[Show abstract][Hide abstract] ABSTRACT: There is an increasing awareness of the role of macrophages in the regulation and maintenance of gastrointestinal function in health and disease. This work has proceeded in the context of an increased understanding of the complex phenotypic variation in macrophages throughout the body and has revealed previously un-identified roles for macrophages in diseases like gastroparesis, post-operative ileus and inflammatory bowel disease. Opportunities for exploiting the phenotypic modulation of tissue resident macrophages have been identified as possible therapies for some of these diseases. In addition, macrophages are an established component of the innate immune system that can respond to variations and changes in the intestinal microbiome and potentially mediate part of the impact of the microbiota on intestinal health. We reviewed the latest work on novel concepts in defining macrophage phenotype, discuss possible mechanisms of action for tissue-resident macrophages in the gut, address the significance of microbiome effects on macrophage phenotype and review the known and possible roles of macrophages in motility disorders of the gastrointestinal tract.
[Show abstract][Hide abstract] ABSTRACT: Background:
The SCN5A-encoded voltage-gated sodium channel NaV 1.5 is expressed in human jejunum and colon. Mutations in NaV 1.5 are associated with gastrointestinal motility disorders. The rat gastrointestinal tract expresses voltage-gated sodium channels, but their molecular identity and role in rat gastrointestinal electrophysiology are unknown.
The presence and distribution of Scn5a-encoded NaV 1.5 was examined by PCR, Western blotting and immunohistochemistry in rat jejunum. Freshly dissociated smooth muscle cells were examined by whole cell electrophysiology. Zinc finger nuclease was used to target Scn5a in rats. Lentiviral-mediated transduction with shRNA was used to target Scn5a in rat jejunum smooth muscle organotypic cultures. Organotypic cultures were examined by sharp electrode electrophysiology and RT-PCR.
We found NaV 1.5 in rat jejunum and colon smooth muscle by Western blot. Immunohistochemistry using two other antibodies of different portions of NaV 1.5 revealed the presence of the ion channel in rat jejunum. Whole cell voltage-clamp in dissociated smooth muscle cells from rat jejunum showed fast activating and inactivating voltage-dependent inward current that was eliminated by Na(+) replacement by NMDG(+) . Constitutive rat Scn5a knockout resulted in death in utero. NaV 1.5 shRNA delivered by lentivirus into rat jejunum smooth muscle organotypic culture resulted in 57% loss of Scn5a mRNA and several significant changes in slow waves, namely 40% decrease in peak amplitude, 30% decrease in half-width, and 7 mV hyperpolarization of the membrane potential at peak amplitude.
Conclusions & inferences:
Scn5a-encoded NaV 1.5 is expressed in rat gastrointestinal smooth muscle and it contributes to smooth muscle electrophysiology.
Full-text · Article · Oct 2015 · Neurogastroenterology and Motility
[Show abstract][Hide abstract] ABSTRACT: Objective To understand motivations, educational needs, and concerns of individuals contemplating whole-exome sequencing (WES) and determine what amount of genetic information might be obtained by sequencing a generally healthy cohort so as to more effectively counsel future patients. Patients and Methods From 2012 to 2014, 40 medically educated, generally healthy scientists at Mayo Clinic were invited to have WES conducted on a research basis; 26 agreed to be in a drawing from which 10 participants were selected. The study involved pre- and posttest genetic counseling and completion of 4 surveys related to the experience and outcomes. Whole-exome sequencing was conducted on DNA from blood from each person. Results Most variants (76,305 per person; range, 74,505-77,387) were known benign allelic variants, variants in genes of unknown function, or variants of uncertain significance in genes of known function. The results of suspected pathogenic/pathogenic variants in Mendelian disorders and pharmacogenomic variants were disclosed. The mean number of suspected pathogenic/pathogenic variants was 2.2 per person (range, 1-4). Four pharmacogenomic genes were included for reporting; variants were found in 9 of 10 participants. Conclusion This study provides data that may be useful in establishing reality-based patient expectations, outlines specific points to cover during counseling, and increases confidence in the feasibility of providing adequate preparation and counseling for WES in generally healthy individuals.
Full-text · Article · Oct 2015 · Mayo Clinic Proceedings
[Show abstract][Hide abstract] ABSTRACT: Anoctamin 1 (Ano1, TMEM16A) is a Ca(2+)-activated Cl(-) channel (CACC) expressed in interstitial cells of Cajal (ICC). The mechanisms by which Ca(2+) regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-exon "0"-upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca(2+) sensitivity and electrophysiological properties of Ano1. Constructs with (+0) or without (-0) the newly identified exon were transfected into HEK293 cells. Voltage clamp electrophysiology was used to determine voltage- and time-dependent parameters of whole cell Cl(-) currents between isoforms with varying concentrations of intracellular Ca(2+), extracellular anions, or Cl(-) channel inhibitors. We found that exon 0 did not change voltage sensitivity and had no impact on relative permeability of Ano1 to most anions. Ano1(+0) exhibited greater changes in current density but lesser changes in kinetics than (-0) in response to varying intracellular Ca(2+). The CACC inhibitor niflumic acid inhibited current with greater efficacy and higher potency against Ano1(+0) compared to Ano1(-0). Likewise, the Ano1 inhibitor T16Ainh-A01 reduced Ano1(+0) more than Ano1(-0). In conclusion, human Ano1 containing exon 0 imparts its Cl(-) current with greater sensitivity to intracellular Ca(2+) and CACC inhibitors.
[Show abstract][Hide abstract] ABSTRACT: Background & aims:
Diabetic gastroparesis is associated with changes in interstitial cells of Cajal (ICC), neurons and smooth muscle cells in both animal models and humans. Macrophages appear to be critical to the development of cellular damage that leads to delayed gastric emptying but the mechanisms involved are not well understood. Csf1(op/op) (Op/Op) mice lack biologically active Csf1, resulting in the absence of Csf1-dependent tissue macrophages. The aim of this study was to use Csf1(op/op) mice to determine the role of macrophages in the development of delayed gastric emptying.
Animals were injected with streptozotocin to make them diabetic. Gastric emptying was determined weekly. Immunohistochemistry was used to identify macrophages and ICC networks in the gastric muscular layers. Oxidative stress was measured by serum malondialdehyde (MDA) levels. Quantitative, reverse transcription PCR was used to measure levels of mRNA.
Csf1(op/op) mice had normal ICC. With onset of diabetes both Csf1(op/op) and wild type Csf1(+/+) mice developed increased levels of oxidative stress (75.8 ± 9.1 and 41.2±13.6 nmol/mL MDA respectively). Wild type Csf1(+/+) mice developed delayed gastric emptying after onset of diabetes (4/13) whereas no diabetic Csf1(op/op) mouse developed delayed gastric emptying (0/15, P=0.035). ICC were disrupted in diabetic wild type Csf1(+/+) mice with delayed gastric emptying but remained normal in diabetic Csf1(op/op) mice.
Cellular injury and development of delayed gastric emptying in diabetes requires the presence of muscle layer macrophages. Targeting macrophages may be an effective therapeutic option to prevent cellular damage and development of delayed gastric emptying in diabetes.
[Show abstract][Hide abstract] ABSTRACT: Abdominal wall pain (AWP) is an important cause of chronic abdominal pain. History and physical examination are critical to the diagnosis of AWP. Trigger point injection (TPI) using either a steroid or a local anesthetic or a combination of both is often used to treat AWP.
To determine the efficacy of ultrasound-guided TPI and to determine the predictors of a successful response.
Patients who received ultrasound-guided TPI between July 2010 and June 2011 were surveyed. The primary outcome was determined using the Treatment Efficacy Questionnaire (TEQ). Electronic medical records were reviewed to determine patient, pain and TPI characteristics. Linear regression was used to determine the predictors of a successful response on the TEQ.
Right upper quadrant was the most common site of AWP, and the median pain duration was 12 months. Pain was rated as >8 (1-10 scale) by 57 % and 30 % described it as an ache. Narcotic use was reported in 38 %, and 73 % had a history of at least one abdominal surgery. Forty-four of the 120 (37 %) patients met the criteria for responder on the TEQ. Compared to before treatment, 36 % reported being "significantly better" and 22 % "slightly better." Multiple linear regression analysis showed that higher somatization negatively predicted response. None of the other historical, examination or TPI characteristics were associated with response to the TPI.
TPI can provide significant, long-term symptom relief in a third of patients with chronic abdominal pain attributed to AWP. Somatization was inversely related to the treatment success.
No preview · Article · Aug 2015 · Digestive Diseases and Sciences
[Show abstract][Hide abstract] ABSTRACT: Whole exome sequencing (WES) is increasingly being used for diagnosis without adequate information on predictive characteristics of reportable variants typically found on any given individual and correlation with clinical phenotype. In this study, we performed WES on 89 deceased individuals (mean age at death 74 years, range 28-93) from the Mayo Clinic Biobank. Significant clinical diagnoses were abstracted from electronic medical record via chart review. Variants [Single Nucleotide Variant (SNV) and insertion/deletion] were filtered based on quality (accuracy >99%, read-depth >20, alternate-allele read-depth >5, minor-allele-frequency <0.1) and available HGMD/OMIM phenotype information. Variants were defined as Tier-1 (nonsense, splice or frame-shifting) and Tier-2 (missense, predicted-damaging) and evaluated in 56 ACMG-reportable genes, 57 cancer-predisposition genes, along with examining overall genotype-phenotype correlations. Following variant filtering, 7046 total variants were identified (~79/person, 644 Tier-1, 6402 Tier-2), 161 among 56 ACMG-reportable genes (~1.8/person, 13 Tier-1, 148 Tier-2), and 115 among 57 cancer-predisposition genes (~1.3/person, 3 Tier-1, 112 Tier-2). The number of variants across 57 cancer-predisposition genes did not differentiate individuals with/without invasive cancer history (P > 0.19). Evaluating genotype-phenotype correlations across the exome, 202(3%) of 7046 filtered variants had some evidence for phenotypic correlation in medical records, while 3710(53%) variants had no phenotypic correlation. The phenotype associated with the remaining 44% could not be assessed from a typical medical record review. These data highlight significant continued challenges in the ability to extract medically meaningful predictive results from WES.
Preview · Article · Aug 2015 · Frontiers in Genetics
[Show abstract][Hide abstract] ABSTRACT: Background & aims:
In gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-α (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1.
We studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA(+) subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit(+/copGFP), Pdgfra(+/eGFP), and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation.
KIT and PDGFRA were coexpressed in 3%-5% of mouse ICC, 35%-44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT(+)ETV1(+)PDGFRA(+) GIST cells (50% maximal inhibitory concentration = 5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (50% maximal inhibitory concentration >230 nM). Crenolanib inhibited phosphorylation of PDGFRA and PDGFRB, but not KIT. However, Kit overexpression sensitized mouse ICC precursors to crenolanib. ETV1 knockdown reduced KIT expression and GIST proliferation. Crenolanib down-regulated ETV1 by inhibiting extracellular-signal-regulated kinase (ERK)-dependent stabilization of ETV1 protein and also reduced expression of KIT and PDGFRA.
In KIT-mutant GIST, inhibition of PDGFRA disrupts a KIT-ERK-ETV1-KIT signaling loop by inhibiting ERK activation. The PDGFRA inhibitor crenolanib might be used to treat patients with imatinib-resistant, KIT-mutant GIST.