Proinflammatory cytokine interferon-γ and microbiome-derived metabolites dictate epigenetic switch between forkhead box protein 3 isoforms in coeliac disease.

Coeliac disease (CD) is an autoimmune enteropathy triggered by gluten and characterized by a strong T helper type 1 (Th1)/Th17 immune response in the small intestine. Regulatory T cells (Treg ) are CD4+ CD25++ forkhead box protein 3 (FoxP3+ ) cells that regulate the immune response. Conversely to its counterpart, FoxP3 full length (FL), the alternatively spliced isoform FoxP3 Δ2, cannot properly down-regulate the Th17-driven immune response. As the active state of CD has been associated with impairments in Treg cell function, we aimed at determining whether imbalances between FoxP3 isoforms may be associated with the disease. Intestinal biopsies from patients with active CD showed increased expression of FOXP3 Δ2 isoform over FL, while both isoforms were expressed similarly in non-coeliac control subjects (HC). Conversely to what we saw in the intestine, peripheral blood mononuclear cells (PBMC) from HC subjects did not show the same balance between isoforms. We therefore hypothesized that the intestinal microenvironment may play a role in modulating alternative splicing. The proinflammatory intestinal microenvironment of active patients has been reported to be enriched in butyrate-producing bacteria, while high concentrations of lactate have been shown to characterize the preclinical stage of the disease. We show that the combination of interferon (IFN)-γ and butyrate triggers the balance between FoxP3 isoforms in HC subjects, while the same does not occur in CD patients. Furthermore, we report that lactate increases both isoforms in CD patients. Collectively, these findings highlight the importance of the ratio between FoxP3 isoforms in CD and, for the first time, associate the alternative splicing process mechanistically with microbial-derived metabolites.

The safety, tolerance, pharmacokinetic and pharmacodynamic effects of single doses of AT-1001 in coeliac disease subjects: a proof of concept study.

BACKGROUND: Lifelong adherence to a strict gluten-free diet is the cornerstone of coeliac disease treatment. Elucidation of disease pathogenesis has created opportunities for novel therapeutic approaches to coeliac disease. AT-1001 is an inhibitor of paracellular permeability whose structure is derived from a protein secreted by Vibrio cholerae. AIM: To determine the safety and tolerability of 12 mg doses of AT-1001 in coeliac disease subjects challenged with gluten. METHODS: An in-patient, double-blind, randomized placebo-controlled safety study utilizing intestinal permeability, measured via fractional excretions of lactulose and mannitol, as an exploratory measure of drug efficacy. RESULTS: Compared to placebo, no increase in adverse events occurred in patients exposed to AT-1001. Following acute gluten exposure, a 70% increase in intestinal permeability was detected in the placebo group, while none was seen in the AT-1001 group. Interferon-gamma levels increased in four of seven patients (57%) of the placebo group, but only in four of 14 patients (29%) of the AT-1001 group. Gastrointestinal symptoms were more frequently detected in the placebo group when compared to the AT-1001 group (P = 0.018). CONCLUSIONS: AT-1001 is well tolerated and appears to reduce intestinal barrier dysfunction, proinflammatory cytokine production, and gastrointestinal symptoms in coeliacs after gluten exposure.

Combined carriership of TLR9-1237C and CD14-260T alleles enhances the risk of developing chronic relapsing pouchitis.

AIM: To investigate the single nucleotide polymorphisms (SNPs) in genes involved in bacterial recognition and the susceptibility to pouchitis or pouchitis severity. METHODS: Analyses of CD14 -260C>T, CARD15/NOD2 3020insC, Toll-like receptor (TLR)4 +896A>G, TLR9 -1237T>C, TLR9+2848G>A, and IRAKM + 22148G>A SNPs were performed in 157 ileal-pouch anal anastomosis (IPAA) patients (79 patients who did not develop pouchitis, 43 infrequent pouchitis patients, 35 chronic relapsing pouchitis patients) and 224 Italian Caucasian healthy controls. RESULTS: No significant differences were found in SNP frequencies between controls and IPAA patients. However, a significant difference in carriership frequency of the TLR9-1237C allele was found between the infrequent pouchitis and chronic relapsing pouchitis groups [P = 0.028, oddos ratio (OR) = 3.2, 95%CI = 1.2-8.6]. This allele uniquely represented a 4-locus TLR9 haplotype comprising both studied TLR9 SNPs in Caucasians. Carrier trait analysis revealed an enhanced combined carriership of the alleles TLR9 -1237C and CD14 -260T in the chronic relapsing pouchitis and infrequent pouchitis group (P = 0.018, OR = 4.1, 95%CI = 1.4 -12.3). CONCLUSION: There is no evidence that the SNPs predispose to the need for IPAA surgery. The significant increase of the combined carriership of the CD14 -260T and TLR9 -1237C alleles in the chronic relapsing pouchitis group suggests that these markers identify a subgroup of IPAA patients with a risk of developing chronic or refractory pouchitis.

Improved method for the isolation and cultivation of human scalp dermal papilla cells.

We present an improved method for the isolation and cultivation of human scalp anagen hair follicle dermal papilla cells. Following treatment of the isolated dermal papilla with collagenase, incubation in Chang's medium mediates accelerated growth of the papilla cells when compared with other media such as DMEM, M199, and EMEM. Upon reaching confluency, the cells cultured in this fashion exhibit a multilayer-forming property that is dependent on normal proteoglycan synthesis. The papilla cells maintain this morphologic behavior for as long as 7 weeks in culture, or after being subcultured six times. During this time, the cells continue to synthesize extracellular matrix components associated with the human anagen follicle in situ. These include chondroitin sulfate, laminin, and type IV collagen. Type III collagen and keratan sulfate are poorly expressed by the papilla both in situ and in vitro. Heparan sulfate proteoglycan, a matrix component of the papilla in situ, is poorly expressed in vitro. Earlier reports suggested that the expression of extracellular matrix components is not maintained in culture. We show that the expression of these molecules is not dependent on the secondary culture medium, but continues in DMEM and M199 after primary culture in Chang's medium. Our results suggest that initial exposure of the dermal papilla to Chang's medium either selectively permits the outgrowth of papilla cells having extracellular matrix components similar to those found in situ, or stabilizes the expression of extracellular matrix components among the entire cultured cell population.

Probiotics and barrier function in colitis.

Probiotic administration may exert a protective effect in colitis by preventing mucosal barrier disruption and influencing the extent of mucosal injury.

Effect of probiotic strains on interleukin 8 production by HT29/19A cells.

OBJECTIVES: Promising results from clinical studies on the effect of probiotics as maintenance therapy in inflammatory bowel disease and in the prevention of onset of pouchitis ask for studies to unravel the still poorly understood mechanism of action of probiotics. METHODS: To evaluate whether the probiotic bacteria that were used in the clinical studies (VSL#3, Escherichia coli Nissle 1917, and Lactobacillus GG) are able to induce chemokine production in epithelial cells, HT29/19A monolayers were incubated with cell debris and cell extract fractions of single strains of the probiotic bacteria in doses ranging from 10(3) to 10(9) colony-forming units/ml for 32 h. Supernatants were measured for interleukin 8 by ELISA. RESULTS: Lactobacilli and bifidobacteria strains from VSL#3 and Lactobacillus GG did not induce interleukin 8, whereas both cell debris and cell extracts from E. coli Nissle 1917 induced interleukin 8 production in a dose-dependent way. Cell extracts from streptococcal strains induced interleukin 8 when applied at high concentrations. CONCLUSIONS: Probiotic Gram-positive bacteria did not induce interleukin 8, whereas the nonpathogenic, Gram-negative E. coli Nissle 1917 strain induced interleukin 8 in a dose-dependent way in this culture model. These results suggest that probiotic Gram-positive bacteria and E. coli Nissle 1917 may exert their beneficial effects on the host by a different mechanism of action.

The effect of transient intestinal ischemia on inflammatory parameters.

BACKGROUND AND AIMS: To determine the early biological changes occurring in intestinal ischemia in vivo. PATIENTS AND METHODS: We studied the effects of acute transient intestinal ischemia in 15 patients undergoing elective open surgery for the treatment of abdominal subrenal aortic aneurysm induced by clamping of the aorta at subrenal level and above the branching of the inferior mesenteric artery. Blocking the blood flow results in hypoperfusion of the inferior mesenteric artery and then to rectal mucosal ischemia. RESULTS: With the introduction of a mucosal ischemic period the basal intestinal mucosal pH decreased during ischemia, and showed a rapid increase during reperfusion to the level preceding ischemia. Parameters were evaluated in blood taken from inferior mesenteric vein. A rectal dialysis was put into the rectum to evaluate eicosanoid concentrations in rectal fluid collected before and during clamping and after declamping. Significant enhancement in plasma level of xanthine, a marker for tissue damage, was observed during reperfusion. Interleukin-6 levels were significantly elevated from 11.28+/-3.4 pg/ml (preischemic) to 109+/-85.9 pg/ml (ischemic) and to 189.33+/-120.24 pg/ml (reperfusion); and tromboxane B(2) levels from 141.57+/-51.20 pg/ml preoperation to 473.01+/-319.01 pg/ml during the surgical procedure. CONCLUSION: These observations indicate that even transient ischemia modifies the inflammatory pattern.

The effects of minoxidil, 1% pyrithione zinc and a combination of both on hair density: a randomized controlled trial.

BACKGROUND: Recent studies of antidandruff shampoos or tonics containing antifungal or antibacterial agents produced effects suggestive of a potential hair growth benefit. OBJECTIVES: The purpose of this 6-month, 200-patient, randomized, investigator-blinded, parallel-group clinical study was to assess the hair growth benefits of a 1% pyrithione zinc shampoo. The efficacy of a 1% pyrithione zinc shampoo (used daily), was compared with that of a 5% minoxidil topical solution (applied twice daily), a placebo shampoo and a combination of the 1% pyrithione zinc shampoo and the 5% minoxidil topical solution. METHODS: Two hundred healthy men between the ages of 18 and 49 years (inclusive) exhibiting Hamilton-Norwood type III vertex or type IV baldness were enrolled. Total hair counts, the primary efficacy measure, were obtained using fibre-optic microscopy and a computer-assisted, manual hair count method. Secondary measures of efficacy included assessments of hair diameter, as well as patient and investigator global assessments of improvement in hair growth. These were based on photographs of the scalp using both midline and vertex views. RESULTS: Hair count results showed a significant (P < 0.05) net increase in total visible hair counts for the 1% pyrithione zinc shampoo, the 5% minoxidil topical solution, and the combination treatment groups relative to the placebo shampoo after 9 weeks of treatment. The relative increase in hair count for the 1% pyrithione zinc shampoo was slightly less than half that for the minoxidil topical solution and was essentially maintained throughout the 26-week treatment period. No advantage was seen in using both the 5% minoxidil topical solution and the 1% pyrithione zinc shampoo. A small increase in hair diameter was observed for the minoxidil-containing treatment groups at week 17. Assessments of global improvements by the patients and investigator generally showed the benefit of 5% minoxidil. The benefit of the 1% pyrithione zinc shampoo used alone tended (P < 0.1) to be apparent only to the investigator. CONCLUSIONS: Hair count results show a modest and sustained improvement in hair growth with daily use of a 1% pyrithione zinc shampoo over a 26-week treatment period.

Polarised interleukin 8 secretion by HT 29/19A cells.

Interleukin 8 is a neutrophil chemotactic and stimulating cytokine induced by various inflammatory stimuli, including tumour necrosis factor, interleukin 1, and endotoxin. The ability of HT 29/19A enterocytes to synthesise interleukin 8 was studied. The results show that interleukin 1 is an important stimulus for interleukin 8 synthesis and secretion by HT 29/19A cells, being more potent than tumour necrosis factor. The tumour necrosis factor and interleukin 1 induced interleukin 8 secretion by HT 29/19A cells was seen to be polarised according to the direction of stimulation. These results support the concept that mucosal cells (enterocytes) may play an important part in initiating mucosal inflammation. Furthermore, it is proposed that HT 29/19A cells constitute a tool to study stimulus directed polarised cytokine secretion.