Society: AGA

Objectives:
Dysphagia and chest pain are the common symptoms in achalasia. The mechanisms underlying such symptoms are not completely understood. The two symptoms can be triggered by mechanical stimulation via the sensory vagal and spinal afferent nerve fibers. We previously reported that superficial nociceptive mucosal afferent nerves (i.e. calcitonin gene-related peptide-immunoreactive (CGRP-IR) nerves) might contribute to acid hypersensitivity in non-erosive reflux disease. The mucosal nerves can also detect mechanical deformation of the mucosa apart from chemical stimulation. Therefore, the aim of this study was to investigate the association between the severity of esophageal symptoms in achalasia patients and the characteristics of mucosal afferent nerve innervation.

Methods:
This was a muti-center prospective study involving six tertiary centers. Patients with predominant dysphagia and/or chest pain referred to the centers were recruited if they underwent upper endoscopy and high-resolution manometry for the examination of their symptoms. Esophageal motility was diagnosed on the basis of Chicago classification ver 4.0. The severity of dysphagia and chest pain were evaluated using Brief Esophageal Dysphagia Questionnaire (BEDQ) and a self-reported chest pain questionnaire respectively. Biopsies were taken from the proximal and distal esophagus for assessment of CGRP-IR mucosal nerves. The position of such nerves was expressed as cell layers from the esophageal luminal surface. We used a historical cohort of 8 asymptomatic healthy volunteers for comparison with the patients.

Results:
Overall, 61 patients with achalasia (n=57) or EGJ outflow obstruction (n=4) were included (median age, 56 years; 24 female (39%)). All the patients had dysphagia with the median of BEDQ score of 19 (11-26), and 39% of them had concomitant strong chest pain. The mucosal afferent nerves were located significantly closer to the lumen in the patients than in HVs (8 vs 11 cell layers, p=0.046 in the proximal, 9 vs 24 cell layers, p<0.001 in the distal). The position of mucosal afferent nerve negatively correlated to BEDQ score both in the proximal (rho=-0.567, p<0.001) and distal esophagus (rho=-0.317, p=0.021). The patients with strong chest pain showed more superficial mucosal afferent in the proximal esophagus (7 cell layers (4-9)) than those with weak chest pain (9 cell layers (7-14), p=0.022). Multivariate analysis found that the position of the afferent nerves in the proximal esophagus were independently and inversely associated with BEDQ score (-0.013 (-0.020, -0.006), <0.001) and chest pain (-0.054 (-0.101, -0.007), p=0.044). However, there was not such relationship in the distal esophagus.

Conclusions:
The superficial afferent nerves in the proximal, not distal esophagus, were associated with severe dysphagia and chest pain.

Background
The oesophageal microbiome is thought to contribute to the pathogenesis of oesophageal cancer. However, investigations using culture and molecular barcodes have provided only a low-resolution view of this important microbial community. We therefore exploited culturomics and metagenomic binning to generate a catalogue of reference genomes from the human oesophageal microbiome using oesophageal brushes from healthy patients, alongside a comparison set from saliva.

Results
Genomes from cultured isolates revealed twenty-two distinct colonial morphotypes from healthy oesophageal samples. These fell into twelve species clusters, eleven of which represented previously defined species. Two isolates belonged to a novel species, which we have named Rothia gullae.
Metagenome-assembled genomes was performed using metagenomic binning of reads generated from UK samples from this study alongside reads generated from Australian samples in the recent study by Deshpande et al., 2018. Metagenomic binning generated 136 medium or high-quality metagenome-assembled genomes (MAGs). MAGs were assigned to 56 species clusters, eight representing novel Candidatus species, which we have named Ca.Granulicatella gullae, Ca. Streptococcus gullae, Ca. Nanosynbacter quadramensis, Ca. Nanosynbacter gullae, Ca.Nanosynbacter colneyensis, Ca. Nanosynbacter norwichensis, Ca. Nanosynococcus oralis and Ca. Haemophilus gullae. Five of these novel species belong to the recently described phylum Patescibacteria. Although members of the Patescibacteria are known to inhabit the oral cavity, this is the first report of their presence in the oesophagus.
Our non-redundant species catalogue, contained 63 species derived from cultured isolates or MAGs. Mapping revealed that these species account for around half of the sequences in the oesophageal and saliva metagenomes. Although no species was present in all oesophageal samples, 60 species occurred in at least one oesophageal metagenome from either study, with 50 identified in both cohorts.

Conclusions
In recovering over a hundred bacterial genomes through culture and metagenomic analysis, we have obtained the first high-resolution view of microbial diversity within this important environment. Remarkably, we have discovered one new cultured species and eight novel Candidatus species, opening up the way for detailed characterisation of these newfound taxa. We have not only discovered new species within well-characterised genera, such as Strepotococcus and Haemophilus, but also found six species from the enigmatic Patescibacteria, which are thought to live as epibionts in close association with other bacteria in this environment . The genes and genomes that we have released into the public domain will provide a base line for future comparative, mechanistic and intervention studies.

Background: Defective secondary peristalsis on manometry has been shown in patients with gastroesophageal reflux disease (GERD) both before and after antireflux surgery. To better understand the role of motility in post-fundoplication symptoms, this study aimed to investigate secondary peristaltic contractile response (CR) patterns on functional lumen imaging probe (FLIP) Panometry and associated clinical parameters in symptomatic patients with history of fundoplication.

Methods: 89 adult patients with a history of fundoplication (age 21-87, 73% female) and completion of 16-cm FLIP Panometry during sedated endoscopy for symptom assessment were included. Secondary peristaltic CR patterns and associated parameters were assessed on FLIP Panometry. CR classifications with antegrade contractions included normal CR (NCR) and borderline CR (BCR). Abnormal CRs included impaired/disordered CR (IDCR), absent CR (ACR), or spastic-reactive CR (SRCR). CR patterns were analyzed against available associated clinical data including high resolution manometry (HRM) metrics per Chicago Classification (CCv4.0), esophagram findings including esophageal body width (diameter), and patient-reported outcomes.

Results: Of the 89 patients, FLIP CR patterns comprised of 14 (16%) NCR, 31 (35%) BCR, 29 (33%) IDCR, 13 (15%) ACR, and 2 (2%) SRCR. Rates of abnormal CRs (IDCR, ACR, SRCR) did not differ between intact (20/44; 45%) and failed (i.e. wrap disrupted or slipped and/or recurrent hiatal hernia: 24/45; 55%) fundoplications (p=0.884). 63 patients completed HRM after fundoplication: 37 (59%) had normal primary peristalsis, 11 (30%) of whom had abnormal CR on FLIP. 26 (41%) had abnormal primary peristalsis (Table 1), 19 (73%) of whom had abnormal CRs on FLIP. Patients with abnormal CRs compared with NCR or BCR demonstrated significantly greater age (p=0.030), time since fundoplication (p=0.002), and esophageal body width on esophagram (n=52; p=0.015) (Figure 1). Patients with abnormal CRs also had greater supine integrated relaxation pressure (IRP) on HRM (p=0.015) and significantly lower EGJ-distensibility index (p=<0.001) and maximum EGJ diameter (p=0.008) on FLIP (Table 1). Symptom severity scores, however, were similar relative to CRs. The rate of re-do antireflux surgery post-FLIP assessment was significantly increased (p=<0.0001) in patients without NCR (26/75 35% vs. 0/14).

Conclusions: Abnormal secondary peristalsis was common after fundoplication and associated with abnormal primary peristalsis. Abnormal CRs were also associated with increased time after fundoplication and greater esophageal width (dilatation), as well as higher IRP and lower EGJ distensibility, suggesting that chronic remodeling of the esophageal body after fundoplication (possibly related to EGJ obstruction) may contribute to esophageal dysmotility in this setting.

<b>Figure 1.</b> Esophageal Body Width (cm) and Time Since Fundoplication (y) in relation to Normal CR vs. Abnormal CR Pattern on FLIP Panometry

Figure 1. Esophageal Body Width (cm) and Time Since Fundoplication (y) in relation to Normal CR vs. Abnormal CR Pattern on FLIP Panometry

Introduction. Retrograde cricopharyngeal dysfunction (R-CPD) is a newly recognized disorder characterized by self-reported inability to belch, accompanied by gastrointestinal symptoms such as chest pain, gurgling noises, bloating and flatulence. It has been linked to ineffective relaxation of the upper esophageal sphincter (UES) in response to gastroesophageal gas reflux, and botulinum toxin (Botox) injection into the UES has demonstrated success in case series. However, in the absence of adequate testing, the diagnosis is often missed. Recently, high resolution impedance manometry (HRiM) with belch provocation was suggested as diagnostic tool (Oude Nijhuis, 2022).
Aim. To confirm manometric findings in patients with R-CPD before and after Botox injection, and to compare with HRiM in control patients. To propose an algorithm for diagnosis and management.
Methods. Retrospective analysis of HRiM in patients with suggestive symptoms and control patients from May 2021 to November 2022. Ten swallows and a rapid drinking challenge with sparkling water (until symptom provocation) were added to the Chicago IV protocol, followed by an observation period. Control patients were referred for HRiM for different symptoms/conditions. In 5 patients, HRiM was repeated 1 to 5 months after Botox injection. Gastroesophageal gas reflux was defined as a rapid increase in impedance (>3000Ω) from distal to proximal channels, air entrapment as a period of high impedance levels (>3000Ω) in ≥ 2 proximal channels (in seconds, divided by the total recording time in minutes), oscillations as a continuous up and down movement of air.
Results. HRiM with belch provocation test was available for 19 patients before, and 5 patients after Botox injection (table 1). Normal UES relaxation occurred after deglutition in all. Mean observation period after belch provocation was 7.8min. We observed a mean of 2 gastroesophageal gas reflux events per minute, which resulted in UES relaxation in 6% of events. Air entrapment occurred in 22 sec/min, oscillations in 28 sec/min. After Botox injection, 4/5 patients improved, 3/5 patients were able to belch during HRiM. Air entrapment time was reduced to 8 sec/min.
In control patients (n=18, table 1), drinking challenge with sparkling water induced gastroesophageal gas reflux with adequate belching in all, with air entrapment/oscillations in 9 sec/min.
Conclusion. We can confirm typical findings on HRiM with belch provocation test, which are not present in control patients. Liquid swallows with sparkling water were unable to differentiate R-CPD patients from controls, however a drinking challenge with sparkling water provoked symptoms and typical manometric anomalies in patients, but not controls. These findings can provide a basis for a diagnostic algorithm (figure 1) and a standardized HRiM protocol for patients presenting with ‘inability to belch’.

Background: Achalasia is an autoimmune disease whose probable causal agent is a neurotropic virus that chronically infects the myenteric plexus of the esophagus and induces the disease in a genetically susceptible host. The association between achalasia and coronaviruses has not been reported.
Aims: To evaluate the presence of the SARS-CoV-2 virus, the ACE2 expression, the tissue architecture, and immune response in the lower esophageal sphincter muscle (LESm) of achalasia patients who posteriorly had SARS-CoV-2 (achalasia-COVID-19) infection before laparoscopic Heller myotomy (LHM) and compare the findings with type II achalasia patients and transplant donors (controls) without COVID-19.
Methods: The LESm of 7 achalasia-COVID-19 patients (diagnosed by PCR), ten achalasia patients, and ten controls without COVID-19 were included. The presence of the virus was evaluated by in situ PCR and immunohistochemistry. ACE2 receptor expression and effector CD4 T cell and regulatory subsets were determined by immunohistochemistry.
Results: Coronavirus was detected in 6/7 patients-COVID-19. The SARS-CoV-2 was undetectable in the LESm of the achalasia patients and controls. ACE2 receptor was expressed in all the patients and controls. One patient developed achalasia type II post-COVID-19. The percentage of Th22/Th17/Th1/pDCreg was higher in achalasia and achalasia-COVID-19 pre-HLM vs. controls. The Th2/Treg/Breg cell percentages were higher only in achalasia vs. controls.
Conclusion: SARS-CoV2 and its receptor expression in the LESm of achalasia patients who posteriorly had COVID-19 but not in the controls suggests that it could affect the myenteric plexus. Unlike achalasia, patients-COVID-19 have an imbalance between effector CD4 T cells and the regulatory mechanisms.

<b>F I G U R E 1</b> <b>(A)</b> Upper endoscopy shows dilated esophagus (patient that developed achalasia post-COVID-19). <b>(B) </b>HRM shows abnormal relaxation of the LES and aperistalsis with panesophageal pressurization in 40% of swallows, corresponding with type II achalasia. <b>(C)</b> Barium esophagram that shows dilation and bird beak sign (arrow). <b>(D)</b> <i>In situ</i> PCR for SARS-CoV2 (arrows indicate positive cells, X600) of the LES muscle of type II achalasia patient (<i>left</i>) and type II achalasia post-COVID-19 patient (<i>right</i>). <b>(E)</b> Immunohistochemistry for SARS-CoV2 (arrows indicate positive spike, X600). <b>(F) </b>Immunohistochemistry for ACE2 expression (X600). <b>(G)</b> Immunohistochemistry for HSV-1 expression (X600). <b>(H)</b> Tissue architecture of the LES muscle. Dot circles depict inflammatory infiltrates (X200).

F I G U R E 1 (A) Upper endoscopy shows dilated esophagus (patient that developed achalasia post-COVID-19). (B) HRM shows abnormal relaxation of the LES and aperistalsis with panesophageal pressurization in 40% of swallows, corresponding with type II achalasia. (C) Barium esophagram that shows dilation and bird beak sign (arrow). (D) In situ PCR for SARS-CoV2 (arrows indicate positive cells, X600) of the LES muscle of type II achalasia patient (left) and type II achalasia post-COVID-19 patient (right). (E) Immunohistochemistry for SARS-CoV2 (arrows indicate positive spike, X600). (F) Immunohistochemistry for ACE2 expression (X600). (G) Immunohistochemistry for HSV-1 expression (X600). (H) Tissue architecture of the LES muscle. Dot circles depict inflammatory infiltrates (X200).

Background: The etiology of achalasia (AC) is unknown. We previously found that the reduction of Transgelin (TAGLN) and HSPB1 induced Ferroptosis in esophageal myenteric plexus in AC patients. Cystathionine-β-synthase (CBS) expression also decreased in AC. Recently, we found Sindbis virus (SINV) infection in the esophageal tissue of AC patients. Based on the previous results, it is speculated that the infection of Sindbis virus-induced inflammatory response and caused Ferroptosis in nerve cells, leading to the occurrence of AC.
Methods: Sindbis virus suspension was prepared, and 2-week-old mice were selected. The primary pressure of lower esophageal sphincter (LES) was measured, and the esophagography was recorded.
The fresh mouse brain virus suspension with TCID₅₀ of 6.67 was perfused orally, and the control group was perfused with the same amount of normal saline. After 48h (or near death) of the infection operation, the mice were again subjected to esophageal manometry and gastrointestinal angiography. After the completion of the operation, the mice were killed, and the LES part specimens of the mice were taken. After 48 hours of SINV infection, the mice were given exogenous H₂S donors and the same amount of normal saline for oral administration. The mice were again subjected to esophageal manometry and gastrointestinal radiography. After that, the mice were killed, and LES part specimens were taken. The expression of CBS/CSE was knocked down in the mice neuron cells, and the H₂S donor was added to transfected cells. The expressions of TG, HSPB1, CBS, and Ferroptosis protein markers were detected, and the content of MDA, ROS, GSH, ferrous iron, and total iron was measured.
Results: After infection with the Sindbis virus, the resting pressure of mice LES was increased, the motor function was impaired, and the number of esophageal myenteric plexus neurons was reduced. The expression of CBS/TAGLN/HSPB1 was reduced, the expression of the ferroptosis marker GPX4 and the ferritin gene FTH1 was downregulated, and COX-2 and ACSL4 were upregulated in LES. The LES relaxation disorder in SINV-infected mice was alleviated after H₂S donor administration. After CBS/CSE was knocked down, the expressions of TAGLN and HSPB1 decreased, and the expressions of COX-2 and ACSL4 were up-regulated while GPX4 and FTH1 were down-regulated. The content of ferrous iron and total iron in neuron cells reduced, the MDA and ROS concentrations decreased and GSH content increased after the hydrogen sulfide donor.
Conclusion: Sindbis virus triggers an inflammatory reaction, which then causes Ferroptosis of nerve cells, leading to the occurrence of AC. Exogenous H₂S donors can treat AC and inhibit Ferroptosis of AC esophageal myenteric plexus nerve cells through CBS/Transgelin.

Xiangxu Kong

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SINDBIS VIRUS INFECTION PROMOTES FERROPTOSIS OF INTERMUSCULAR PLEXUS CELLS AND CAUSES ACHALASIA

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