Farrugia, Gianrico

AbstractBackgroundAutoimmunity may play a role in the pathogenesis of gastroparesis in a subset of patients. Antinuclear antibody (ANA) testing is often used to screen for autoimmune disorders.Aims1) Determine prevalence of a positive ANA in patients with gastroparesis; 2) Describe characteristics of idiopathic gastroparesis patients with positive ANA.MethodsPatients were assessed with gastric emptying scintigraphy (GES), symptom assessment via Patient Assessment of Upper GI Symptoms [PAGI‐SYM], and blood tests—ANA, erythrocyte sedimentation rate (ESR), C‐reactive protein (CRP).ResultsPositive ANA was seen in 148 of 893 (17%) patients with gastroparesis, being similar in idiopathic (16% of 536 patients), T1DM (16% of 162), T2DM (18% of 147), and postfundoplication (19% of 48 patients) gastroparesis. Among 536 patients with idiopathic gastroparesis, ANA titer 1:40–1:80 was seen in 33 (6%) patients, 1:160–1:320 in 36 (7%) patients, and ≥1:640 in 17 (3%) patients. Increasing ANA titer was associated with female gender (p = 0.05), Hispanic ethnicity (p = 0.02), comorbid rheumatoid arthritis (p = 0.02), systemic sclerosis (p = 0.004), and elevated ESR (p = 0.007). ANA positivity was associated with lower total GCSI (p = 0.007) and lower nausea/vomiting subscale (p = 0.0005), but not related to gastric emptying.ConclusionsThe prevalence of a positive ANA in patients with gastroparesis was high at ~17% and did not differ significantly based on etiology. In idiopathic patients, ANA positivity was associated with rheumatoid arthritis, systemic sclerosis, and elevated ESR. ANA‐positive gastroparesis represents a subset who often have other autoimmune symptoms or disorders, but less severe nausea and vomiting.

Background: Genes encoding sodium channel interacting proteins (ChIPs) have been identified as novel long QT syndrome (LQTS)-susceptibility genes. Approximately 25% of patients with LQTS remain genotype negative. ChIPs comprising the sodium channel macromolecular complex represent attractive candidates for disease susceptibility. Previously, in human jejunal circular smooth muscle, we demonstrated that syntrophin γ2 regulates sodium channel gating by a PDZ-domain interaction involving the C-terminus. In the heart, syntrophin α is more highly expressed than γ2, therefore, syntrophin α was evaluated as a possible LQTS-susceptibility ChIP. Methods: Comprehensive open reading frame/splice mutational analysis of syntrophin α was performed using PCR, DHPLC, and direct DNA sequencing on a cohort of 50 unrelated patients with LQT1–10 negative/phenotype positive LQTS (34 females; average age at diagnosis 26 ± 16 years; average QTc 531 ± 60.4 ms; QTc range 480 –759 ms). Mutations were engineered by site-directed mutagenesis and transiently expressed in HEK293 cells containing the stably-expressed SCN5A- encoded sodium channel α subunit (hNaV1.5). Results: Analysis of syntrophin α, encoded by SNTA1 , revealed 3 missense mutations in 2 patients (4%). A female diagnosed with LQTS at 13 years (QTc, 480 ms) with family history of autopsy-negative unexplained sudden death, was compound heterozygous for P74L/A257G while a male, diagnosed with LQTS at 18 years following syncopal episodes (QTc, 529 ms), hosted the A390V mutation. These mutations were absent in 600 reference alleles and A257G and A390V involved highly conserved residues. Functional studies revealed a marked leftward shift in sodium current activation for A257G and a significant rightward shift in channel inactivation for A390V-SNTA1. Both kinetic perturbations precipitated a ``gain-of-function” increase in the sodium channel window current. Conclusion: We provide the sentinel report of SNTA1 as a novel LQTS-susceptibility gene with a putative pathogenic mechanism involving a secondary ``gain-of-function” in the NaV1.5 sodium channel. Thus, CAV3-LQTS (LQT9), SCN4B-LQTS (LQT10), and now SNTA1-LQTS represent functional homologues with LQT3-like perturbations.

NBCe1 is an electrogenic Na + bicarbonate cotransporter expressed as three isoforms. NBCe1A is mainly found in the kidney (100% activity), NBCe1B is a general isoform (~25% activity), and NBCe1C is found in the nervous system (20% activity). This project was to characterize mice with loss of NBCe1 in the kidney and urogenital tract and to test isoform activity ± IRBIT. Previous work has shown that NBCe1‐B is activated when IRBIT is present and interacts with the N‐terminus (Nt). Even though NBCe1C shares this Nt, the effects of IRBIT on human NBCe1C are unknown. Interstitial Cells of Cajal (ICC)pacemaker cells in the gastrointestinal tract ( c‐kit + ) have NBCe1C, IRBIT and require bicarbonate for normal gut activity. Developmentally similar ICC cells are found in the urogenital tract; however, NBCe1C locations within the kidney and urogenital tract are unknown. Using c‐kit ‐copGFP and NBCe1A‐knockout mice, kidneys, ureters, prostate, bladder, and urethra were dissected and analyzed via direct imaging, sectioning, and immunofluorescence. Oocytes injected with NBCe1C ± IRBIT were voltage clamped to determine the cotransporter's activity. “Strings” of c‐kit + ‐cells were found in the copGFP & nbce1A kidneys. In nbce1 (+/−) ‐(Het) mice (Figure), prostate ducts appeared enlarged, and in nbce1A − / − kidney, c‐kit protein was broadly expressed, showing cluster colocalization with NBCe1C. NBCe1C is not obviously expressed in WT or copGFP mice. Electrophysiology data showed that IRBIT activates NBCe1C by ~10‐fold. As c‐kit is also a marker of stem cells, NBCe1C/c‐kit association and c‐kit increase in nbce1A − / − mice, could suggest that NBCe1A loss elicits kidney repair itself (proliferation) and causes expression of other NBCe1‐isoforms. Support or Funding Information R25‐DK101405, R01‐DK057061, U54‐DK100227 (Mayo) and U54‐DK104310 (UW‐Madison) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

BACKGROUND Enteroendocrine cells (EECs) in the gastrointestinal (GI) epithelium are specialized sensors of luminal chemicals and forces. Upon stimulation, EECs release a variety of neurotransmitters and hormones, but cellular mechanisms of excitation‐secretion coupling are poorly understood. We recently discovered that a population of EECs expresses the mechanosensitive ion channel Piezo2, which is necessary for force‐dependent intracellular Ca2+ increase and serotonin release. However, the molecular mechanism that links Piezo2 and serotonin release is unknown. In other sensory systems, the Cacna1h ‐encoded t‐type voltage‐gated calcium channel Cav3.2 is important in mechanotransduction for linking mechanical activation with neurotransmitter release. AIM to determine if Cav3.2 is expressed and functionally relevant in EECs. METHODS Quantitative RT‐PCR for Cacna1h mRNA expression was evaluated in colon epithelium of control and NeuroD1 Cre x RiboTag mice, in which actively transcribed mRNA specifically from EECs is tagged with hemagglutinin (HA). NeuroD1 Cre x tdTomato colon cross‐sections were fixed and immunolabeled with anti‐Cav3.2 antibody. Single primary NeuroD1 cre x GCaMP5 EECs were mechanically stimulated and Cav3.2 role in mechanotransduction was tested by pharmacology (Ca2+ free, nickel, mibefradil, ω‐agatoxin IVA) and Cacna1h siRNA knockdown. RESULTS Murine colon epithelium expressed Cav3.2 mRNA. In NeuroD1 Cre x RiboTag mouse, HA co‐localized with EEC marker Chromogranin A (Chga). NeuroD1+ EEC ribosomal trapped mRNA was enriched for markers of epithelium (Vil1), EEC (Chga), and enriched 7.0‐fold for Cav3.2 (n=3, p<0.05 compared to whole epithelium). Immunohistochemistry in NeuroD1 Cre x tdTomato showed that Cav3.2 co‐localized specifically with tdTomato (NeuroD1+) EECs. In primary NeuroD1+ EECs, Cav3.2 inhibition by all pharmacologic agents and siRNA knockdown inhibited mechanically induced Ca2+ response (96±1% for Ca2+ free, 71±9% nickel, 61±6% mibefradil, 80±4% ω‐agatoxin IVA and 96±1% Cav3.2 siRNA, n=3–18, p<0.05 compared to control untreated cells or cells treated with non‐targeted siRNA). CONCLUSIONS Cav3.2 is densely and specifically expressed in mouse colon EECs and it is important for regulating Ca2+ in EEC mechanotransduction. Future studies will determine how Cav3.2 channels contribute to EEC electrical excitability, excitation‐secretion coupling, and ultimately EEC Cav3.2 roles in GI physiology. Support or Funding Information Supported by NIH DK106456, NIH DK084567, and AGA RSA This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Electrical slow wave activity, generated by interstitial cells of Cajal (ICC), is essential for efficient gastrointestinal (GI) transit. Electrophysiology of slow waves has been extensively studied; inclusion of bicarbonate (HCO 3 − ) in extracellular solutions is required for recording normal slow wave activity. HCO 3 − transport contributes to regulation of intracellular pH, which has multiple consequences for normal cellular function. Previous transcriptional profiling found that the Na + / HCO 3 − cotransporter (NBCe1/Slc4a4) is enriched in pacemaker myenteric ICC (ICC‐MY) compared to non pacemaker ICC in the deep muscular plexus (ICC‐DMP) of the small intestine. Our aim was to determine the distribution and identity of Slc4a4/NBCe1 variants in ICC in the GI tract. NBCe1 immunoreactivity (IR) was detected by fluorescence labeling of parafomaldehyde‐fixed cross sections and whole mounts of adult mouse small intestine, colon and stomach using primary antibodies directed against a 329 amino acid sequence in the N terminus of the NBCe1 protein. Labeling of Kit receptor tyrosine kinase with a goat anti‐Kit polyclonal antibody was used to detect ICC. Slc4a4 knockout (KO) mice were bred from heterozygous founders. Slc4a4 fl/fl mice were bred with ETV1 CreERT2/− mice to allow targeted, conditional KO of Slc4a4 in ICC in response to tamoxifen treatment (130 mg/Kg). mRNA transcripts for Slc4a4 in Kit expressing cells were isolated from Kit‐ribotag mice, which were generated by breeding Kit creERT2 with Rpl22 tm1.1Psam mice. The abundance of specific Slc4a4 transcripts was measured by quantitative real time PCR (qRT‐PCR). Green fluorescent protein (GFP)‐labeled ICC were dissociated from the small intestine of neonatal Kit copGFP mice and RNA expression levels were determined using the Single Cell‐to‐CT™ qRT‐PCR Kit from Ambion. NBCe1‐IR was absent in all cells in Slc4a4‐KO tissues but co‐localized with Kit in ICC‐MY, but not in ICC‐DMP, in small intestine of wild type mice. In the proximal and distal colon, pacemaker submucosal ICC (ICC‐SMP) but not intramuscular ICC (ICC‐IM) or ICC‐MY showed NBCe1‐IR. In the corpus and antrum of the stomach NBCe1‐IR was detected in ICC‐MY but not in ICC‐IM. NBCe1‐IR did not co‐localize with Kit in the gastric fundus. mRNA for variant C, but not variants A or B of Slc4a4, was detected in small intestinal ICC‐MY. Reduced NBCe1‐IR was restricted to small intestinal ICC‐MY of Slc4a4 conditional KO mice. NBCe1 is enriched in subtypes of ICC responsible for pacemaker activity in all regions of the GI tract indicating a role for this transporter in slow wave generation. The presence of the C variant of Slc4a4 in ICC‐MY suggests dynamic regulation of NBCe1 activity in ICC by the I P 3 R b inding protein released with i nositol 1, 4, 5‐ t risphosphate (IRBIT). Support or Funding Information Supported by NIH R01 DK57061. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background Microbial conversion of dietary substrate to bioactive molecules represents a potential regulatory mechanism by which gut microbes can alter intestinal physiology. Tryptamine, a monoamine structurally similar to serotonin (5‐HT), can be generated via decarboxylation of tryptophan by the gut bacteria but its physiological effect is not known. Aim To determine the effect of bacterial metabolite tryptamine on colonic secretion. Methods We mounted proximal colon mucosa‐submucosa preparations from germ free (GF), humanized (HM; ex‐GF colonized with human gut bacteria for 4–5 weeks), conventionally raised WT and 5‐HT4 receptor knockout (5‐HT4R −/− ) male mice in the Ussing chamber. The change in short circuit current (ΔIsc) was determined in response to application of increasing concentrations of tryptamine (3μM – 3mM) on the submucosal (basolateral) side either alone or in the presence of 5‐HT3R or 5‐HT4R antagonist, ondansetron (100nM) and GR‐113808 (30nM) respectively, with and without TTX (500nM). Colonoids (3‐dimensional epithelial organoids derived from colon) from GF and HM were used for swelling assay and ELISA based cAMP measurement. Results Tryptamine application significantly increased Isc in both GF and HM mice (n=6, P < 0.05, two‐way ANOVA with Bonferroni post‐hoc). Tryptamine mediated ΔIsc response was not significantly affected by amiloride, an inhibitor of epithelial sodium channels and Na + /H + exchangers, which are primarily responsible for absorption but was significantly decreased by 5‐Nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB; anion blocker), a calcium‐sensitive chloride channel and sodium bicarbonate exchanger inhibitor responsible for anionic secretion in both GF and HM mice (n=4, P < 0.05, two‐way ANOVA with Bonferroni post‐hoc). Tryptamine evoked ΔIsc was not inhibited by 5‐HT3R (n=4, P > 0.05) but completely blocked by 5‐HT4R antagonist and was absent in 5HT4R knock out mice suggesting that the effect of tryptamine is mediated via 5HT4R. The increase in Isc in response to tryptamine and its inhibition by 5‐HT4R antagonist was also seen in presence of TTX (neuronal blocker; n=4, one‐way ANOVA , P < 0.05) suggesting that the epithelial 5‐HT4R alone can mediate the effect of tryptamine. Application of tryptamine (1mM) for 30 minutes led to a significant increase in the surface area of fluorescein labelled colonoids (n=5, one‐way ANOVA , P < 0.05), similar to forskolin, when compared to vehicle treated and 5HT4R antagonist pre‐treated GF and HM colonoids. Colonoids incubated with tryptamine (1mM) for 60 minutes had significantly higher cAMP levels (normalized to forskolin) compared to vehicle treated GF and HM colonoids, confirming that tryptamine activates 5‐HT4 GPCR (n=3, student's t‐test, P < 0.05). Conclusion Tryptamine, a tryptophan‐derived microbial metabolite, increases colonic secretion via 5HT4 GPCR activation. The identification of a bacterial metabolite activating a host GPCR to alter GI function represents an important advance in the field, and provides a distinct example of small molecule mediated host‐microbe interaction. Engineered probiotics producing tryptamine could be used as therapeutics in diseases associated with constipation. Support or Funding Information NIH DK111850, DK114007 (PCK) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

INTRODUCTION Intestinal enterochromaffin (EC) cells are the specialized epithelial mechanosensors of the gastrointestinal (GI) epithelium and are functionally and developmentally like Merkel cells in the skin. EC cells release serotonin (5‐HT) in response to mechanical stimuli. Recently, we discovered that like Merkel cells, human and mouse EC cells express the mechanosensitive (MS) ion channel Piezo2. However, little is known about the functional properties of this channel in primary EC cells. AIM To determine the role of Piezo2 in primary EC cells. METHODS The enteroendocrine cell transcription factor NeuroD1 was used to mark the late stage of EC cell development and generate NeuroD1‐cre::GCaMP5/tdTomato mouse to identify EC cells and examine their Ca 2+ dynamics. We examined colocalization of tdTomato with 5‐HT and Piezo2 by immunohistochemistry. We established and used primary colon epithelium cultures for whole‐cell patch clamp and Ca 2+ imaging experiments and mechanically stimulated by membrane displacement with a piezoelectrically driven glass probe or shear flow. RESULTS Immunostaining showed no tdTomato labeling outside of the GI epithelium, and epithelial tdTomato cells were 5‐HT+ and Piezo2+. Mechanical stimulation of primary EC cells by membrane displacement produced inward MS currents (20.8±4.9 pA/pF, C m =3.5 pF, n=6) with fast activation and inactivation kinetics (τ inact 11.1±2.8 ms), steep mechanosensitivity (Boltzmann function fit with a mid‐point of 3.46±0.80 μm and slope 0.79±0.27 μm) and a linear current‐voltage relationship (slope 0.61±0.17 pA/mV, x‐intercept −4.26±1.34 mV, n=5). Shear flow (20 s) transiently increased intracellular calcium ((Ca 2+ ) i ) (2.15±0.5 ΔF/F0, upstroke 3–5 s, return to baseline in 60 s, n=12). MS currents were inhibited by the MS ion channel blocker Gd 3+ (20.8±4.9 pA/pF versus 5.3±1.9 pA/pF), Piezo channel blocker GsMTx‐4 (20.8±4.9 pA/pF versus 2.5±0.9 pA/pF) and Piezo2 siRNA (20.8±4.9 pA/pF versus 1.1±0.3 pA/pF), but not non‐targeted (NT) siRNA (20.8±4.9 pA/pF versus 16.0±5.2 pA/pF and) (n=4–6 for each condition, p <0.05 for all except NT siRNA). Similarly, the (Ca 2+ ) i responses to force were inhibited by Gd 3+ (2.28±0.7 ΔF/F0 to 0.09±0.05 ΔF/F0), GsMTx‐4 (2.0±0.6 ΔF/F0 to 0.3±0.1 ΔF/F0), Piezo2 siRNA (2.15±0.5 ΔF/F0 to 0.08±0.05 ΔF/F0), but not NT siRNA (2.15±0.5 ΔF/F0 to 2.7±0.3 ΔF/F0) (n=4–6, p <0.05 for all except NT siRNA). CONCLUSIONS In the mouse epithelium, Piezo2+ EC cells were specifically marked by NeuroD1. Primary colonic NeuroD1+ cells had inward and non‐rectifying MS currents that could be blocked by Gd 3+ , GsMTx‐4 and Piezo2 siRNA. Similarly, shear flow evoked changes in (Ca 2+ ) i that were also blocked by Piezo2 inhibitors and dependent on extracellular Ca 2+ . These results suggest that the MS ion channel Piezo2 acts as the primary EC cell mechanosensor, whose activation by force leads to MS ionic current and (Ca 2+ ) i increase that may mediate mechanically‐induced 5‐HT release. Support or Funding Information This work was supported by NIH K08 DK106456, AGA Research Scholar Award, Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567) and DK52766. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Ano1 is a Ca 2+ activated Cl − channel expressed in several proliferating cell types including various epithelia, gastrointestinal stromal tumors, interstitial cells of Cajal (ICC) and CFPAC‐1 cells. The aim of this study was to determine if Ano1 regulates proliferation. ICC from Ano1 (−/−) and Ano1 (+/+) mice were cultured with Cl − channel inhibitors or in low Cl − media. CFPAC‐1 cells grown in low Cl − media were pulsed with EdU for 1 h. Incorporation was measured by flow cytometry. For immunoblots on mouse jejunum, antibodies to pRb, phospho(Ser773)‐pRb and GAPDH were used. Fewer proliferating ICC were detected in Ano1 (−/−) cultures (15 ± 3.5% Ki67 + ICC vs 25 ± 4% in Ano1 (+/+) , mean ± SEM, n = 4, p < 0.05). Cl − channel inhibitors DIDS (10 μM), niflumate (10 μM) and tamoxifen (1 μM) decreased ICC proliferation by 53, 53 and 59%, respectively ( p < 0.05). This effect was reduced in cultures from Ano1 (−/−) mice (29% and 19 % decrease, 22% increase, p > 0.05). Fewer proliferating ICC and CFPAC‐1 cells were detected in low Cl − media (% EdU + ICC: 120 mM; 19.8 ± 5.3, 12 mM; 8.5 ± 3.2, n = 4, and % EdU + CFPAC‐1: 120 mM; 29.5, 12 mM; 14.3 p < 0.05). Proportionally less Ser773‐phosphorylated pRb was detected in jejunum from Ano1 (−/−) mice (Ano1 (+/+) ; 5.98 ± 0.78, Ano1 (−/−) ; 3.60 ± 0.49, mean ± SEM, n = 7, p < 0.05). In conclusion, Ano1 regulates proliferation possibly by promoting the G 1 /S transition of the cell cycle. Support: DK57061 and DK68055.

Gut microbes play a significant role in development and maintenance of intestinal functions, but can contribute to pathogenesis of gastrointestinal (GI) disorders when host‐microbiome relationship is perturbed. In this study we sought to examine how constipation affects microbiome, and how these alterations in turn affect the host. Adult C57Bl/6 mice were treated with 0.1% Loperamide in drinking water for 7 days to induce constipation. Loperamide‐treated mice had increased GI transit time, indicating constipation. Bacterial DNA analysis by HiSeq sequencing showed decreased Firmicutes and increased Bacteroidetes in constipated mice. Microbiome metabolic potential was measured with tetrazolium dye assay and showed shift toward increased sugar metabolism in Loperamide‐treated mice. Microbial metabolites were measured by GC‐MS, and showed lower butyrate concentration in constipated mice. To determine constipation effects on the host, we introduced microbiota from constipated donors to germ‐free recipients. These mice had delayed GI transit time. To determine mechanism for delayed GI transit we analyzed colon tissues by qRT‐PCR and western blotting analysis, and determined lower expression of nNos mRNA and protein levels in these mice. Overall, these results suggest that delayed GI transit shifts microbiome taxonomic and metabolic profile, and these changes further contribute to development of constipation. Grant Funding Source : NIH NIDDK T32 DK07074

Objective Haemorrhoidal disease (HEM) affects a large and silently suffering fraction of the population but its aetiology, including suspected genetic predisposition, is poorly understood. We report the first genome-wide association study (GWAS) meta-analysis to identify genetic risk factors for HEM to date. Design We conducted a GWAS meta-analysis of 218 920 patients with HEM and 725 213 controls of European ancestry. Using GWAS summary statistics, we performed multiple genetic correlation analyses between HEM and other traits as well as calculated HEM polygenic risk scores (PRS) and evaluated their translational potential in independent datasets. Using functional annotation of GWAS results, we identified HEM candidate genes, which differential expression and coexpression in HEM tissues were evaluated employing RNA-seq analyses. The localisation of expressed proteins at selected loci was investigated by immunohistochemistry. Results We demonstrate modest heritability and genetic correlation of HEM with several other diseases from the GI, neuroaffective and cardiovascular domains. HEM PRS validated in 180 435 individuals from independent datasets allowed the identification of those at risk and correlated with younger age of onset and recurrent surgery. We identified 102 independent HEM risk loci harbouring genes whose expression is enriched in blood vessels and GI tissues, and in pathways associated with smooth muscles, epithelial and endothelial development and morphogenesis. Network transcriptomic analyses highlighted HEM gene coexpression modules that are relevant to the development and integrity of the musculoskeletal and epidermal systems, and the organisation of the extracellular matrix. Conclusion HEM has a genetic component that predisposes to smooth muscle, epithelial and connective tissue dysfunction.