Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. 427: 355C360. IgA production in dogs. Subsequently, the intestinal S1P concentration and the expression of S1P-related molecules were measured in dogs with IBD and healthy dogs. The intestinal concentration of S1P was significantly lower in dogs with IBD than in healthy dogs. In addition, the gene expression levels of S1P receptor (and was detected using an automatic electrophoresis system (QIAxceL Advanced System, QIAGEN, Hulsterweg, Netherlands). In addition, CD3+ Briciclib T cells and CD21+ B cells were sorted from the canine PBMCs using a cell sorter (BD FACSAriaTM cell sorter, Becton, Dickinson and Co., Franklin Lakes, NJ, U.S.A.). CD21 is used as B cell maker in canine peripheral blood. Intestinal expression of and was also examined in duodenal samples obtained using endoscopic biopsy from healthy dogs. The primer pair sequences used in this experiment are shown in Supplementary Table 1. Study population The study included dogs diagnosed with IBD that underwent endoscopic examination in the Veterinary Medical Center of the University of Tokyo (VMC-UT). Informed consent was obtained from all owners, and the study protocols were approved by the Animal Care Committee of the VMC-UT. The case selection criteria for IBD have been previously described [22]. Diagnosis of IBD was based on: (1) exclusion of other identifiable causes of chronic gastrointestinal signs by complete blood count, serum biochemistry, urinalysis, parasitic and bacterial PCR analyzes of fecal samples, and abdominal ultrasonography, (2) histopathologic evidence of lymphocytic-plasmacytic enteritis, (3) the absence of clonal gene rearrangement of or in the PCR for antigen receptor gene rearrangement (PARR), and (4) exclusion of food- and antibiotic-responsive enteritis. The cases with a discrepancy between histopathology and PARR results were excluded. In addition, dogs that had been treated with corticosteroids during the 2 weeks before the study were also excluded. For S1P ELISA, 25 dogs with Briciclib IBD were included in the study. The median age of these dogs was 88 months (range: 38C139 months); seven of the dogs were female (3 intact and 4 neutered) and the remaining 18 dogs were male (11 intact and 7 neutered). The median body weight was 6.06 kg (range: 2.12C9.5 kg). The following breeds with more than two individuals were included: French Bulldog (of PBS and centrifuged, and the supernatant was used for the ELISA. Absorbance was recorded at 450 nm using a microplate reader (Bio Rad Laboratories) and the results were analyzed using Microplate Manager software, version 5.2.1 (Bio Rad Laboratories). All the samples were tested in duplicate, and the mean optical density (OD) was calculated. S1P concentration was expressed as nanograms per 100 and and were chosen as the reference genes in this study. The primer pair sequences used in this study are shown in Supplementary Table 3. All the samples were examined in duplicate and the mean Ct value was calculated. The relative expression of the target gene is reported as the and and relative to the Briciclib CD3+ T cells (Fig. 6). These results suggest that the effect of FTY720 was induced through blockade of S1P1 and/or S1P4. To examine the expression pattern of S1P receptors in canine intestines, conventional PCR was performed. Of the five S1P receptors, only was detected in the canine SLI intestine (Fig. 6). Open in a separate window Fig. 6. Gene expression of in lymphocytes, monocytes, T cells, B cells, and duodenum of a representative healthy dog. Lym, lymphocytes. Mono, monocytes. T, T cells. B, B cells. Duo, duodenum. Intestinal S1P concentration in healthy dogs and dogs with IBD In healthy dogs, the duodenal S1P concentration varied widely among the individuals (Fig. 7A). In contrast, duodenal S1P concentration in all the cases with IBD was very.
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