Search

Background Idiopathic achalasia is a disease of the esophagus causing impaired peristalsis and absent lower esophageal sphincter relaxation. The etiopathogenesis of the disease is unclear but most histopathologic studies from surgical resections show an absence of nitric oxide-producing neurons in the myenteric plexus. The peroral endoscopic myotomy (POEM) procedure has evolved in the last decade to treat achalasia and has provided a unique way to sample diseased tissue from an otherwise inaccessible tissue compartment. Aims To study the effects of achalasia on innervation of smooth muscle and smooth muscle phenotype. Methods Patients with a diagnosis of achalasia based on high resolution manometry undergoing a POEM at Kingston general hospital were approached for enrollment between June 2017 to September 2018. Demographic information including age, symptom duration, previous treatments, and Eckhardt score was collected. Intraoperatively, biopsies of the circular smooth muscle (CSM) layer were taken at the proximal and distal extents of the myotomy and placed in formalin. Tissue was embedded in wax, sectioned and stained using immunocytochemistry (ICC) for neuronal, axonal and smooth muscle cell markers. This was compared to control tissue from patients undergoing gastroesophagectomy for adjacent malignancy. Results Control tissue from 3 separate esophagectomies were obtained. Samples were obtained from a total of 25 patients (13 males), with a median age of 50 (IQR 38–67). Most patients had Type 2 achalasia (19 [76%]) followed by Type 1 and 3 (2, [8%] respectively). Two cases had conflicting manometric findings. The median duration of symptoms was 3 years (IQR 1.5–10.5) and the median Eckhardt score was 6.5 (IQR 5–9). In sample tissues, no neuronal cell bodies were detected in the CSM layer. ICC for the axonal marker PGP9.5 showed that CSM of achalasia samples were almost completely devoid of axon structure, independent of the subtype of achalasia, compared to abundant axon presence in control tissues. In parallel, ICC showed that cholinergic (ChAT) or nitrergic (nNOS) axonal subtypes were absent in biopsy CSM while abundant in controls. CSM cells displayed hypertrophy with no detection of proliferation by ICC (KI67) or alterations in the phenotypic marker SM-22. Conclusions Preliminary results from advanced immunohistochemical techniques show the absence of functional innervation of the CSM layer of all patients with achalasia. This is characterized by a depleted excitatory and inhibitory axon population. Further studies are focused on defining differences in smooth muscle phenotype and the presence or absence of inflammatory cells within the CSM. Funding Agencies None

Background Chronic inflammation in inflammatory bowel disease (IBD) causes structural alterations of the intestine. In Crohn’s disease, this can lead to stricture formation, arising from unclear interactions among local inflammatory cells, cytokines, and mesenchymal intestinal cells. Specifically, proliferation of smooth muscle and increased deposition of collagen-rich extracellular matrix leads to fibrosis and obstructive wall thickening. Since there are minimal treatment options, we explored the effects of two multimodal tyrosine kinase inhibitors, nintedanib and pirfenidone, which were recently approved for idiopathic pulmonary fibrosis (IPF), a chronic lung condition resembling Crohn’s disease. Aims To understand the basic pharmacology of two novel anti-fibrotic tyrosine kinase inhibitors and their effects on cell proliferation and collagen deposition. Methods In vitro model systems of adult rat colonic circular smooth muscle cells (CSMS), rat IEC-18 intestinal epithelial cells or mouse 3T3 were assessed for response to serum or the mesenchymal growth factor PDGF-BB, evaluating growth responses by proliferation assay, and type I collagen expression by immunocytochemistry and western blotting. Programmed cell death was detected by ICC and western blotting for caspase-dependent apoptotic markers. Results Both 3T3 fibroblasts and rat ISMC showed concentration-dependent proliferation in response to serum or PDGF application. Nintedanib reduced this response to baseline at EC100 of 2.3 ± 0.7 (n=5) µM, and EC50 of 0.3 ± 0.1 (n=6) µM without cytotoxicity, while pirfenidone was ineffective at levels ≤ 5mM. Nintedanib (10 µM) was ineffective against serum-induced growth of rat IEC-18 cells while blocking growth of rat CSMC or mouse 3T3 fibroblasts in parallel assays, suggesting a selective effect on mesenchymal cell types. In nintedanib-treated cultures, nuclear staining showed concentration-dependent reduction of mitotic figures with proportional appearance of nuclear fragmentation, typical of apoptosis. Western blotting for collagen I identified at 125kD band for both CSMC and 3T3 cells and suggested down-regulation with both nintedanib and pirfenidone. Conclusions Novel anti-fibrotic therapies for chronic idiopathic pulmonary disease display distinctive effects on ISMC proliferation and extracellular matrix production. These address molecular mechanisms of fibrosis that are in common with IBD, and so the translation of therapeutic approaches may be a promising treatment option. Detecting specific mechanisms of action of nintedanib, such as apoptosis, leads to better targets for therapeutic options. Funding Agencies NSERC

Inflammation of the human intestine causesthickening of the smooth muscle layers, and studies inrats infected with Trichinella spiralis (Tsp) have shownhyperplasia of the intestinal smooth muscle cells (ISMC). We have shown that Tsp-inducedinflammation caused a fivefold increase in total proteinper ISMC over control, while ISMC from the noninflameddistal ileum also showed a threefold increase. The amount of α-smooth muscle (SM) actin perISMC increased nearly 500% over control by postinfection(PI) day 6. The proportion of α-SM actin in thetotal cellular protein increased 200% by day 6 PI, indicating a higher density of α-SM actinin the hypertrophied ISMC. γ-SM actin mRNAincreased sharply and was matched by an increasedfractional content of γ-SM actin protein. Theseincreases in the smooth muscle-specific actins may affectforce production and further demonstrate the plasticityof smooth muscle in the inflamed intestine.