Citations

The binding of Steel factor (SF) to c-kit initiates a signaling pathway essential for development and maintenance of interstitial cells of Cajal (ICC). Soluble and membrane-bound isoforms of SF are expressed in the gastrointestinal tract, but the role for either isoform in supporting ICC development is unknown. The aim of this study was to determine the role of SF in supporting ICC in culture. ICC were cultured from dissociated mouse jejunum and grown with fibroblast cell lines that produced either soluble, membrane-bound or membrane-restricted SF. ICC were identified and counted by c-kit immunoreactivity. The number of c-kit immunoreactive cells was greater in the coculture system compared with cultures grown without SF-producing fibroblasts. All forms of SF-producing fibroblasts increased ICC number in culture but physical separation of the fibroblasts from the c-kit immunoreactive cells, the addition of exogenous SF to the culture medium, or fibroblast-conditioned media did not. These results are consistent with the hypothesis that the membrane-bound form of SF preferentially contributes to expression of c-kit-positive ICC under cell culture conditions.

Previous classification systems of failure of limb salvage focused primarily on endoprosthetic failures and lacked sufficient depth for the effective study of the causes of failure. In order to address these inadequacies, the International Society of Limb Salvage (ISOLS) formed a committee to recommend revisions of the previous systems. The purpose of this study was to report on their recommendations. The modifications were prepared using an earlier, evidence-based model with subclassification based on the existing medical literature. Subclassification for all five primary types of failure of limb salvage following endoprosthetic reconstruction were formulated and a complementary system was derived for the failure of biological reconstruction. An additional classification of failure in paediatric patients was also described. Limb salvage surgery presents a complex array of potential mechanisms of failure, and a complete and precise classification of types of failure is required. Earlier classification systems lacked specificity, and the evidence-based system outlined here is designed to correct these weaknesses and to provide a means of reporting failures of limb salvage in order to allow the interpretation of outcome following reconstructive surgery. Cite this article: Bone Joint J 2014;96-B:1436–40.

Anoctamin‐1 (Ano1) is a widely expressed protein responsible for endogenous Ca 2+ ‐activated Cl 2 currents. Ano1 is overexpressed in cancer. Differential expression of transcriptional variants is also found in other diseases. However, the mechanisms underlying regulation of Ano1 are unknown. This study identifies the Ano1 promoter and defines a mechanism for regulating its expression. Next‐generation RNA sequencing (RNA‐seq) analysis in human gastric muscle found a new exon upstream of the reported exon 1 and identified a promoter proximal to this new exon. Reporter assays in human embryonic kidney 293 cells showed a 6.7 ± 2.1fold increase in activity over empty vector. Treatment with a known regulator of Ano1 expression, IL‐4, increased promoter activity by 1.6 ± 0.02‐fold over untreated cells. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 (STAT6), a downstream effector of IL‐4. Chromatin immunoprecipitation (ChIP) experiments on T84 cells, which endogenously express Ano1, showed a 2.1 ± 0.12‐fold increase in binding of STAT6 to P0 after IL‐4 treatment. These results were confirmed by mutagenesis, expression, and RNA interference techniques. This work allows deeper understanding of the regulation of Ano1 in physiology and as a potential therapeutic target in a variety of diseases—Mazzone, A., Gibbons, S. J., Bernard, C. E., Nowsheen, S., Middha, S., Almada, L. L., Ordog, T., Kendrick, M. L., Reid Lombardo, KM., Shen, K. R., Galietta, L. J. V., Fernandez‐Zapico, M. E., and Farrugia, G., Identification and characterization of a novel promoter for the human ANO1 gene regulated by the transcription factor signal transducer and activator of transcription 6 (STAT6). FASEB J. 29, 152–163 (2015). www.fasebj.org

Abstract Background Diabetic gastroparesis results in significant morbidity for patients and major economic burden for society. Treatment options for diabetic gastroparesis are currently directed at symptom control rather than the underlying disease and are limited. The pathophysiology of diabetic gastroparesis includes damage to intrinsic and extrinsic neurons, smooth muscle, and interstitial cells of C ajal ( ICC ). Oxidative damage in diabetes appears to be one of the primary insults involved in the pathogenesis of several complications of diabetes, including gastroparesis. Recent studies have highlighted the potential role of macrophages as key cellular elements in the pathogenesis of diabetic gastroparesis. Macrophages are important for both homeostasis and defense against a variety of pathogens. Heme oxygenase 1 ( HO 1), an enzyme expressed in a subset of macrophages has emerged as a major protective mechanism against oxidative stress. Activation of macrophages with high levels of HO 1 expression protects against development of delayed gastric emptying in animal models of diabetes, while activation of macrophages that do not express HO 1 are linked to neuromuscular cell injury. Targeting macrophages and HO 1 may therefore be a therapeutic option in diabetic gastroparesis. Purpose This report briefly reviews the pathophysiology of diabetic gastroparesis with a focus on oxidative damage and how activation and polarization of different subtypes of macrophages in the muscularis propria determines development of delay in gastric emptying or protects against its development.

AbstractBackgroundHeme oxygenase 1 (HO‐1) degrades heme and protects against oxidative stress. In vitro and animal models suggest that HO‐1 is beneficial in several diseases (e.g., postoperative ileus, gastroparesis, acute pancreatitis, and colitis). However, the only drugs (i.e., hemin and heme arginate) which pharmacologically upregulate HO‐1 in humans are expensive and can only be administered intravenously. Our aims were to compare the effects of placebo, aspirin, and simvastatin alone, and with α‐lipoic acid, on HO‐1 protein concentration and activity in humans.MethodsThis randomized, double‐blind, placebo‐controlled study compared the effects of three oral regimens administered for 7 days, i.e., placebo; aspirin (325 mg twice daily) and simvastatin (40 mg twice daily); aspirin, simvastatin, and the sodium salt of R‐ α‐lipoic acid (NaRLA, 600 mg three times daily) on markers of HO‐1 activation (i.e., plasma HO‐1 protein concentration and venous monocyte HO‐1 protein activity) in 18 healthy subjects (14 females). Markers of HO‐1 activation were evaluated at baseline, days 2, and 7.Key ResultsBaseline HO‐1 protein concentrations and activity were similar among the three groups. Compared to placebo, aspirin and simvastatin combined, or together with NaRLA did not affect HO‐1 protein concentration or activity at 2 or 7 days. HO‐1 protein concentrations and activity were correlated on day 7 (r = 0.75, p = 0.0004) but not at baseline and on day 2.Conclusions & InferencesAt therapeutic doses, aspirin, simvastatin, and α‐lipoic acid do not increase plasma HO‐1 protein concentration or venous monocyte HO‐1 activity in healthy humans.

Aims: Establishing epicardial flow with percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction (STEMI) is necessary but not sufficient to ensure nutritive myocardial reperfusion. We evaluated whether adding myocardial blush grade (MBG) and quantitative reperfusion ventricular arrhythmia “bursts” (VABs) surrogates provide a more informative biosignature of optimal reperfusion in patients with Thrombolysis in Myocardial Infarction (TIMI) 3 flow and ST-segment recovery (STR). Methods and results: Anterior STEMI patients with final TIMI 3 flow had protocol-blinded analyses of simultaneous MBG, continuous 12-lead electrocardiogram (ECG) STR, Holter VABs, and day 5–14 SPECT imaging infarct size (IS) assessments. Over 20 million cardiac cycles from >4500 h of continuous ECG monitoring in subjects with STR were obtained. IS and clinical outcomes were examined in patients stratified by MBG and VABs. VABs occurred in 51% (79/154) of subjects. Microcirculation (MBG 2/3) was restored in 75% (115/154) of subjects, of whom 53% (61/115) had VABs. No VABs were observed in subjects without microvascular flow (MBG of 0). Of 115 patients with TIMI 3 flow, STR, and MBG 2/3, those with VABs had significantly larger IS (median: 23.0% vs 6.0%, p=0.001). Multivariable analysis identified reperfusion VABs as a factor significantly associated with larger IS ( p=0.015). Conclusions: Despite restoration of normal epicardial flow, open microcirculation, and STR, concomitant VABs are associated with larger myocardial IS, possibly reflecting myocellular injury in reperfusion settings. Combining angiographic and ECG parameters of epicardial, microvascular, and cellular response to STEMI intervention provides a more predictive “biosignature” of optimal reperfusion than do single surrogate markers.

Key points Ano1, a Ca2+‐activated Cl− channel, is expressed in interstitial cells of Cajal (ICC) throughout the gut. We report here that it is required to maintain coordinated Ca2+ transients within myenteric ICC of mouse small intestine. Ca2+ transients in Ano1 WT mice were rhythmic and coordinated whereas uncoordinated Ca2+ transients were seen in knockout mice. Ca2+ transients were un‐coordinated following pharmacological block of Ano1 in WT mice using niflumic acid, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid and 4,4′‐diisothiocyanato‐2,2′‐stilbenedisulfonic acid disodium salt. Transient knockdown of Ano1 in organotypic cultures with short hairpin RNA to Ano1 in WT tissues also caused loss of coordinated Ca2+ transients. Contractility of Ano1 knockout mouse intestinal segments in organ bath experiments was significantly decreased, less coordinated and non‐rhythmic. Spatiotemporal maps from knockout mouse small intestine also showed loss of phasic contractile activity. This study provides important information on the basic mechanisms driving coordinated contractile activity in the gastrointestinal tract. AbstractInterstitial cells of Cajal (ICC) are pacemaker cells that generate electrical activity to drive contractility in the gastrointestinal tract via ion channels. Ano1 (Tmem16a), a Ca2+‐activated Cl− channel, is an ion channel expressed in ICC. Genetic deletion of Ano1 in mice resulted in loss of slow waves in smooth muscle of small intestine. In this study, we show that Ano1 is required to maintain coordinated Ca2+ transients between myenteric ICC (ICC‐MY) of small intestine. First, we found spontaneous Ca2+ transients in ICC‐MY in both Ano1 WT and knockout (KO) mice. However, Ca2+ transients within the ICC‐MY network in Ano1 KO mice were uncoordinated, while ICC‐MY Ca2+ transients in Ano1 WT mice were rhythmic and coordinated. To confirm the role of Ano1 in the loss of Ca2+ transient coordination, we used pharmacological inhibitors of Ano1 activity and shRNA‐mediated knock down of Ano1 expression in organotypic cultures of Ano1 WT small intestine. Coordinated Ca2+ transients became uncoordinated using both these approaches, supporting the conclusion that Ano1 is required to maintain coordination/rhythmicity of Ca2+ transients. We next determined the effect on smooth muscle contractility using spatiotemporal maps of contractile activity in Ano1 KO and WT tissues. Significantly decreased contractility that appeared to be non‐rhythmic and uncoordinated was observed in Ano1 KO jejunum. In conclusion, Ano1 has a previously unidentified role in the regulation of coordinated gastrointestinal smooth muscle function through coordination of Ca2+ transients in ICC‐MY.

Abstract Background There is increasing evidence for specific cellular changes in the stomach of patients with diabetic ( DG ) and idiopathic ( IG ) gastroparesis. The most significant findings are loss of interstitial cells of Cajal ( ICC ), neuronal abnormalities, and an immune cellular infiltrate. Studies done in diabetic mice have shown a cytoprotective effect of CD 206+ M2 macrophages. To quantify overall immune cellular infiltrate, identify macrophage populations, and quantify CD 206+ and i NOS + cells. To investigate associations between cellular phenotypes and ICC . Methods Full thickness gastric body biopsies were obtained from non‐diabetic controls (C), diabetic controls ( DC ), DG , and IG patients. Sections were labeled for CD 45, CD 206, Kit, i NOS , and putative human macrophage markers ( HAM 56, CD 68, and EMR 1). Immunoreactive cells were quantified from the circular muscle layer. Key Results Significantly fewer ICC were detected in DG and IG tissues, but there were no differences in the numbers of cells immunoreactive for other markers between patient groups. There was a significant correlation between the number of CD 206+ cells and ICC in DG and DC patients, but not in C and IG and a significant correlation between i NOS + cells and ICC in the DC group, but not the other groups. CD 68 and HAM 56 reliably labeled the same cell populations, but EMR 1 labeled other cell types. Conclusions & Inferences Depletion of ICC and correlation with changes in CD 206+ cell numbers in DC and DG patients suggests that in humans, like mice, CD 206+ macrophages may play a cytoprotective role in diabetes. These findings may lead to novel therapeutic options, targeting alternatively activated macrophages.

Our aim was to conduct a pilot case-control study of RNA expression profile using RNA sequencing of rectosigmoid mucosa of nine females with -diarrhea-predominant irritable bowel syndrome (IBS-D) with accelerated colonic transit and nine female healthy controls. Mucosal total RNA was isolated and purified, and next-generation pair-end sequencing was performed using Illumina TruSeq. Analysis was carried out using a targeted approach toward 12 genes previously associated with IBS and a hypothesis-generating approach. Of the 12 targeted genes tested, patients with IBS-D had decreased mRNA expression of TNFSF15 (fold change controls to IBS-D: 1.53, P = 0.01). Overall, up- and downregulated mRNA expressions of 21 genes ( P = 10−5 to 10−8; P values with false detection rates are shown) were potentially relevant to IBS-D including the following: neurotransmitters [ P2RY4 ( P = 0.001), vasoactive intestinal peptide ( VIP, P = 0.02)]; cytokines [ CCL20 ( P = 0.019)]; immune function [ C4BPA complement cascade ( P = 0.0187)]; interferon-related [ IFIT3 ( P = 0.016)]; mucosal repair and cell adhesion [trefoil protein ( TFF1, P = 0.012)], retinol binding protein [ RBP2 ( P = 0.017)]; fibronectin ( FN1, P = 0.009); and ion channel functions [guanylate cyclase ( GUCA2B, P = 0.017), PDZ domain-containing protein 3 ( PDZD3, P = 0.029)]. Ten genes associated with functions related to pathobiology of IBS-D were validated by RT-PCR. There was significant correlation in fold changes of the selected genes ( Rs = 0.73, P = 0.013). Up- or downregulation of P2RY4, GUC2AB, RBP2, FNI, and C4BPA genes were confirmed on RT-PCR, which also revealed upregulation of farnesoid X receptor ( FXR) and apical sodium-coupled bile acid transporter ( IBAT/ASBT). RNA-Seq and RT-PCR analysis of rectosigmoid mucosa in IBS-D show transcriptome changes that provide the rationale for validation studies to explore the role of mucosal factors in the pathobiology of IBS-D.