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Achalasia is a rare esophageal motility disorder for which the etiology is not fully understood. Evidence suggests that autoimmune inflammatory infiltrates, possibly triggered by a viral infection, may lead to a degeneration of neurons within the myenteric plexus. While the infection is eventually resolved, genetically susceptible individuals may still be at risk of developing achalasia. This study aimed to determine whether immunological and physiological networks differ between male and female patients with achalasia. This cross-sectional study included 189 preoperative achalasia patients and 500 healthy blood donor volunteers. Demographic, clinical, laboratory, immunological, and tissue biomarkers were collected. Male and female participants were evaluated separately to determine the role of sex. Correlation matrices were constructed using bivariate relationships to generate complex inferential networks. These matrices were filtered based on their statistical significance to identify the most relevant relationships between variables. Network topology and node centrality were calculated using tools available in the R programming language. Previous occurrences of chickenpox, measles, and mumps infections have been proposed as potential risk factors for achalasia, with a stronger association observed in females. Principal component analysis (PCA) identified IL-22, Th2, and regulatory B lymphocytes as key variables contributing to the disease. The physiological network topology has the potential to inform whether a localized injury or illness is likely to produce systemic consequences and the resulting clinical presentation. Here we show that immunological involvement in achalasia appears localized in men because of their highly modular physiological network. In contrast, in women the disease becomes systemic because of their robust network with a larger number of inter-cluster linkages.

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Background/Aims: The evidence suggests that a shorter esophageal length (EL) in gastroesophageal reflux disease (GERD) patients is associated with the presence of hiatal hernia (HH). However, there are no reports of this association in patients with achalasia. The aim is to (1) determine the prevalence of hiatal hernia in achalasia patients, (2) compare achalasia EL with GERD patients and healthy volunteers (HV), (3) measure achalasia manometric esophageal length to height (MELH) ratio, and (4) determine if there are differences in symptoms between patients with and without hiatal hernia. Methods: This retrospective and cross-sectional study consist of 87 pre-surgical achalasia patients, 22 GERD patients, and 30 HV. High-resolution manometry (HRM), barium swallow, and upper endoscopy were performed to diagnose HH. The EL and MELH ratio were measured by HRM. Symptoms were assessed with Eckardt, Eating Assessment Tool, and GERD-health-related quality of life questionnaires. Results: The HH in GERD's prevalence was 73% vs 3% in achalasia patients (P < 0.001). Achalasia patients had a longer esophagus and a higher MELH ratio than HV and GERD patients (P < 0.001). GERD patients had a lower MELH ratio than HV (P < 0.05). EAT-10 (P < 0.0001) and Eckardt (P < 0.05) scores were higher in achalasia without HH vs HH. Conclusions: The prevalence of HH in achalasia is significantly lower than in GERD. The longer EL and the higher MELH ratio in achalasia could explain the lower prevalence of HH. Despite the low prevalence of HH in achalasia patients, the surgeon should be encouraged not to rule out HH since the risk of postoperative reflux may increase if this condition is not identified and corrected.

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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. KEY 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 & INFERENCES: 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.

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Background: Episodic angina-like retrosternal pain is a prevalent symptom for achalasia patients pre- and post-treatment. The cause of postoperative chest pain remains poorly understood. Moreover, there are no reports on their predictive value for chest pain in the long-term post-treatment. The effect of laparoscopic Heller myotomy (LHM) and fundoplication techniques (Dor vs. Toupet) is unclear. Methods: We analyzed a cohort of 129 achalasia cases treated with LHM and randomly assigned fundoplication technique. All the patients were diagnosed with achalasia by high-resolution manometry (HRM). Patients were followed up at 1-, 6-, 12-, and 24-month post-treatment. We implemented unadjusted and adjusted logistic regression analyses to evaluate the predictive significance of pre- and post-operative clinical factors. Results: Preoperative chest pain with every meal was associated with an increased risk of occasional postoperative chest pain [unadjusted model: odds ratio (OR) = 12, 95% CI: 2.2-63.9, P = 0.006; adjusted model: OR = 26, 95% CI: 2.6-259.1, P = 0.005]. In type II achalasia, hypercontraction was also associated with an increased risk of chest pain (unadjusted model: OR = 2.6 e9 in all the patients). No significant differences were associated with age, type of achalasia, dysphagia, esophageal shape, and integrated relaxation pressure (IRP) with an increased risk of occasional postoperative chest pain. Also, there was no significant difference between fundoplication techniques or surgical approaches (e.g., length of myotomy). Conclusion: Preoperative chest pain with every meal was associated with a higher risk of occasionally postoperative chest pain.

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OBJECTIVES: Autosomal dominant polycystic kidney disease (ADPKD), the most frequently diagnosed hereditary disease affecting Persian cats, is caused by a cytosine-to-adenine transversion (10063C>A) in PKD1, the gene that codes for polycystin-1. The objective of this study was to provide a preliminary estimate of the frequency of the pathogenic 10063C>A single nucleotide polymorphism (SNP) of PKD1 in Persian and Persian-related cat breeds in western Mexico. METHODS: Blood samples were collected from 104 cats (89 Persian, seven Persian crossbreed, five Siamese and three Himalayan cats). Genotyping was performed with our proposed PCR restriction fragment length polymorphism (RFLP) assay, as well as a previously established PCR-RFLP method for validation. The genotypes of control cats were corroborated by a commercial veterinary genetics laboratory. RESULTS: Our proposed PCR-RFLP assay and the validated PCR-RFLP methodology indicated that 24/104 (23.1%) cats in this study were heterozygous carriers of the 10063C>A SNP, including 23/89 Persian cats (25.8%) and 1/7 Persian crossbreed cats (14.3%). No Siamese or Himalayan cats were carriers. There were no discrepancies between the results obtained with our proposed assay and those obtained with the validation method or with commercial laboratory results. CONCLUSIONS AND RELEVANCE: The carrier frequency of the PKD1 10063C>A SNP in Persian and Persian-related cat breeds in western Mexico was found to be 23.1%. ADPKD frequencies among cat populations in Mexico have not been published previously. Genotyping assays can be used to facilitate the selection of breeding stocks by local breeders and veterinarians to avoid propagation of ADPKD.

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BACKGROUND: Serum anti-myenteric autoantibodies define autoimmune achalasia and tissue MMP-9 activity may locally process autoantigenic proteins in the muscle of the lower esophageal sphincter (LES) of achalasia patients. METHODS: Biopsies of the LES muscle from 36 achalasia patients, 6 esophagogastric junction outflow obstruction (EGJOO) patients, and 16 transplant donors (TD) were compared in a blind cross-sectional study. Histological characteristics such as inflammation, fibrosis, presence of ganglion cells, cells of Cajal, GAD65, PNMA2, S-100, P substance, and MMP-9 proteoforms in tissue were assessed by H&E and Picrosirius Red staining and immunohistochemistry analysis. Anti-neuronal antibodies, onconeural antigens, recoverin, SOX-1, titin, zic4, GAD65, and Tr were evaluated by immunoblot/line assay. KEY RESULTS: Tissue of achalasia patients had heterogeneous inflammatory infiltrates with fibrosis and contrasting higher levels of activated MMP-9, as compared with EGJOO and TD. Moreover, lower ganglion cell percentages and cell of Cajal percentages were determined in esophageal tissues of achalasia patients versus TD. The tissues of achalasia versus EGJOO patients had higher GAD65 and PNMA2 protein expression. Unexpectedly, these proteins were absent in TD tissue. S-100 and P substance had similar expression levels in tissues of achalasia patients versus TD and EGJOO. Most of the achalasia sera had anti-GAD65 (83%) and anti-PNMA2 (90%) autoantibodies versus EGJOO (17% and 33%, respectively) and healthy volunteers (10% and 0%, respectively). CONCLUSIONS AND INFERENCES: Tissue-specific ectopic expression of GAD65 and PNMA/Ta2 and active MMP-9, associated with the presence of specific autoantibodies directed against these proteins, might participate in the pathophysiology of achalasia triggering and/or perpetuating autoimmune disease.

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BACKGROUND: Laparoscopic Heller myotomy fails in approximately 3.5% to 15% of patients. Evidence of successful laparoscopic reoperation is limited to a few studies. METHODS: This case-control study was conducted in patients who underwent laparoscopic Heller myotomy reoperation (LHM-R) from 2008 to 2016. The operative outcomes, preoperative and last follow-up manometric parameters, and symptom questionnaire results, including the Eckardt, Gastroesophageal Reflux Disease-Health Related Quality of Life (GERD-HRQL) and eating assessment tool (EAT-10) scores, were obtained. The data were compared with those of patients who underwent primary laparoscopic Heller myotomy (LHM-1). RESULTS: Thirty-five patients who underwent LHM-R and 35 patients who underwent LHM-1 were included. The reasons for failure in the LHM-R patient group included incomplete myotomy (71.4%), myotomy fibrosis (25.7%) and structural alterations in fundoplication (2.9%). The follow-up duration was 34 months for the LHM-R group and 24 months for the LHM-1 group (p = 0.557). The procedure was performed by laparoscopy in 100% of the patients in the two groups. No differences were found regarding surgical morbidity (11.4% LHM-R vs. 2.9% LHM-1, p = 0.164). The symptomatic outcomes were equivalent between groups (Eckardt p = 0.063, EAT-10 p = 0.166, GERD-HRQL p = 0.075). An IRP < 15 mmHg was achieved in 100% of the LHM-R and LHM-1 patients. At the last follow-up, 82.1% of the LHM-R patients and 91.4% of the LHM-1 patients were in symptomatic remission (p = 0.271). CONCLUSION: The results achieved with LHM-R are similar to those achieved with LHM-1. Laparoscopic reoperation should be considered an effective and safe treatment after a failed Heller myotomy.

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OBJECTIVE: To determine the differences between clinical, manometric, and neuroimmunological profile of esophagogastric junction outflow obstruction (EGJOO) and achalasia patients. METHODS: Seven EGJOO and 27 achalasia patients were enrolled in a blind cross-sectional study. Peripheral blood (PB) of 10 healthy donors and 10 lower esophageal sphincter (LES) muscle biopsies from organ transplant donors were included as controls. The presence of ganglion cells, cells of Cajal, Th22/Th7/Th2/Th1/Tregs/Bregs/pDCregs in tissue, and PB was assessed by immunohistochemistry and flow cytometry. Serum concentration of IL-22/IL-17A/IL-17F/IL-4/IFN-γ/IL-1ß/IL-6/IL-23/IL-33/TNF-α/IL-10 was determined using bioplex plates. ANAs and antineuronal antibodies were evaluated by immunofluorescence and Western blot. KEY RESULTS: EGJOO and achalasia patients had lower ganglion cells and cells of Cajal percentage vs. controls, while fibrosis was present only in achalasia patients. EGJOO and controls had lower cell percentage of Th22/Th17/Th2 vs. achalasia. EGJOO tissue had lower Th1/Treg cell number vs. achalasia, but higher levels vs. control group. Bregs and pDCregs percentage was higher in EGJOO vs. control group. Percentage of PB subpopulations in EGJOO was not significantly different from control group. Serum cytokine levels were higher for IL-1ß/IL-6/TNF-α, while IL-17A levels were lower in EGJOO vs. achalasia and control group. EGJOO group was negative for ANAs, while in achalasia group, 54% were positive. GAD65 and PNMa/Ta2 antibodies were present in achalasia, whereas Yo and recoverin were positive in EGJOO group. CONCLUSIONS AND INFERENCES: Although EGJOO shares some clinical characteristics with achalasia, the neuroimmunological profile is completely different, suggesting that EGJOO might be a different entity.

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Complete blood count (CBC)-derived parameters such as neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), eosinophil-to-lymphocyte (ELR) ratio, and platelet-to-lymphocyte ratio (PLR) are sensitive markers of occult inflammation and disease activity for systemic lupus erythematosus, rheumatoid arthritis, psoriasis, esophageal cancer, etc. We assessed NLR, PLR, MLR, and ELR as indicators of inflammation in achalasia patients.This cross-sectional study included 103 achalasia patients and 500 healthy blood donor volunteers (HD). Demographic, clinical and laboratory information was collected. NLR, MLR, ELR and PLR were calculated. Peripheral Th22, Th17, Th2 and Th1 subsets were determined by flow cytometry. Correlation between hematologic indices and clinical questionnaires scores, HRM parameters and CD4+ T-cells were assessed. Hematologic parameters associated with the different achalasia subtypes were evaluated by logistic regression analysis.Hemoglobin, leukocytes, lymphocytes, monocytes, and platelets counts were significantly lower in achalasia patients vs controls. NLR (P = .006) and ELR (P < .05) were higher in achalasia patients vs controls. NLR was significantly associated with achalasia in multivariate analysis (P < .001). Compared to HD, the achalasia group was 1.804 times more likely to have higher NLR (95% CI 1.287-2.59; P < .001). GERD-HRQL score had statistically significant correlations with PLR (Pearson's rho:0.318, P = .003), and ELR (Pearson's rho:0.216; P = .044). No correlation between CD4+ T-cells and hematologic indices were determined. NLR with a cut-off value of ≥2.20 and area under the curve of 0.581 yielded a specificity of 80% and sensitivity of 40%, for the diagnosis of achalasia.NLR is increased in achalasia patients vs HD. Sensitivity and specificity achieved by NLR may contribute to a clinical and manometric evaluation. We suggest these indices as potential indicators of silent inflammation and disease activity.

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BACKGROUND: Idiopathic achalasia is an uncommon esophageal motor disorder. The disease involves interaction between inflammatory and autoimmune responses. However, the antigens related to the disease are still unknown. AIM: To identify the possible antigen targets in muscle biopsies from lower esophageal sphincter (LES) of achalasia patients. METHODS: Esophageal biopsies of patients with type I and type II achalasia and esophagogastric junction outflow obstruction (EGJOO) were analyzed. Lower esophageal sphincter muscle biopsy from a Healthy organ Donor (HD) was included as control for two-dimensional gel electrophoresis. Immunoblotting of muscle from LES lysate with sera of type I, type II achalasia, or type III achalasia, sera of EGJOO and sera of healthy subjects (HS) was performed. The target proteins of the serum were identified by mass spectrometry Matrix-assited laser desorption/ionization time-of-flight (MALDI-TOF). KEY RESULTS: The proteomic map of muscle from LES tissue lysates of type I, and type II achalasia, EGJOO, and HD were analyzed and divided into three important regions. We found a difference in the concentration of certain spots. Further, we observed the serum reactivity of type I achalasia and type II achalasia against 45 and 25 kDa bands of type I achalasia tissue. Serum of type III achalasia and EGJOO mainly recognized 25 kDa band. Bands correspond to triosephosphate isomerase (TPI) (25 kDa), carbonic anhydrase (CA) (25 kDa) and creatinine kinase-brain (CKB) isoform (45 kDa). CONCLUSIONS AND INFERENCES: We identify three antigen targets, TPI, CA, and CKB isoform, which are recognized by sera from patients with achalasia.

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