Flow cytometry detection of vitamin D receptor changes during vitamin D treatment in Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory disease associated with a dysregulated T cell response towards intestinal microflora. Vitamin D has immune modulatory effects on T cells through the nuclear vitamin D receptor (VDR) in vitro. It is unclear how oral vitamin D treatment affects VDR expression. The aim of this study was to establish a flow cytometry protocol, including nuclear and cytoplasmic VDR expression, and to investigate the effects of vitamin D treatment on T cell VDR expression in CD patients. The flow cytometry protocol for VDR staining was developed using the human acute monocytic leukaemia cell line (THP-1). The protocol was evaluated in anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) from vitamin D3- (n = 9) and placebo-treated (n = 9) CD patients. Anti-VDR-stained PBMCs were examined by flow cytometry, and their cytokine production was determined by cytokine bead array. VDR, CYP27B1 and RXRα mRNA expression levels in CD4(+) T cells were measured by quantitative reverse transcriptase polymerase chain reaction. The flow cytometry protocol enabled detection of cytoplasmic and nuclear VDR expression. The results were confirmed by confocal microscopy and supported by correlation with VDR mRNA expression. VDR expression in CD4(+) T cells increased following stimulation. This VDR up-regulation was inhibited with 30% by vitamin D treatment compared to placebo in CD patients (P = 0027). VDR expression was correlated with in-vitro interferon-γ production in stimulated PBMCs (P = 0.01). Flow cytometry is a useful method with which to measure intracellular VDR expression. Vitamin D treatment in CD patients reduces T cell receptor-mediated VDR up-regulation.

Long-term effect of medical treatment of diarrhoea in 377 patients with SeHCAT scan diagnosed bile acid malabsorption from 2003 to 2016; a retrospective study.

BACKGROUND: Excessive amounts of bile acids entering the colon due to bile acid malabsorption cause chronic bile acid diarrhoea. Diagnosis is possible by measuring the retention fraction of orally ingested 75 Selenium homotaurocholic acid (SeHCAT). The knowledge of long-term effects of medical treatment is sparse. AIM: To describe diarrhoea, adherence to treatment, treatment effects and quality of life in a large, well-defined cohort of patients with bile acid diarrhoea. METHODS: A retrospective survey was performed among 594 patients with bile acid malabsorption verified by SeHCAT scans at our unit between 2003 and 2016. Questionnaires about medical history, diarrhoea, use of medication, and quality of life scores were mailed to all patients. RESULTS: Among 594 patients 377 (69%) responded. Among respondents, 121 (32%) had bile acid diarrhoea due to ileal disease or resection (type 1), 198 (52%) idiopathic bile acid diarrhoea (type 2) and 58 (16%) bile acid diarrhoea due to other non-ileal disease, mainly cholecystectomy (type 3). At follow-up, half of the patients, 184 (50%), reported improvement of diarrhoea. However, 273 patients (74%) still reported diarrhoea and 234 (62%) regularly used anti-diarrhoeal medication. In spite of treatment, 235 (64%) considered reduced quality of life by diarrhoea and 184 (50%) reported that diarrhoea was unaltered or worse than before established diagnosis. CONCLUSION: Many patients with bile acid diarrhoea continue to have bothersome diarrhoea in spite of correct diagnosis and treatment.

Inhibition of gastric secretion by exogenous secretin and cholecystokinin in normals and duodenal ulcer patients.

The inhibitory effect of combined secretin (0.5 and 1.0 clin. unit/kg b.w./hour) and cholecystokinin (0.5 and 1.0 IDU/kg b.w./hour on the pentagastrin-stimulated (0.15--1.0 and 6.0 ug/kg b.w./hour) gastric secretion was investigated in twelve volunteers and in twelve duodenal ulcer patients. Regardless of the dose level no difference was found between normals and duodenal ulcer patients, indicating that a decreased sensitivity to secretin and cholecystokinin is not a pathogenetic factor for the development of duodenal ulcer.

Combined effect of glucagon and cholecystokinin on gastric secretion in man.

The inhibition of the pentagastrin-stimulated (1 ug/kg b.w./hour) gastric secretion by glucagon (20 ug/kg b.w./hour), cholecystokinin (0.5 IDU/kg b.w./hour), and a combination of these were investigated in 7 volunteers. Glucagon as well as CCK given isolated inhibited the gastric secretion, but no augmentation of inhibition was demonstrated when the hormones were in combination.

Vitamin D3 treatment of Crohn's disease patients increases stimulated T cell IL-6 production and proliferation.

BACKGROUND: Vitamin D3 has shown immune-modulating effects in CD4+ T cells from Crohn's disease patients in vitro. AIM: To investigate the effects of in vivo vitamin D3 treatment on T cells in Crohn's disease patients. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated at week 0 and at week 26 from 10 vitamin D3- and 10 placebo-treated Crohn's disease patients participating in a randomized, placebo-controlled, clinical trial study. Monocyte-depleted PBMC were stimulated with anti-CD3 and anti-CD28, and cultured for 7, days, to investigate CD4+ T-cell proliferation and T-cell cytokine production. RESULTS: In vitamin D3-treated patients, the median 25-hydroxyvitamin D3 levels increased 70 nmol/L compared with -5 nmol/L in the placebo group. Vitamin D3 treatment increased interleukin-6 production (delta = 188 pg/mL, range: -444 to 4071) compared with a decrease in the placebo group (delta = -896 pg/mL, range: -3841 to 1323) (P < 0.02, Wilcoxon rank sum test). Interestingly, vitamin D3 increased the amount of proliferating stimulated CD4+ T cells from median 41% (range: 10-75%) to 56% (range: 26-77%) (P = 0.02, Wilcoxon rank sum test). CONCLUSIONS: Vitamin D3 treatment of Crohn's disease patients increased the IL-6 levels. Interestingly, vitamin D3 treatment enhanced the CD4+ T cell proliferation.

Clinical trial: vitamin D3 treatment in Crohn's disease - a randomized double-blind placebo-controlled study.

BACKGROUND: Vitamin D has immune-regulatory functions in experimental colitis, and low vitamin D levels are present in Crohn's disease. AIM: To assess the effectiveness of vitamin D3 treatment in Crohn's disease with regard to improved disease course. METHODS: We performed a randomized double-blind placebo-controlled trial to assess the benefits of oral vitamin D3 treatment in Crohn's disease. We included 108 patients with Crohn's disease in remission, of which fourteen were excluded later. Patients were randomized to receive either 1200 IU vitamin D3 (n = 46) or placebo (n = 48) once daily during 12 months. The primary endpoint was clinical relapse. RESULTS: Oral vitamin D3 treatment with 1200 IU daily increased serum 25OHD from mean 69 nmol/L [standard deviation (s.d.) 31 nmol/L] to mean 96 nmol/L (s.d. 27 nmol/L) after 3 months (P < 0.001). The relapse rate was lower among patients treated with vitamin D3 (6/46 or 13%) than among patients treated with placebo (14/48 or 29%), (P = 0.06). CONCLUSIONS: Oral supplementation with 1200 IE vitamin D3 significantly increased serum vitamin D levels and insignificantly reduced the risk of relapse from 29% to 13%, (P = 0.06). Given that vitamin D3 treatment might be effective in Crohn's disease, we suggest larger studies to elucidate this matter further. ClinicalTrial.gov(NCT00122184).

Septic complications after appendicectomy for perforated appendicitis. A controlled clinical trial metronidazole and topical ampicillin.

A prospective, controlled trial of antibiotic therapy was carried out in 90 patients with perforated appendicitis. One randomly selected group received systemic metronidazole, started peroperatively, plus locally instilled ampicillin. The other group, also randomly selected, received only local ampicillin. There was no statistically significant difference in the overall frequency of postoperative septic complications (wound infection or intra-abdominal abscess) between the two groups, but wound infections were significantly fewer in the patients given metronidazole. There was no intergroup difference in hospitalization time. Treatment with systemic metronidazole and local ampicillin is recommended in patients operated on for perforated appendicitis.