Guanylate cyclase-C/cGMP: an emerging pathway in the regulation of visceral pain

Activation of guanylate cyclase-C (GC-C) expressed predominantly on intestinal epithelial cells by guanylin, uroguanylin or the closely related GC-C agonist peptide, linaclotide, stimulates generation and release of cyclic guanosine-3’,5’-monophosphate (cGMP). Evidence that the visceral analgesic effects of linaclotide are mediated by a novel, GC-C-dependent peripheral sensory mechanism was first demonstrated in animal models of visceral pain. Subsequent studies with uroguanylin or linaclotide have confirmed the activation of a GC-C/cGMP pathway leading to increased submucosal cGMP mediated by cGMP efflux pumps, which modulates intestinal nociceptor function resulting in peripheral analgesia. These effects can be reproduced by the addition of exogenous cGMP and support a role for GC-C/cGMP signaling in the regulation of visceral sensation, a physiological function that has not previously been linked to the GC-C/cGMP pathway. Notably, targeting the GC-C/cGMP pathway for treatment of gastrointestinal pain and abdominal sensory symptoms has now been validated in the clinic. In 2012, linaclotide was approved in the United States and European Union for the treatment of adult patients with irritable bowel syndrome with constipation

Genetically engineered probiotic E. coli Nissle to consume amino acids associated with orphan metabolic diseases

Orphan metabolic diseases are rare genetic defects that interfere with metabolism due to ineffective or missing enzymes. Two of them, Phenylketonuria (PKU) and Maple Syrup Urine Disease (MSUD) are defined by accumulation of amino acids to toxic levels due to defective metabolism of protein break down products. PKU is caused by a defect in the gene encoding phenylalanine hydroxylase (PAH). MSUD is caused by a defect in a multi-enzyme complex found in mitochondria called branched chain ɑ-ketoacid dehydrogenase “BCKDH”. Without the activity of these enzymes, the amino acid phenylalanine (Phe) in the case of PKU or the branched-chain amino acids leucine (Leu), isoleucine and valine for MSUD build up to neurotoxic levels in the blood and brain, leading to neurological deficits. Current treatment options focus on dietary protein restriction, are insufficient and, unfortunately, can lead to a failure to thrive. Lifelong compliance with a prescription diet is also a concern. We have genetically engineered Nissle, a probiotic strain of E. coli, to reduce serum phenylalanine and leucine levels in patients with PKU or MSUD; preclinical data supporting the activity of these strains are described. Please click Additional Files below to see the full abstract

The Effect of Psyllium Husk on Intestinal Microbiota in Constipated Patients and Healthy Controls

Psyllium is a widely used treatment for constipation. It traps water in the intestine increasing stool water, easing defaecation and altering the colonic environment. We aimed to assess the impact of psyllium on faecal microbiota, whose key role in gut physiology is being increasingly recognised. We performed two randomised, placebo-controlled, double-blinded trials comparing 7 days of psyllium with a placebo (maltodextrin) in 8 healthy volunteers and 16 constipated patients respectively. We measured the patients’ gastrointestnal (GI) transit, faecal water content, short-chain fatty acid (SCFA) and the stool microbiota composition. While psyllium supplement had a small but significant effect on the microbial composition of healthy adults (increasing Veillonella and decreasing Subdoligranulum), in constipated subjects there were greater effects on the microbial composition (increased Lachnospira, Faecalibacterium, Phascolarctobacterium, Veillonella and Sutterella and decreased uncultured Coriobacteria and Christensenella) and alterations in the levels of acetate and propionate. We found several taxa to be associated with altered GI transit, SCFAs and faecal water content in these patients. Significant increases in three genera known to produce butyrate, Lachnospira, Roseburia and Faecalibacterium, correlated with increased faecal water. In summary, psyllium supplementation increased stool water and this was associated with significant changes in microbiota, most marked in constipated patients

Genetically engineered E. coli Nissle attenuates hyperammonemia and prevents memory impairment in bile‐duct ligated rats

Hyperammonemia associated with chronic liver disease (CLD) is implicated in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia production that contributes to hyperammonemia in CLD and HE and remains the primary therapeutic target for lowering hyperammonemia. As an ammonia‐lowering strategy, Escherichia coli Nissle 1917 bacterium was genetically modified to consume and convert ammonia to arginine (S‐ARG). S‐ARG was further modified to additionally synthesize butyrate (S‐ARG+BUT). Both strains were evaluated in bile‐duct ligated (BDL) rats; experimental model of CLD and HE. Methods One‐week post‐surgery, BDLs received non‐modified EcN (EcN), S‐ARG, S‐ARG+BUT (3x1011 CFU/day) or vehicle until sacrifice at 3‐ or 5‐weeks. Plasma (ammonia/pro‐inflammatory/liver‐function), liver fibrosis (hydroxyproline), liver mRNA (pro‐inflammatory/fibrogenic/anti‐apoptotic) and colon mRNA (pro‐inflammatory) biomarkers were measured post‐sacrifice. Memory, motor‐coordination, muscle‐strength, and locomotion were assessed at 5‐weeks. Results In BDL‐Veh rats, hyperammonemia developed at 3‐ and further increased at 5‐weeks. This rise was prevented by S‐ARG and S‐ARG+BUT, whereas EcN was ineffective. Memory impairment was prevented only in S‐ARG+BUT vs BDL‐Veh. Systemic inflammation (IL‐10/MCP‐1/endotoxin) increased at 3‐ and 5‐weeks in BDL‐Veh. S‐ARG+BUT attenuated inflammation at both timepoints (except 5‐week endotoxin) vs BDL‐Veh, whereas S‐ARG only attenuated IP‐10 and MCP‐1 at 3‐weeks. Circulating (ALT/AST/ALP/GGT/albumin/bilirubin) and gene expression liver‐function markers (IL‐10/IL‐6/IL‐1β/TGF‐β/α‐SMA/collagen‐1α1/Bcl‐2) were not normalized by either strain. Colonic mRNA (TNF‐α/IL‐1β/occludin) markers were attenuated by synthetic strains at both timepoints vs BDL‐Veh. Conclusion S‐ARG and S‐ARG+BUT attenuated hyperammonemia, with S‐ARG+BUT additional memory protection likely due to greater anti‐inflammatory effect. These innovative strategies, particularly S‐ARG+BUT, have potential to prevent HE

The Grizzly, November 25, 1996

Honors Pilot Program a Success • Pass-Fail Forum Set for Dec. 4 • Opinion: What About Easter?; It Hurts; Jesus is Dead?; Thanksgiving; Is Ursinus a Disposable Society?; Never-Never Land • Final Exam Schedule • Bears Suffer 31-24 Defeat in NCAA Playoffs • Men\u27s Basketball Opens with an Overtime Win • Bears Place Nine On All-Conference Team • Wrestling Opens with Two Winshttps://digitalcommons.ursinus.edu/grizzlynews/1393/thumbnail.jp

The Grizzly, December 9, 1996

34 Students Arrested in One Month • College Choir Performs Messiah • Dr. Lentz Departing • Opinion: A Question of Security, Take Two; Ending the Bulletin Crusade?; Objects in the Rear View Mirror; Minority Tours Lack Diversity; Crossing the Bridge • An Inconclusive Wrap-Up • Men and Women Swimmers Split • Men\u27s Basketball Team Hits Rough Waters • Coach Gilbert Honored by AFCA • Lady Bears Off to 4-2 Start • Eight Bears Selected by ECAC • Torsone Starts at Petrofes Invitationalhttps://digitalcommons.ursinus.edu/grizzlynews/1394/thumbnail.jp

The Grizzly, October 28, 1996

Alice Parker Conducts College Choir • A Question on Diversity • Security Flash • New Curriculum for Comm. Arts • Opinions: More on the Gun Control Debate; The Debates of Nothingness; How Open Are You? Voting for Dole in \u2796; I\u27m Voting for Dennis Miller • Letters from Ireland • University of Pennsylvania Anthropologist to Speak on Maya and Aztecs • Spotlight: Karl Yergey • Plugging-In to the Benefits of E-mail • Looking for a few Good Dangerous Minds: Education Club Re-activates • Field Hockey Drops One To American • Women\u27s Soccer Nets First Conference Win • Volleyball Loses Two • Men\u27s Soccer Defeats Dickinson 2-0 • Kings Point Trips Up Ursinushttps://digitalcommons.ursinus.edu/grizzlynews/1389/thumbnail.jp

Engineering and manufacturing of probiotic E. Coli to treat metabolic disorder

The fields of synthetic biology and microbiome research developed greatly over the last decade. The convergence of those two disciplines is now enabling the development of new therapeutic strategies, using engineered microbes that operate from within the gut as living medicines. Inborn errors of metabolism represent candidate diseases for these therapeutics, particularly those disorders where a toxic metabolite causing a syndrome is also present in the intestinal lumen. Phenylketonuria (PKU), a rare inherited disease caused by a defect in phenylalanine hydroxylase (PAH) activity, is one such disease and is characterized by the accumulation of systemic phenylalanine (Phe) that can lead to severe neurological deficits unless patients are placed on a strict low-Phe diet. As an alternative treatment, Escherichia coli Nissle (EcN), a well-characterized probiotic, was genetically modified to efficiently import and degrade Phe (SYNB1618). The coupled expression of a Phe transporter with a Phe ammonia lyase (PAL) allows rapid conversion of Phe into trans-cinnamic acid (TCA) in vitro, which is then further metabolized by the host to hippuric acid (HA) and excreted in the urine. Experiments conducted in the enu2-/- PKU mouse model showed that the oral administration of SYNB1618 is able to significantly reduce blood Phe levels triggered by subcutaneous Phe injection. Decreases in circulating Phe levels were associated with proportional increases in urinary HA, confirming that Phe metabolism was caused by the engineered pathway in SYNB1618. Subsequent studies have shown that SYNB1618 is similarly operative in a non-human primate model, providing a translational link to inform future human clinical studies. Consistent with preclinical studies, recent Phase 1/2a clinical data demonstrate that oral administration of SYNB1618 resulted in significant dose-dependent production of biomarkers specifically associated with SYNB1618 activity, demonstrating proof-of-mechanism of this cell therapy

Demonstration of differences in colonic volumes, transit, chyme consistency and response to psyllium between healthy and constipated subjects using magnetic resonance imaging

Background: In functional gastrointestinal disorders a lack of objective biomarkers limits evaluation of underlying mechanisms. We aimed to demonstrate the utility of Magnetic Resonance Imaging (MRI) for this task using psyllium, an effective constipation treatment, in patients and controls. Methods: Two crossover studies: 1) adults without constipation (controls, n=9) took three treatments in randomised order for 6 days - maltodextrin (placebo), psyllium 3.5g t.d.s and 7g t.d.s.; 2) adults with chronic constipation (patients, n=20) took placebo and psyllium 7g t.d.s. for 6 days. MRI was performed fasting and postprandially on day 6. Measurements included small bowel and ascending colon water content, colonic volume, transit time and MR relaxometry (T1, T2) to assess colonic chyme. Stool water percentage was measured. Results: 7g psyllium t.d.s. increased fasting colonic volumes in controls from median 372mL (IQR 284-601) to 578 mL (IQR 510-882), and in patients from median 831mL (IQR 745–934) to 1104mL (847–1316),

Engineering of probiotic E.coli to treat metabolic disorders

The fields of synthetic biology and microbiome research developed greatly over the last decade. The convergence of those two disciplines is now enabling the development of new therapeutic strategies, using engineered microbes that operate from within the gut as living medicines. Inborn errors of metabolism represent candidate diseases for these therapeutics, particularly those disorders where a toxic metabolite causing a syndrome is also present in the intestinal lumen. Phenylketonuria (PKU), a rare inherited disease caused by a defect in phenylalanine hydroxylase (PAH) activity, is one such disease and is characterized by the accumulation of systemic phenylalanine (Phe) that can lead to severe neurological deficits unless patients are placed on a strict low-Phe diet. As an alternative treatment, Escherichia coli Nissle (EcN), a well-characterized probiotic, was genetically modified to efficiently import and degrade Phe (SYN-PKU). The coupled expression of a Phe transporter with a Phe ammonia lyase (PAL) allows rapid conversion of Phe into trans-cinnamic acid (TCA) in vitro, which is then further metabolized by the host to hippuric acid (HA) and excreted in the urine. Experiments conducted in the enu2-/- PKU mouse model showed that the oral administration of SYN-PKU is able to significantly reduce blood Phe levels triggered by subcutaneous Phe injection. Decreases in circulating Phe levels were associated with proportional increases in urinary HA, confirming that Phe metabolism was caused by the engineered pathway in SYN-PKU. Subsequent studies have shown that SYN-PKU is similarly operative in a non-human primate model, providing a translational link to inform future human clinical studies. In addition to SYN-PKU, a second EcN strain was genetically engineered to rapidly import and degrade branched-chain amino acids (BCAAs) for the treatment of maple syrup urine disease (SYN-MSUD). MSUD, similar to PKU, is a rare genetic disorder caused by a defect in branched-chain ketoacid dehydrogenase activity leading to the toxic accumulation of BCAAs, particularly leucine, and their ketoacid derivatives. The controlled expression in SYN-MSUD of two BCAA transporters, a leucine dehydrogenase, a ketoacid decarboxylase and an alcohol dehydrogenase, result in the efficient degradation of BCAAs into branched-chain alcohols. In a mouse model of MSUD, the oral delivery of SYN-MSUD suppressed the increase in blood BCAAs level induced by a high-protein challenge and prevented the associated moribund phenotype, as measured by locomotor activity. In conclusion, the therapeutic effects observed with SYN-PKU and SYN-MSUD in pre-clinical studies support the further evaluation of engineered microbes as promising approaches for serious inborn errors of metabolism