorative proctocolectomy. METHODS: The trial was designed as a single-centre, pre-operatively randomized, controlled trial using a two-group parallel superiority design. Eligible for participation were patients scheduled for restorative proctocolectomy either for FAP or for UC. Patients and outcome assessors were blinded to group assignment. The primary endpoint was defined as the amount of blood loss. Statistical analyses were explorative since the trial had to be stopped prematurely. RESULTS: A total of 42 patients (21 LAP (50.0 %); 21 CON (50.0 %)) were randomized. The trial had to be stopped prematurely due to insufficient patient recruitment. There was no difference in the amount of blood loss between both groups: LAP 261.5 ± 195.4 ml, CON 228.1 ± 119.5 ml. Secondary endpoints differ in both groups. Laparoscopic surgery was superior regarding the length of skin incision; in contrast, the conventional approach was superior in duration of operation. There were no discrepancies in length of hospital stay, postoperative pain, bowel function, and quality of life between both approaches. The conversion rate from LAP to CON approach was 23.8 %. CONCLUSION: There was no difference with respect to blood loss between the LAP and the CON group. The LAP approach is feasible for restorative proctocolectomy, and IPAA seems at least as safe as CON surgery. The most obvious advantage of the minimal invasive technique is the improved cosmesis.

Subsequent gene expression pattern in dendritic cells following multiple trauma.

PURPOSE: Major trauma initiates a systemic inflammatory response, which is characterized by systemic release of various chemokines. There is growing evidence for the extraordinary role of dendritic cells (DC) as professional antigen-presenting cells and activators of the immune response. Recently, the impact of severe trauma on DC transcriptomic activation was demonstrated. The purpose of the present study was to evaluate gene expression pattern in DC following multiple trauma to gain further understanding of the mechanisms of posttraumatic immune response. METHODS: Ten patients with multiple injuries aged 20 to 46 years (mean 30 ± 9.2 years) were included in this study. The mean injury severity score (ISS) was 36 ± 10.4 points. Repeated blood samples were taken on the day of admission (day 0) and on five consecutive days (day 1 to day 5). Microarray analysis and RT-qPCR were performed in primary isolated DC. RESULTS: A mean of 116,000 ± 21,466 DC with a purity of 96 ± 0,8 % were harvested. Gene expression of CCL5 and CXCL5 as well as TIMP1 and GUCY1B3 showed a significant increase within the first 4 days after trauma. The time-dependent increase of these genes correlated significantly with serum CRP concentration and the total number of DC but neither with age nor with injury severity. CONCLUSIONS: Our study provides new data regarding temporal expression patterns of CCL5, CXCL5, TIMP1, and GUCY13B in multiple trauma. DC activation following trauma may follow a uniform pattern early after admission, eventually leading to cell recruitment.

Significant decline of peripheral myeloid dendritic cells following multiple trauma.

BACKGROUND: Dendritic cells (DC) represent an important and integral part of the immune system and are potent initiators of inflammation. Two distinct subsets of DC have been identified: myeloid DC (MDC) and plasmacytoid DC (PDC), which differ widely in many respects. Despite the importance of the DC in the inflammatory response that occurs after severe multiple injury, there is a profound lack of information regarding the distribution and regulation of DC subtypes following multiple trauma. The main goal of this study was to assess whether the normal distribution of circulating DC subpopulations is altered during the first 5 d after multiple trauma. PATIENTS AND METHODS: Sixty-three patients with multiple trauma (ISS 31 +/- 15 points) and 11 healthy volunteers (control group) were enrolled. Blood samples were taken on admission (D0) and daily for the following 5 d. The percentages of MDC and PDC were determined by flow cytometry. RESULTS: A significant decline of the MDC concentration was observable on days 3 to 5 after admission in comparison to the values obtained on the day of admission. The ratio of MDC to PDC decreased significantly (3-fold, P < 0.05). This reduction correlated significantly with changes observed in the plasma concentrations of IL-10 (r = 0.5; P < 0.05). DISCUSSION: Our data demonstrate that multiple trauma is followed by a marked change in the subpopulation composition of the DC compartment, and that these changes are inversely associated with enhanced IL-10 plasma concentrations. This imbalance in the DC compartment favoring PDC concentrations may contribute to the immunological alterations that are observed following multiple trauma.