Mucosal protection by phosphatidylcholine

The colonic mucus serves a first barrier towards invasion of commensal bacteria in stools to prevent inflammation. One essential component of intestinal mucus is phosphatidylcholine (PC) which represents more than 90% of the phospholipids in mucus indicative for a selective transport of PC into this compartment. It is arranged in lamellar structures as surfactant-like particles which provide a hydrophobic surface on top of the hydrated mucus gel to prevent the invasion of bacteria from intestinal lumen. In ulcerative colitis (UC), the mucus PC content is reduced by 70%, irrespective of the state of inflammation. Thus, it could represent an intrinsic primary pathogenetic condition predisposing to bacterial invasion and the precipitation of inflammation. Since PC was shown to be mainly secreted by the ileal mucosa from where it is assumed to move distally to the colon, the PC content along the colonic wall towards the rectum gradually thins, with the least PC content in the rectum. This explains the start of the clinical manifestation of UC in the rectum and the expansion from there to the upper parts of the colon. In three clinical trials, when missing mucus PC in UC was supplemented by an oral, delayed release PC preparation, the inflammation improved and even resolved after a 3-month treatment course. The data indicate the essential role of the mucus PC content for protection against inflammation in colon. Copyright (C) 2012 S. Karger AG, Base

Nuclear hormone 1α,25-dihydroxyvitamin D3 elicits a genome-wide shift in the locations of VDR chromatin occupancy

A global understanding of the actions of the nuclear hormone 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and its vitamin D receptor (VDR) requires a genome-wide analysis of VDR binding sites. In THP-1 human monocytic leukemia cells we identified by ChIP-seq 2340 VDR binding locations, of which 1171 and 520 occurred uniquely with and without 1α,25(OH)2D3 treatment, respectively, while 649 were common. De novo identified direct repeat spaced by 3 nucleotides (DR3)-type response elements (REs) were strongly associated with the ligand-responsiveness of VDR occupation. Only 20% of the VDR peaks diminishing most after ligand treatment have a DR3-type RE, in contrast to 90% for the most growing peaks. Ligand treatment revealed 638 1α,25(OH)2D3 target genes enriched in gene ontology categories associated with immunity and signaling. From the 408 upregulated genes, 72% showed VDR binding within 400 kb of their transcription start sites (TSSs), while this applied only for 43% of the 230 downregulated genes. The VDR loci showed considerable variation in gene regulatory scenarios ranging from a single VDR location near the target gene TSS to very complex clusters of multiple VDR locations and target genes. In conclusion, ligand binding shifts the locations of VDR occupation to DR3-type REs that surround its target genes and occur in a large variety of regulatory constellations

A Novel Epidermal Growth Factor-Containing Wound Dressing for the Treatment of Hard-to-Heal Venous Leg Ulcers

OBJECTIVE: To evaluate the efficacy, tolerability, and safety of a novel wound dressing containing epidermal growth factor (EGF) in a collagen-gel matrix on hard-to-heal venous leg ulcers. PATIENTS AND METHODS: The authors included 33 hard-to-heal venous leg ulcers found on 31 patients. The EGF-containing dressing was applied 3 times while best practice conservative wound treatment was continued. Patients were followed up with after 1, 2, and 3 months to evaluate (a) the wound size, (b) the ease of application and dissolution of the dressing, and (c) the wound dressing by means of a scale ranging from 1 to 5 (1 = best, 5 = worst). RESULTS: The protocol was completed by 25 of 31 patients. The reasons for discontinuation were wound infection, pain, and lost to follow-up (n = 2 each, respectively). After 3 months, the average wound surface was significantly reduced (from 33.69 cm(2) to 18.94 cm(2), P = .023). On a scale from 0 to 100, the wound dressing was evaluated as very easy to apply and highly dissolvable (mean value of 97.14 and 98.11, respectively; 100 = very easy to apply or 100% dissolution). The dressing was generally well tolerated and scored a mean overall rating of 2.16 by healthcare specialists and 2.40 by patients. CONCLUSION: The authors' results demonstrate that the novel EGF-containing wound dressing was generally well tolerated and safe. Combined with the significant wound surface reduction, it can be regarded as an adequate novel treatment option for patients with hard-to-heal venous leg ulcers

Ergothioneine stands out from hercynine in the reaction with singlet oxygen: Resistance to glutathione and TRIS in the generation of specific products indicates high reactivity

The candidate vitamin ergothioneine (ET), an imidazole-2-thione derivative of histidine betaine, is generally considered an antioxidant. However, the precise physiological role of ET is still unresolved. Here, we investigated in vitro the hypothesis that ET serves specifically to eradicate noxious singlet oxygen (O-1(2)). Pure O-1(2) was generated by thermolysis at 37 degrees C of N, N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide 1,4-endoperoxide (DHPNO2). Assays of DHPNO2 with ET or hercynine (= ET minus sulfur) at pH 7.4 were analyzed by LC-MS in full scan mode to detect products. Based on accurate mass and product ion scan data, several products were identified and then quantitated as a function of time by selected reaction monitoring. All products of hercynine contained, after a [4 + 2] cycloaddition of O-1(2), a carbonyl at position 2 of the imidazole ring. By contrast, because of the doubly bonded sulfur, we infer from the products of ET as the initial intermediates a 4,5-dioxetane (after [2 + 2] cycloaddition) and hydroperoxides at position 4 and 5 (after Schenck ene reactions). The generation of single products from ET, but not from hercynine, was fully resistant to a large excess of tris (hydroxymethyl) aminomethane (TRIS) or glutathione (GSH). This suggests that O-1(2) markedly favors ET over GSH (at least 50-fold) and TRIS (at least 250-fold) for the initial reaction. Loss of ET was almost abolished in 5 mM GSH, but not in 25 mM TRIS. Regeneration of ET seems feasible, since some ET products - by contrast to hercynine products - decomposed easily in the MS collision cell to become aromatic again

Intestinal manipulation affects mucosal antimicrobial defense in a mouse model of postoperative ileus

<div><p>Aim</p><p>To explore the effects of abdominal surgery and interleukin-1 signaling on antimicrobial defense in a model of postoperative ileus.</p><p>Methods</p><p>C57BL/6 and Interleukin-1 receptor type I (IL-1R1) deficient mice underwent intestinal manipulation to induce POI. Expression of mucosal IL-1α, IL-1β and IL-1R1 and several antimicrobial peptides and enzymes were measured by quantitative PCR or ELISA, western blotting or immunohistochemistry. Bacterial overgrowth was determined by fluorescent in-situ hybridization and counting of jejunal luminal bacteria. Translocation of aerobic and anaerobic bacteria into the intestinal wall, mesenteric lymph nodes, liver and spleen was determined by counting bacterial colonies on agar plates 48h after plating of tissue homogenates. Antimicrobial activity against <i>E</i>. <i>coli</i> and <i>B</i>. <i>vulgatus</i> was analyzed in total and cationic fractions of small bowel mucosal tissue homogenates by a flow cytometry-based bacterial depolarization assay.</p><p>Results</p><p>Jejunal bacterial overgrowth was detected 24h after surgery. At the same time point, but not in the early phase 3h after surgery, bacterial translocation into the liver and mesenteric lymph nodes was observed. Increased antimicrobial activity against <i>E</i>. <i>coli</i> was induced within early phase of POI. Basal antimicrobial peptide and enzyme gene expression was higher in the ileal compared to the jejunal mucosa. The expression of lysozyme 1, cryptdin 1, cryptdin 4 and mucin 2 were reduced 24h after surgery in the ileal mucosa and mucin 2 was also reduced in the jejunum. Postoperative IL-1α and IL-1β were increased in the postoperative mucosa. Deficiency of IL-1R1 affected the expression of antimicrobial peptides during homeostasis and POI.</p><p>Conclusion</p><p>Small bowel antimicrobial capacity is disturbed during POI which is accompanied by bacterial overgrowth and translocation. IL-1R1 is partially involved in the gene expression of mucosal antimicrobial peptides. Altered small bowel antimicrobial activity may contribute also to POI development and manifestation in patients undergoing abdominal surgery.</p></div

Mucosal IL-1R1 signaling is involved in POI.

<p>(A-C) WT mice underwent IM. Gene expression of (A) IL-1R1; (B) IL-1α and (C) IL-1β was analyzed in mucosal tissue of jejunal and ileal bowel segments after indicated time. (D) Basal gene expression of antimicrobial proteins was quantified in IL-1R1 deficient compared to control mice of jejunal and ileal mucosa. n = 5 for all groups. Statistical analysis was done by 1-way ANOVA, followed by Bonferroni post hoc test or t-test, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 vs. indicated groups.</p

Primers used for qPCR.

<p>Primers used for qPCR.</p

Translocation of luminal bacteria and increase of bacterial killing occurs during POI.

<p>(A) WT mice underwent IM. Representative photomicrographs of bacterial internalization into the post-operated gut mucosa (magnification ×200). Jejunal segments of intestinal manipulated mice and controls were subjected to FISH with universal bacterial DNA probes. DAPI-stained cell nuclei (blue) are shown for orientation. Cy2-staining shows intestinal auto-fluorescence (AF), whereby Cy3-labelled probe EUB338, complementary to the 16S rRNA in most microorganisms was combined with Cy5-labelled FISH probe NON-EUB338 to exclude unspecific probe binding. Photo images were obtained from at least three mice per group. (B) Bacteria (Cy3⁺) were analyzed in jejunal cross sections at different postoperative time points and in unmanipulated control mice. (C) Colony forming units (CFU) of MLN, liver, lung and spleen of manipulated WT mice were determined postoperatively and compared to WT naïve controls. (D+E) Flow cytometric analysis of the antimicrobial activity of the total protein [D] or the cationic fraction [E] of the mucosal tissue of intestinal manipulated mice against <i>E</i>. <i>coli</i> and <i>B</i>. <i>vulgatus</i>. Cell damage leads to an uptake of the membrane potential-sensitive dye DiBAC₄ (3) in the bacteria and therefore to an increasing depolarization [%] compared to that of the untreated control. n = 5 for all groups. For all experiments, statistical analyses were performed with a 1-way ANOVA, followed by Bonferroni post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. controls.</p

Basal antimicrobial proteins gene expression is differently regulated in the jejunum and ileum.

<p>Gene expression of antimicrobial proteins was quantified in ileal mucosa compared to jejunal mucosa of naïve control mice. (A) lysozyme 1; (B) β-defensin 3; (C) cryptdin 1; (D) cryptdin 4; (E) REG3γ; (F) mucin 2. n = 5 for all groups. Statistical analysis was done by unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. jejunum.</p