Introduction

Neurokinin A (NKA) and substance P (SP) may be responsible for airway hyperreactivity and hypersensitization. Animal studies showed an increase of their blood concentration in response to acid installation into the esophagus, suggesting the presence of neurogenic inflammation in pathogenesis of asthma and chronic cough. However, little is known about association between serum concentration of these cytokines and esophageal function testing parameters in patients with gastroesophageal reflux disease (GERD).

Aims & Methods

Aim of the study was to assess serum concentration of neurokinin A and substance P in patients with erosive esophagitis and non-erosive form of GERD and perform correlation analysis between concentrations of these substances and data of multichannel esophageal pH-impedance (MII-pH) and high-resolution esophageal manometry (HREM).
Methods: The data of examination of 88 subjects (31with erosive esophagitis (EE); 57 with non-erosive form of GERD (NERD)) served as a source for the study. Diagnosis of GERD was established per Lyon criteria. Subjects with autoimmune disorders, receiving immunosuppressants, antisecretory agents and medications able to affect esophageal motility were not eligible. Patients underwent HREM with the use of 36-channel solid-state catheters and MII-pH with 2-pH, 6-impedandce sensors catheters. Serum concentrations of NKA and SP were measured with Sunrise analyzer and ELISA kits (Biochemmack, USA). Non-parametric statistics was used to compare the results obtained in EE and NERD groups and to perform correlation analysis.

Results

Acid exposure time (AET), and number of high gastroesophageal refluxes were higher in EE group compared to NERD, while number of refluxes and concentrations of NKA and SP were similar. Mean, minimal and residual lower esophageal sphincter resting pressures (mean by 10 water swallows) were lower in EE compared to NERD. There was direct correlation between serum concentration of SP and number of gastroesophageal refluxes (Spearman rank R=0.258), AET (R=0.252), and number of refluxes lasted >5 min (R=0.353). Serum concentration of NKA correlated directly with number of refluxes >5 min long (R=0.275), and inversely with levels of integrated relaxation pressure (R=-0.321), and contractile front velocity (R=-0.379). Other correlations were not statistically significant.
Table 1. Correlation coefficients (by Spearman rank R) between serum concentrations of Neurokinin A, Substance P and esophageal function testing parameters.

Conclusion

In subjects with GERD, serum concentrations of Neurokinin A and Substance P do not depend on the presence of esophagitis and probably are not caused by the influence of reflux content to sensory neurons of esophageal mucosa. Established correlations may support the involvement of neurokinin A and substance P in pathogenesis of gastroesophageal reflux disease through disturbance of esophageal motility.
This work was funded by research grant #FGMF-2022-0005 by Ministry of Science and Higher Education of Russia

Introduction

Volatile organic compounds (VOC) in stool samples may reflect functional activity of gut microbiome. Previous studies showed that gut flora may influence transient relaxations of lower esophageal sphincter through short-chain fatty acids production and thus - participate in pathogenesis of gastroesophageal reflux disease (GERD). However, the role of VOC in pathogenesis of GERD is poorly studied yet.

Aims & Methods

The aim of this study (FGMF-2025-0003) was to perform correlation analysis between concentrations of VOC in stool samples and GERD-Q score.
We enrolled willing to participate patients referred to perform multichannel 24-h pH-impedance recording (Ohmega, Laborie). According to the Lyon 2.0 criteria patients were allocated to the GERD or the control group. GERD-Q questionnaire was filled in by the participants on the same day as stool samples were collected. Thermo TSQ 8000 triple quadrupole mass-spectrometer (GC/MS) was used for the measurement of VOCs in stool samples were analyzed with. Correlation analysis (Spearman rank R) was performed between values of 80 VOCs in stool samples and the results of GERD-Q questionnaire.

Results

Data of 76 patients were eligible: 42 with GERD and 34 - of the control group. Participants in the GERD group were older (54.7±12.2 y.o. vs 46±14.9 y.o., P<0.05) and their BMI was greater (30.9±5.2 vs 27.2±6.5 kg/m², P<0.05). GERD-Q score was logically greater in GERD group (11.0±2.5 vs 5.2±1.2, p<0.01). Correlation analysis revealed significant correlations between GERD-Q score and concentrations of 2-methylbutyric acid (Spearman R =-0.31), caproic acid (0.34) and enantiic acid (0.396) in stool samples.

Conclusion

GERD-Q score positively correlates with concentrations of Caproic acid (0.34) and Enantiic acid (0.396) in stool samples and negatively – with 2-Methylbutyric acid. The data of this pilot study may serve as a basis for further research on the role of functional activity of gut microbiome in pathogenesis of GERD.

Introduction

Gastro-oesophageal reflux disease (GORD) is characterised by visceral pain hypersensitivity, manifesting as heartburn, and distal oesophageal inflammation. In the oesophageal mucosa of GORD patients, even without visible inflammation (non-erosive reflux disease; NERD) there is increased innervation of sensory nerve fibres containing Substance P (SP) (Xu et al., 2013). SP may be released in response to luminal refluxate or inflammatory mediators to potentiate neuro-immune pathways relevant to GORD pathogenesis. We aimed to investigate the contribution of SP and its receptor NK1R to GORD symptoms.

Aims & Methods

To assess expression of NK1R on native tissue epithelium, biopsies were collected from healthy controls (HC), functional heartburn (FH), NERD and erosive reflux disease (ERD) patients following informed consent (Royal London Hospital ethics number: 19/LO/1506) and prepared for immunohistochemistry. NK1R was co-stained with epithelial cell marker E-Cadherin, pan-leukocyte marker CD45, and general T lymphocyte marker CD3. NK1R protein level was determined by mean fluorescence intensity of 3 FOVs. Cells of the NE-1 oesophageal epithelial cell line were incubated with media containing 100 nM and 300 nM SP for 30 minutes. Cells were lysed and intracellular inflammatory pathway activation was determined by p-NF-κB/NF-κB ratio through Western blot. mRNA levels of the inflammatory cytokine CXCL8 were determined via qPCR with 18S rRNA as a housekeeping gene.

Results

Quantification of mucosal tissue samples showed NK1R mean fluorescence intensity was significantly higher in NERD (fold-change 1.52; p<0.05) and ERD (fold change 1.70; p<0.01) oesophageal mucosa compared to healthy control (n=6-8). Interestingly, NK1R was also found expressed in a sub-group of CD3+ T lymphocytes specifically in the papillae and epithelium of GORD patients. in NE-1 oesophageal epithelial cells, 30 minutes exposure to 100 nM (fold change 1.41; p<0.05) and 300 nM (fold change 1.92; p<0.0001) SP induced phosphorylation of NF-κB in a dose dependent manner (n=7-8). CXCL8 expression in NE-1 cells exposed to 30 nM, 100 nM, or 300 nM SP for 30 minutes resulted in significantly higher mRNA expression at the highest concentration (fold change 6.29; p<0.05), although a dose-dependent trend was observed at lower concentrations (n=3).

Conclusion

The significant increase in NK1R expression in NERD and ERD patients compared to healthy volunteers, combined with expression on T lymphocytes provides evidence, for the first time, that SP may have a critical role in potentiating pain sensation in the oesophagus. Indeed, SP directly induces NF-κB phosphorylation and increases transcription of inflammatory cytokine IL-8 in oesophageal epithelial cells. This demonstrates a novel neuroimmune pathway regulated by SP and NK1R that may be critical in GORD symptom generation.

References

Xu, X., Li, Z., Zou, D., Yang, M., Liu, Z., & Wang, X. (2013). High expression of calcitonin gene-related peptide and substance P in esophageal mucosa of patients with non-erosive reflux disease. Dig Dis Sci, 58(1), 53-60. doi:10.1007/s10620-012-2308-z

Introduction

Gastroesophageal reflux disease (GERD) and asthma are commonly co-occurring conditions, with shared genetic factors identified. However, the specific loci and the influence of common genetic architecture remain undefined.

Aims & Methods

We obtained genome-wide association study (GWAS) summary statistics for GERD (71,522 cases, 261,079 controls) and asthma (56,167 cases, 352,255 controls). Using linkage disequilibrium score regression (LDSC), we assessed genetic correlations between GERD and asthma. Bidirectional mendelian randomization (MR) was performed to investigate potential causal relationships, followed by cross-trait GWAS meta-analysis and colocalization analysis to identify shared risk loci. Additionally, summary-data-based mendelian randomization (SMR) and transcriptome-wide association study (TWAS) were conducted to pinpoint common functional genes. Finally, we analyzed gene expression profiles in both healthy individuals and GERD patients using esophageal single-cell RNA sequencing (scRNA-seq) data.

Results

We identified a significant genetic correlation between GERD and asthma (r_g = 0.37, P = 6.19×10^–38) and a significant causal effect of GERD on asthma (OR = 1.22, P = 1.54×10^-5). Cross-trait meta-analyses revealed 56 shared risk loci between GERD and asthma, including 51 loci that were newly identified. Three loci (rs61937247, rs7960225, and rs769670) exhibited evidence of colocalization. Gene-level analyses pinpointed three novel shared genes(RBM6, SUOX, and MPHOSPH9) between GERD and asthma. Single-cell RNA sequencing analysis uncovered heightened expression of these genes in immune cells of patients diagnosed with GERD.

Conclusion

Our study has discovered novel shared genetic loci and candidate genes between GERD and asthma, providing further insights into the genetic susceptibility of comorbidity and potential mechanisms of the two diseas​​​​es.

References

1. Broers C, Tack J, Pauwels A. Review article: gastro-oesophageal reflux disease in asthma and chronic obstructive pulmonary disease. Aliment Pharmacol Ther 2018;47:176–191.
2. Eapen AA, Gupta MR, Lockey RF, et al. Gastroesophageal reflux disease, laryngopharyngeal reflux, and vocal cord dysfunction/inducible laryngeal obstruction-overlapping conditions that affect asthma. J Allergy Clin Immunol. 2024;154(6):1369-1377.
3. Bulik-Sullivan B, Finucane HK, Anttila V, et al. An atlas of genetic correlations across human diseases and traits. Nat Genet. 2015;47(11):1236-1241.
4. Morrison J, Knoblauch N, Marcus JH, et al. Mendelian randomization accounting for correlated and uncorrelated pleiotropic effects using genome-wide summary statistics. Nat Genet 2020;52:740–747.
5. Purcell S, Neale B, Todd-Brown K, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 2007;81:559–575.
6. Stuart T, Butler A, Hoffman P, et al. Comprehensive Integration of Single-Cell Data. Cell 2019;177:1888-1902.e21.
7. Finucane HK, Bulik-Sullivan B, Gusev A, et al. Partitioning heritability by functional annotation using genome-wide association summary statistics. Nat Genet 2015;47:1228–1235.
8. Freuer D, Linseisen J, Meisinger C. Asthma and the risk of gastrointestinal disorders: a Mendelian randomization study. BMC Med 2022;20:82.

Disclosure

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Introduction

Esophageal mucosal admittance (MA) is a promising diagnostic method for gastroesophageal reflux disease (GERD), however, only some studies were done and mainly in Japan.

Aims & Methods

We conducted a study to describe the esophageal MA in patients with reflux symptoms and determine its diagnostic accuracy. We recruited 92 patients with ambulatory pH-impedance monitoring, upper gastrointestinal endoscopy, and MA measured by the tissue conductance meter. MA was measured during endoscopy at 5cm (distal esophagus) and 15cm above the Z line (middle esophagus), repeated at least five times at each position, and median MA was obtained. Afterwards, two biopsies were taken 5cm above the Z line for histopathological evaluation using the Esohisto criteria. Patients were classified as GERD or non-GERD according to the 2018 Lyon consensus.

Results

The mean age was 43.2 years, and 42 patients were males. The most common symptoms were regurgitation (75.0%), belching (65.2%), and heartburn (46.7%). Twenty-three (32.3%) were diagnosed with GERD using the Lyon consensus, and 24 (26.1%) had esophagitis on histopathology. The median MA at the distal and middle esophagus was moderately correlated. The median MA at both positions was higher in the GERD group but only statistically significant in the middle esophagus. MA was not associated with pH-impedance parameters and esophagitis on histopathology. The diagnostic model developed using the logistic regression did not have good accuracy.

Conclusion

MA was not different between GERD and non-GERD patients. The diagnostic model integrating MA did not have high accuracy in diagnosing GERD.

Introduction

The etiology of gastroesophageal reflux disease (GERD) is unknown but reflux-induced esophageal injury as a result of cytokine-mediated inflammation has been implicated (1). There is emerging evidence duodenal immune activation may be associated with an increased risk of GERD (2). Celiac disease is strongly associated with GERD which improves upon removal of gluten (3). The duodenum is the first major part of the gut to process food and swallowed aerosol antigens, and is a master controller of upper gut motility. The role of duodenal inflammation in GERD, if any, remains understudied. Assessing T-helper (Th) subsets using chemokine staining has been previously documented and allows the use of archived gut samples (4). We aimed to identify if there is a duodenal Th signal in GERD.

Aims & Methods

Two retrospective case control studies. Study 1; Duodenal biopsies of 3 groups: 1) GERD (n=22), defined as abnormal acid exposure time on 24-pH monitoring, with or without positive symptom association probability (GERD-SAP+ and GERD-SAP-). 2) Healthy controls (n=17): no symptoms, endoscopic or histological abnormalities. 3) Disease controls: Eosinophilic duodenitis (EoD): (n=8). Blinded histological review of duodenal samples to assess cellular and architectural changes. Dual Immunohistochemistry (IHC) was optimized to identify CD4-Th subsets using the chemokine staining patterns: Th1(CD4+CCR4-CCR5+CCR6-), Th2 (CD4+CCR4+CCR5-CCR6-) and Th17(CD4+CCR4+CCR5-CCR6+). Immunofluorescence confirmed co-staining. Dual IHC and blinded quantitative intensity measuring (H-score) were performed using HALO software. Clinical and histological data were collected. One-way Anova was used to compare signal intensity to controls. A positive signal is defined as a statistically significant higher co-staining of CD4-chemokine stain compared to controls. Study 2: Same methods used to assess esophageal T-helper signal in reflux esophagitis (LA Grade B and higher) (RE) (n=16) compared to eosinophilic esophagitis (EoE) (n=31), lymphocytic esophagitis (LE) (n=12) and healthy controls (n=11).

Results

Considering duodenal histopathology (study 1), no differences were found in any of the mean inflammatory cell counts and no architectural abnormalities comparing GERD to healthy controls. Considering duodenal IHC, the CD4 and CD8 intensities were not significantly higher in GERD compared to controls (p=0.96 and 0.96, respectively). GERD- SAP+ demonstrated a significantly higher Th2 intensity than controls (p=0.01), with no difference in Th1(p=0.55) and Th17 intensities (p= 0.27). A significantly higher Th17 intensity was found in EoD (p=0.006). Duodenal Th2 intensity was significantly higher in GERD with a history of atopy (p=0.035), upright reflux (p=0.037), low esophageal contraction amplitude (p=0.0025) and failed peristalsis (p=0.048). Th1 and Th17 intensities were not higher in any of the GERD subgroups compared to controls. Considering esophageal IHC (study 2), CD8 intensity was significantly elevated in RE (p=0.002), but the CD4 intensity was not (p=0.059). RE had a higher Th2 intensity with no difference in other Th markers (p<0.0001). Th-17 intensity was higher in EoE and LE (p=0.0017 and p<0.0001).

Conclusion

A significantly increased duodenal Th-2 signal is found in established GERD when present with a positive symptom association probability, while an increased esophageal Th-2 signal is found in documented reflux esophagitis. The role of immune activation in GERD deserves further investigation.

References

1. Souza RF, Huo X, Mittal V, et al. Gastroesophageal reflux might cause esophagitis through a cytokine-mediated mechanism rather than caustic acid injury.
2. Ronkainen J, Aro P, Walker MM, et al.. Duodenal eosinophilia is associated with functional dyspepsia and new onset gastro-oesophageal reflux disease. Aliment Pharmacol Ther. 2019 Jul;50(1):24-32.
3. Irani MZ, Eslick GD, Burns GL, et al. Coeliac disease is a strong risk factor for Gastro-oesophageal reflux disease while a gluten free diet is protective: a systematic review and meta-analysis. EClinicalMedicine. 2024;71
4.Oki M, Ohtani H, Kinouchi Y, et al. Accumulation of CCR5+ T cells around RANTES+ granulomas in Crohn's disease: a pivotal site of Th1-shifted immune response? Laboratory investigation. 2005;85(1):137-45.

Disclosure

Nicholas J. Talley: Disclosures: GutSee Ltd (consulting microbiome, 2025), Brown University, Agency for Health Care Research and Quality (fiber and laxation) (2024), Rome Foundation (member gastroduodenal committee) (present) Biocodex (FD diagnostic tool) (present) Microba (consulting microbiome - commencing 2025) Comvita Manuka Honey (FD trial consulting) (2025), BluMaiden outside the submitted work. In addition, Dr. Talley has a patent Nepean Dyspepsia Index (NDI) 1998, a patent Licensing Questionnaires Talley Bowel Disease Questionnaire licensed to Mayo/Talley, “Diagnostic marker for functional gastrointestinal disorders” Australian Provisional Patent Application
2021901692, ”Methods and compositions for treating age-related neurodegenerative disease associated with dysbiosis” US Patent Application No. 63/537,725. Boards: The Gut Foundation Board (2025-present), Asia Pacific Association of Medical Editors (APAME) Board (President 2023-2025), Doctors for the Environment (Chair 2024-present), Luminal Faculty GESA (Chair) (2023-2025) Editorial: UptoDate (Section Editor), Mayo Clinic Proceeding Associate Editor (2023-present)
Financial support: Dr. Talley is supported by funding from the National Health and Medical Research Council (NHMRC) to the Centre for Research Excellence in Digestive Health and he holds an NHMRC Investigator grant.