Three-dimensional high-resolution anorectal manometry can predict response to biofeedback therapy in defecation disorders.

PURPOSE: Biofeedback therapy (BT) is a simple and effective technique for managing outlet constipation and fecal incontinence. Several clinical factors are known to predict BT response, but a 50% failure rate persists. Better selection of BT responsive patients is required. We aimed to determine whether the defecation disorder type per high-resolution manometry (HRM) was predictive of BT response. METHODS: We analyzed clinical, manometric, and ultrasound endoscopic data from patients who underwent BT in our department between January 2015 and January 2016. Patients were classified into four groups per the following defecation disorder classification criteria: rectal pressure > 40 mmHg and anal paradoxical contraction (type I); rectal pressure < 40 mmHg and anal paradoxical contraction (type II); rectal pressure > 40 mmHg and incomplete anal relaxation (type III); and rectal pressure < 40 mmHg and incomplete anal relaxation (type IV). An experienced single operator conducted ten weekly 20-min sessions. Efficacy was evaluated with the visual analog scale. RESULTS: Of 92 patients, 47 (50.5%) responded to BT. Type IV and type II defecation disorders were predictive of success (p = 0.03) (OR = 5.03 [1.02; 24.92]) and failure (p = 0.05) (OR = 0.41 [0.17; 0.99]), respectively. The KESS score severity before BT (p = 0.03) (OR = 0.9 [0.81; 0.99]) was also predictive of failure. CONCLUSION: The manometry types identified according to the defecation disorder classification criteria were predictive of BT response. Our data confirm the role of three-dimensional HRM in the therapeutic management of anorectal functional disorders.

Sacral nerve stimulation can improve continence in patients with Crohn's disease with internal and external anal sphincter disruption.

PURPOSE: Sacral nerve stimulation is a technique commonly used for the treatment of idiopathic incontinence. This study was designed to assess the efficiency of sacral nerve stimulation as a means of treating fecal incontinence in patients with Crohn's disease with disrupted internal and external anal sphincters. METHODS: Five patients (3 women) with fecal incontinence suffering from Crohn's disease-related anoperineal lesions were treated by applying three weeks of sacral nerve stimulation and then by permanent sacral nerve stimulation implantation. Endoanal ultrasonography showed that all of these patients had disrupted external and internal anal sphincters. RESULTS: Continence was improved in all treated patients. The median follow-up time was 14 (range, 3-36) months. At the end of the follow-up period, the median Wexner's score significantly improved from 15 to 6 and the median number of daily stools decreased from 7 to 2. The patients' quality of life also increased significantly. CONCLUSIONS: Sacral nerve stimulation improves fecal continence in patients suffering from Crohn's anoperineal lesions with internal and external anal sphincters disruption.

Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells.

Bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments revealed that heterotrimeric G proteins and their effectors were found in stable complexes that persisted during signal transduction. Adenylyl cyclase, Kir3.1 channel subunits and several G-protein subunits (Galpha(s), Galpha(i), Gbeta(1) and Ggamma(2)) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically Gbeta(1)-YFP(1-158) and Ggamma(2)-YFP(159-238), which heterodimerize to produce fluorescent YFP-Gbeta(1)gamma(2)). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-Ggamma(2), GFP-Gbeta(1) or YFP-Gbeta(1)gamma(2). Galpha subunits were also stably associated with both effectors regardless of whether or not signal transduction was initiated by a receptor agonist. Although BRET between effectors and Gbetagamma was increased by receptor stimulation, our data indicate that these changes are likely to be conformational in nature. Furthermore, receptor-sensitive G-protein-effector complexes could be detected before being transported to the plasma membrane, providing the first direct evidence for an intracellular site of assembly.

High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay.

In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of beta-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with beta-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- beta-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and beta-arrestin2- Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compounds were screened for inhibition of the agonist-promoted beta-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced beta-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1-beta-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists.