Mercaptopurine for the Treatment of Ulcerative Colitis: A Randomized Placebo-Controlled Trial.

BACKGROUND AND AIMS: Scepticism about the efficacy of thiopurines for ulcerative colitis [UC] is rising. This study aimed to evaluate mercaptopurine treatment for UC. METHODS: In this prospective, randomized, double-blind, placebo-controlled trial, patients with active UC, despite treatment with 5-aminosalicylates [5-ASA], were randomized for therapeutic drug monitoring [TDM]-guided mercaptopurine treatment or placebo for 52 weeks. Corticosteroids were given in the first 8 weeks and 5-ASA was continued. Proactive metabolite-based mercaptopurine and placebo dose adjustments were applied from week 6 onwards by unblinded clinicians. The primary endpoint was corticosteroid-free clinical remission and endoscopic improvement [total Mayo score ≤2 points and no item >1] at week 52 in an intention-to-treat analysis. RESULTS: Between December 2016 and April 2021, 70 patients were screened and 59 were randomized at six centres. In the mercaptopurine group, 16/29 [55.2%] patients completed the 52-week study, compared to 13/30 [43.3%] on placebo. The primary endpoint was achieved by 14/29 [48.3%] patients on mercaptopurine and 3/30 [10%] receiving placebo (Δ = 38.3%, 95% confidence interval [CI] 17.1-59.4, p = 0.002). Adverse events occurred more frequently with mercaptopurine [808.8 per 100 patient-years] compared to placebo [501.4 per 100 patient-years]. Five serious adverse events occurred, four on mercaptopurine and one on placebo. TDM-based dose adjustments were executed in 22/29 [75.9%] patients, leading to lower mercaptopurine doses at week 52 compared to baseline. CONCLUSIONS: Optimized mercaptopurine treatment was superior to placebo in achieving clinical, endoscopic and histological outcomes at 1 year following corticosteroid induction treatment in UC patients. More adverse events occurred in the mercaptopurine group.

Faster recovery of gastrointestinal transit after laparoscopy and fast-track care in patients undergoing colonic surgery.

BACKGROUND & AIMS: Postoperative ileus is characterized by delayed gastrointestinal (GI) transit and is a major determinant of recovery after colorectal surgery. Both laparoscopic surgery and fast-track multimodal perioperative care have been reported to improve clinical recovery. However, objective measures supporting faster GI recovery are lacking. Therefore, GI transit was measured following open and laparoscopic colorectal surgery with or without fast-track care. METHODS: Patients (n = 93) requiring elective colonic surgery were randomized to laparoscopic or conventional surgery with fast-track multimodal management or standard care, resulting in 4 treatment arms. Gastric emptying and colonic transit were scintigraphically assessed from days 1 to 3 in 78 patients and compared with clinical parameters such as time to tolerance of solid food and/or bowel movement and time until (ready for) discharge. RESULTS: A total of 71 patients without mechanical bowel obstructions or surgical complications requiring intervention were available for analysis. No differences in gastric emptying 24 hours after surgery between the different groups were observed (P = .61). However, the median colonic transit of patients undergoing laparoscopic/fast-track care was significantly faster compared with the laparoscopic/standard, open/fast-track, and open/standard care groups. Multiple linear regression analysis showed that both laparoscopic surgery and fast-track care were significant independent predictive factors of improved colonic transit. Both were associated with significantly faster clinical recovery and shorter time until tolerance of solid food and first bowel movement. CONCLUSIONS: Colonic transit recovers significantly faster after laparoscopic surgery and the fast-track program; laparoscopy and fast-track care lead to faster recovery of GI motility and improve clinical recovery.

Nicotinic acetylcholine receptor expression and susceptibility to cholinergic immunomodulation in human monocytes of smoking individuals.

OBJECTIVE: Smoking is generally accepted as a factor that affects the disease course in inflammatory bowel disease patients. Whether these effects can be contributed to the immunomodulatory effects of nicotine via nicotinic acetylcholine receptor (nAChR) activation is unclear. As previous data suggest that the α7 nicotinic acetylcholine receptor (CHRNA7) and its duplicated variant CHRFAM7A may specifically participate in the inflammatory response of monocytes, we evaluated whether repeated nicotine exposure or smoking affects monocyte CHRNA7 and CHRFAM7A expression and cholinergic immunomodulation. METHODS: The human monocyte cell line THP-I was incubated with nicotine for different time points before endotoxin exposure. In a pilot volunteer study using smoking (n = 4) and nonsmoking (n = 7) individuals, vagal output was stimulated by olive oil administration after which monocytes were analyzed for nicotinic receptor expression. Serum tumor necrosis factor (TNF) levels were determined using ELISA and expression levels of the nAChR subunits CHRNA7, CHRNB2 or CHRFAM7A were analyzed using QPCR. RESULTS: Repeated nicotine exposure upregulated CHRNA7 expression on THP-I monocytes and led to an enhanced potential of α7 nAChR agonist GSK1345038A to reduce TNF levels. Furthermore, CHRNA7 was only detectable in isolated blood monocytes of smokers. On the other hand, the expression of CHRFAM7A and CHRNB2 was not affected by nicotine exposure. Lipopolysaccharides-induced TNF secretion was inhibited by nicotinic receptor activation in THP-I monocytes, but this response was not consistently seen in blood monocytes from smoking individuals. CONCLUSIONS: We conclude that CHRNA7 expression on blood monocytes is upregulated in smoking individuals, which may contribute to cholinergic immunomodulation.

Unphosphorylated STAT3 modulates alpha 7 nicotinic receptor signaling and cytokine production in sepsis.

The role of STAT3 in infectious diseases remains undetermined, in part because unphosphorylated STAT3 has been considered an inactive protein. Here, we report that unphosphorylated STAT3 contributes to cholinergic anti-inflammation, prevents systemic inflammation, and improves survival in sepsis. Bacterial endotoxin induced STAT3 tyrosine phosphorylation in macrophages. Both alpha 7 nicotinic receptor (alpha 7nAChR) activation and inhibition of JAK2 blunt STAT3 phosphorylation. Inhibition of STAT3 phosphorylation mimicked the alpha 7nAChR signaling, inhibiting NF-kappaB and cytokine production in macrophages. Transfection of macrophages with the dominant-negative mutant STAT3F, to prevent its tyrosine phosphorylation, reduced TNF production but did not prevent the alpha 7nAChR signaling. However, inhibition of STAT3 protein expression enhanced cytokine production and abrogated alpha 7nAChR signaling. Alpha 7nAChR controls TNF production in macrophages through a mechanism that requires STAT3 protein expression, but not its tyrosine phosphorylation. In vivo, inhibition of STAT3 tyrosine phosphorylation by stattic prevented systemic inflammation and improved survival in experimental sepsis. Stattic also prevented the production of late mediators of sepsis and improved survival in established sepsis. These results reveal the immunological implications of tyrosine-unphosphorylated STAT3 in infectious diseases.

Vagus nerve activity augments intestinal macrophage phagocytosis via nicotinic acetylcholine receptor alpha4beta2.

BACKGROUND & AIMS: The vagus nerve negatively regulates macrophage cytokine production via the release of acetylcholine (ACh) and activation of nicotinic acetylcholine receptors (nAChR). In various models of intestinal inflammation, vagus nerve efferent stimulation ameliorates disease. Given the actively constrained cytokine responses of intestinal macrophages, we explored the effect of nAChR activation on endocytosis and phagocytosis by macrophages residing in the peritoneal and mucosal compartment. METHODS: The phagocytic uptake by intestinal and peritoneal macrophages was measured by fluorescence-activated cell sorter analysis, and the nAChR involved was determined by pharmacologic blockade, short hairpin RNA-assisted gene knockdown, and the use of specific nAChR knockout mice. The effect of electrical vagus nerve stimulation on epithelial translocation and macrophage uptake of luminal particles was studied in mice. RESULTS: In isolated intestinal and peritoneal macrophages, nAChR activation enhanced endocytosis and phagocytosis. This effect was mediated via stimulated recruitment of GTPase Dynamin-2 to the forming phagocytic cup. These effects involve nAChR alpha4/beta2, rather than nAChR alpha7. Despite enhanced bacterial uptake, acetylcholine reduced NF-kappaB activation and pro-inflammatory cytokine production, while stimulating anti-inflammatory interleukin-10 production. Vagus nerve stimulation in mice altered mucosal immune responses by augmenting epithelial transport and uptake of luminal bacteria by lamina propria macrophages. CONCLUSIONS: ACh enhances phagocytic potential while inhibiting immune reactivity via nAChR alpha4/beta2 in mouse macrophages. Hence, vagus nerve efferent activity may stimulate surveillance in the intestinal mucosa and peritoneal compartment.

Comparison of Adalimumab Serum Drug Levels When Delivered by Pen Versus Syringe in Patients With Inflammatory Bowel Disease. An International, Multicentre Cohort Analysis.

BACKGROUND: Adalimumab is administered via a pre-filled syringe or spring-loaded pen. In a previous study in Crohn's disease, higher drug levels were observed in syringe users. The aim of this study was to evaluate the impact of delivery device on adalimumab drug levels in patients with Crohn's disease. METHODS: Consecutive Crohn's disease patients treated with maintenance adalimumab [40 mg fortnightly] were recruited from five centres. The first recorded drug level with matched clinical and biochemical markers of disease activity was compared between pen and syringe users. RESULTS: Of 218 patients, 64% used pen, with a median faecal calprotectin 110 µg/g and serum C-reactive protein 4 mg/L. In comparison to pen, syringe users had higher albumin [39 vs 42 g/L; p = 0.016], lower Harvey-Bradshaw Index [2 vs 1; p = 0.017], and higher rates of concomitant immunomodulation [54% vs 71%; p = 0.014]. Drug levels were equivalent between pen and syringe users [median 5.3 vs 5.2 µg/ml; p = 0.584], even after controlling for disease activity and immunomodulation. Syringe users at Alfred Health had higher drug levels than pen [6.1 vs 4.5 µg/ml; p = 0.039]; a greater proportion achieved therapeutic levels [75% vs 44%; p = 0.045]. A higher proportion of pen users from Saint-Étienne had therapeutic levels [79% vs 42%; p = 0.027], yet no significant difference in drug levels [7.9 vs 4.5 µg/ml; p = 0.119]. CONCLUSIONS: Delivery device does not appear to significantly affect adalimumab drug levels. Given differences between study sites, studies evaluating administration education and technique are warranted.

Selective alpha7 nicotinic acetylcholine receptor agonists worsen disease in experimental colitis.

BACKGROUND AND PURPOSE: In various models vagus nerve activation has been shown to ameliorate intestinal inflammation, via nicotinic acetylcholine receptors (nAChRs) expressed on immune cells. As the alpha7 nAChR has been put forward to mediate this effect, we studied the effect of nicotine and two selective alpha7 nAChR agonists (AR-R17779, (-)-spiro[1-azabicyclo[2.2.2] octane-3,5'-oxazolidin-2'-one and GSK1345038A) on disease severity in two mouse models of experimental colitis. EXPERIMENTAL APPROACH: Colitis was induced by administration of 1.5% dextran sodium sulphate (DSS) in drinking water or 2 mg 2,4,6-trinitrobenzene sulphonic acid (TNBS) intrarectally. Nicotine (0.25 and 2.50 micromol.kg(-1)), AR-R17779 (0.6-30 micromol.kg(-1)) or GSK1345038A (6-120 micromol.kg(-1)) was administered daily by i.p. injection. After 7 (DSS) or 5 (TNBS) days clinical parameters and colonic inflammation were scored. KEY RESULTS: Nicotine and both alpha7 nAChR agonists reduced the activation of NF-kappaB and pro-inflammatory cytokines in whole blood and macrophage cultures. In DSS colitis, nicotine treatment reduced colonic cytokine production, but failed to reduce disease parameters. Reciprocally, treatment with AR-R17779 or GSK1345038A worsened disease and led to increased colonic pro-inflammatory cytokine levels in DSS colitis. The highest doses of GSK1345038A (120 micromol.kg(-1)) and AR-R17779 (30 micromol.kg(-1)) ameliorated clinical parameters, without affecting colonic inflammation. Neither agonist ameliorated TNBS-induced colitis. CONCLUSIONS AND IMPLICATIONS: Although nicotine reduced cytokine responses in vitro, both selective alpha7 nAChR agonists worsened the effects of DSS-induced colitis or were ineffective in those of TNBS-induced colitis. Our data indicate the need for caution in evaluating alpha7 nAChR as a drug target in colitis.

The alpha7 nicotinic acetylcholine receptor on fibroblast-like synoviocytes and in synovial tissue from rheumatoid arthritis patients: a possible role for a key neurotransmitter in synovial inflammation.

OBJECTIVE: Recent studies have suggested an important role for neurotransmitters as modulators of inflammation. Therefore, we undertook this study to investigate the expression of the alpha7 subunit of the nicotinic acetylcholine receptor (alpha7nAChR) and its function in rheumatoid arthritis (RA). METHODS: The potential role of the alpha7nAChR in modulating proinflammatory cytokine expression in fibroblast-like synoviocytes (FLS) was identified by screening an adenoviral short hairpin RNA (Ad.shRNA) library. An alpha7-specific antibody was used for immunohistochemistry, and fluorescein isothiocyanate-labeled alpha-bungarotoxin, which binds specifically to the alpha7nAChR, was used for immunofluorescence. Gene expression in FLS was determined by quantitative polymerase chain reaction with primers specific for the alpha7nAChR. In addition, we analyzed messenger RNA (mRNA) expression of dupalpha7, a variant alpha7 transcript. Next, we studied the functional role of the alpha7nAChR in RA FLS by examining the effects of alpha7-specific agonists on the production of interleukin-6 (IL-6) and IL-8 by activated FLS. RESULTS: A screen using an Ad.shRNA library against 807 transcripts revealed that a specific alpha7nAChR shRNA potently modulated IL-8 and matrix metalloproteinase expression in FLS. The alpha7nAChR was expressed in the inflamed synovium from RA patients, predominantly in the intimal lining layer. We found alpha7nAChR expression at both the mRNA and protein level in cultured RA FLS. FLS also constitutively expressed dupalpha7 mRNA. Specific alpha7nAChR agonists reduced tumor necrosis factor alpha-induced IL-6 and IL-8 production by FLS. CONCLUSION: The alpha7nAChR and its dupalpha7 variant are expressed in RA synovium, where they may play a critical role in regulating inflammation. Targeting the alpha7nAChR could provide a novel antiinflammatory approach to the treatment of RA.

Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway.

Acetylcholine released by efferent vagus nerves inhibits macrophage activation. Here we show that the anti-inflammatory action of nicotinic receptor activation in peritoneal macrophages was associated with activation of the transcription factor STAT3. STAT3 was phosphorylated by the tyrosine kinase Jak2 that was recruited to the alpha7 subunit of the nicotinic acetylcholine receptor. The anti-inflammatory effect of nicotine required the ability of phosphorylated STAT3 to bind and transactivate its DNA response elements. In a mouse model of intestinal manipulation, stimulation of the vagus nerve ameliorated surgery-induced inflammation and postoperative ileus by activating STAT3 in intestinal macrophages. We conclude that the vagal anti-inflammatory pathway acts by alpha7 subunit-mediated Jak2-STAT3 activation.