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Background & Aims: Microbial dysbiosis and aberrant hostâmicrobe interactions in the gut are believed to contribute to the development and progression of Crohnâs disease (CD). Microbiome studies in CD typically have focused on microbiota in feces or superficial mucosal layers of the colon because accessing DNA from deeper layers of the bowel is challenging. In this study, we analyzed the deep tissue microbiome in patients who underwent surgical resection of the small intestine. Methods: Paraffin blocks were obtained from 12 CD patients undergoing ileocecal resection, and healthy ileum samples (inflammatory bowel diseaseâfree controls) were obtained from 12 patients undergoing surgery for right-sided colon cancer. Diseased and healthy-appearing ileum was identified using microscopy, and paraffin blocks were macrodissected using a core needle to specifically isolate DNA. Illumina Whole Genome Sequencing was used for microbial sequence identification and subsequent taxonomic classification using the PathSeq tool. Results: We observed significant differences between the microbiome of CD samples vs inflammatory bowel diseaseâfree controls, including depletion of Bacteroidetes and Clostridia. Notably, microbial composition at the phyla level did not differ markedly between healthy and diseased areas of CD patients. However, we observed enrichment of potentially pathogenic organisms at the species level. Conclusions: Our study showed dysbiosis within deeper layers of the ileum of CD patients, specifically enrichment of enterotoxigenic Staphylococcus aureus and an environmental Mycobacterium species not described previously. Future studies with larger cohort sizes are warranted to confirm these findings. Studies would benefit from effective microbial DNA extraction methods from paraffin sections and host nucleic acid depletion approaches to increase microbial read coverage. Keywords: Crohn's Disease, Deeper Mucosal Layers, Microbial Dysbiosis

Purpose The purpose of the study was to compare urinary retention rates following orthopaedic surgery in patients who received low-dose intravenous naloxone while receiving morphine patient-controlled analgesia with patients who did not receive naloxone. Design Randomized controlled trial without blinding. Sample There were 97 participants consenting to the study, 45 were randomly assigned to a control group and 52 assigned to an experimental group. Forty-three patients in the control group and 47 in the experimental group (90 total) completed the study protocol. Findings Postoperative urinary residuals were lower, patients voided more frequently, and fewer catheterizations were needed when given low-dose naloxone while receiving morphine patient-controlled analgesia. At the same time, naloxone in small doses was found to have negligible effect on overall patient pain control.

Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) are mitogens for bipotential oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells. We investigated the mitogenic effect of these growth factors on quiescent mature oligodendrocytes (OL) expressing myelin basic protein (MBP) in OL cultures that were treated for 3 days with cytosine arabinoside (ARA-C) in order to kill O-2A precursors which divide in chemically defined medium. After treatment with ARA-C proliferation decreased and O-2A precursors identified with A2B5 monoclonal antibody were nearly undetectable. After exposure of mature OL to bFGF, cell proliferation increased markedly within 24 hr. PDGF had a much weaker effect. Cultures treated with ARA-C for 3 days and then with bFGF for the next 24 hr and incubated with BrdU for the last 2 hr before the end of the experiment were immunolabeled with anti-MBP or A2B5 and anti-bromodeoxyuridine (BrdU) antibodies. Eighty-seven percent of the cells were MBP+, 10% were both MBP+ and BrdU+, and none was A2B5+ BrdU+, showing that at least a part of the population of mature MBP+ OL retains the ability to reenter the cell cycle in vitro. Since mature OL did not proliferate in response to bFGF in the cultures not treated with ARA-C, i.e., in the presence of O-2A progenitors, we assumed that these precursors were responsible for the lack of mitogenic effect of bFGF on MBP+ OL in such conditions. Conditioned medium from O-2A precursors almost halved the bFGF-induced OL proliferation after treatment with ARA-C, suggesting that O-2A progenitors control the proliferation of a subpopulation of mature OL (possibly young mature OL) via the secretion of active molecule(s).

PAF-acether can be transformed into analogs by the phospholipase D enzyme activity of Streptomyces sp. In this reaction choline is replaced by primary cyclic alcohols (acceptors). The reaction products, cyclic phospholipid and phosphatidic acid, were separated by silicic acid chromatography. This procedure enabled us to synthetize five analogs of PAF-acether, with a cyclic ring structure. The primary cyclic alcohols used in this work were: 3-(2-hydroxyethyl)-indol, OH-Et-I; 2-(hydroxymethyl)-1,4-benzodioxan, OH-Met-BZD; N -(2-hydroxyethyl)-phthalimide, OH-Et-PHT; 2-(2-thienyl)-ethanol, Th-EtOH; (1-R)-(−)-Nopol, R-NOP.

Mouse peritoneal macrophages synthesize 6 monohydroxylated eicosatetraenoic acids when incubated with exogenous arachidonic acid. These compounds were identified by chromatographic techniques (high pressure liquid chromatography and high efficiency glass capillary column gas chromatography and mass spectrometry. The chromatographic and spectrometric data are presented. These results show that peritoneal macrophages constitute one of the best systems to study in evaluating the metabolism of oxygenated products of arachidonic acid.

Mouse peritoneal macrophages were incubated at 37°C for 30 min with arachidonic acid (all- cis -5,8,11,14-eicosatetraenoic acid). Oxygenation of arachidonic acid in mouse peritoneal macrophages occurs by two major pathways: fatty acid cyclooxygenase and lipoxygenase. The major metabolite of the latter is 12-hydroxy-5,8,10,14-eicosatetraenoic acid which was identified by gas liquid chromatography on high resolution glass capillary column and mass spectrometry.

Radioimmunoassays of platelet prostaglandins E1 and F1 alpha in platelet rich plasma or platelet suspension, demonstrate that both PGE1 and PGF1 alpha are present at higher concentrations than prostaglandins E2 and F2 alpha. Gas chromatography--mass spectrometry determinations of prostaglandins E1 and E2 in resting washed platelets confirm this difference. Lastly, there is a greater incorporation of [1--14C] acetate into prostaglandins E1 and F1 alpha compared to that into prostaglandins E2 and F2 alpha.

When ticlopidine is added to macrophages cultures, in the absence of exogenous arachidonic acid, there is a production of both“prostanoids” and“eicosanoids” in the culture medium. These products have been measured using glass capillary gas chomatography prior to multiple ion mass spectrometry. The quantitative determinations are made 5, 15, 25, 35 and 45 minutes after the drug was added to the macrophages cultures. The three drug concentrations used (10-4M, 5.10-5M and 2.5 10-5M) induce a liberation of 6-keto-PGF1α in the culture medium. As in our system 6-keto-PGF1α seems to be the major metabolite of PGI2, ticlopidine is likely to act by releasing important quantities of PGI2 in macrophages. These results suggest an increase of liberation of the endogenous arachidonic acid from the membrane phospholipids of the macrophages or a lack in the acyltransferase system of the cell membranes. The lipoxygenasic pathway was also studied. When ticlopidine is added to macrophages, two products are liberated: . 12-HETE as measured by single iondetection . and 10-hydroxy-ll-12-epoxy, -5, 8, 14-eicosatrienoic acid which comes from an internal rearrangement of the 12-HPETE. In these results, there is a discrepancy between the fact that ticlopidine increases the concentration of 12-HETE and surely its precursor the 12-HPETE and the fact that the synthesis of PGI2 is not inhibited by these high concentrations of hydroperoxide. To understand this phenomenon we studied the production of hydroperoxide when arachidonic acid is incubated with soybean lipoxygenase. When ticlopidine (10-4M) is added to the reaction mixture (AA + soy lipoxygenase) there is an increase of the initial rate and an extent of the reaction as if the enzyme irreversible deactivation was postponed. Ticlopidine could act by suppressing the classical inhibition of PGI2 synthetase by hydroperoxides, in particular 12-HPETE and 15-HPETE, both produced by mammalian cells.

Prostacyclin (PGI2) synthesis seems to be one of the major physiological mechanisms involved in regulating platelet and vessel wall interactions. PGI2 is produced in large amounts by vascular endothelial cells, and vascular smooth muscle cells (SMC) also produce significant quantities. The capacity of SMC to produce PGI2, especially after endothelial injury, seems to be of importance. It is probably this type of situaton that is involved in the atherosclerotic process: experimental atherosclerosis in rabbits has been associated with a severe decrease in PGI, synthesis by arteries. Lipid peroxide accumulation within the arterial wall or in the plasma may also be involved in this process. Using arterial SMC in culture, we demonstrate here that, in comparison with healthy cultured cells, cells originating from atherosclerotic aorta have a decreased capacity to produce PGI2. The results were obtained using biological and radiochemical techniques and were confirmed by GC-MS. They suggest a potential role for PGI2 in inhibiting the atherosclerotic process.

The analytical approach described allows for the rapid screening and concurrent quantitative determination of all of the major metabolites of the cyclooxygenase pathway of arachidonic acid, including the primary prostaglandins PGE2, PGD2 and PGF2 alpha in addition to the stable end-products of short-lived prostacyclin and thromboxane A2 (6-keto-PGF1 alpha and TXB2, respectively), generated by incubating arachidonic acid with mouse peritoneal macrophage cells. After derivatization into the corresponding methyl ester-methoxime-TMS derivative, the extracted endogenous compounds were analysed by combined high efficiency glass capillary column chromatography and selected ion monitoring. Comparatively lower detection limits can be achieved with the methyl ester-butylboronate-TMS derivatives (e.g. 10 pg for 6-keto-PGF1 alpha). A brief account is also presented on a new type of mixed methyl ester-pentafluorobenzyloxime-butylboronate-TMS derivative of TXB2 and 5-keto-PGF1 alpha.

Arachidonic acid can be transformed into a series of HPETEs by the lipoxygenase enzyme activity of mouse peritoneal marcrophages. These resulting HPETEs inhibit some mouse lymphocyte responses. When mice are injected with 15- l -HPETE, their splenocytes show a decreased [ 3 H]thymidine uptake after lectin stimulation.