OBJECTIVETo characterize in sera anti-myenteric autoantibody profiles and in tissues MMP-9 proteoforms towards the identification of possible autoantigenic proteins in the muscle of the lower esophageal sphincter (LES) of achalasia patients.METHODSBiopsies 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, S100, P substance, and MMP-9 proteforms in tissue were assessed by H&E and Picro-Sirius Red stainings, and immunohistochemistry analysis. Antineuronal antibodies (amphiphysin, CV2, and PNMA2 (Ma2/Ta)), onconeural antigens (Ri, Yo, and Hu), recoverin, SOX-1, titin, zic4, GAD65, and Tr (DNER) were evaluated by immunoblot/line assay.RESULTSTissue of achalasia patients had heterogeneous inflammatory infiltrates with fibrosis and contrasting higher levels of activated MMP-9 compared with EGJOO and TD. Moreover, lower ganglion cell percentages and cell of Cajal percentages were determined in esophageal tissues of achalasia patientsvs. TD. In addition, tissue of achalasia patients had higher GAD65 and PNMA2 protein expressionvs. EGJOO. Unexpectedly, these proteins were absent in TD tissue. S100 and P substance had similar expression levels in tissues of achalasia patientsvs.TD and EGJOO. Most of the achalasia sera had anti-GAD65 (83%) and anti-PNMA2 (90%) autoantibodiesvs. EGJOO (17% and 33%, respectively) and healthy volunteers (10% and 0%, respectively).CONCLUSIONTissue-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.WHAT YOU NEED TO KNOW?BACKGROUND AND CONTEXTAchalasia is a primary esophageal motility disorder associated with a selective loss of inhibitory neurons in the myenteric esophageal plexus.An inflammatory/autoimmune response seems involved in pathophysiology.NEW FINDINGSMost of the achalasia sera have anti-GAD65 and anti-PNMA2 autoantibodies vs. EGJOO and healthy volunteers.Esophageal tissues of achalasia patients have higher ectopic GAD65 and PNMA2 protein expression vs. EGJOO. These proteins are absent in healthy donors with non-inflamed muscle of the LES.Activated MMP-9 is detectedin situtogether with GAD65 and PNMA2 substrates.LIMITATIONSBy its design, our study does not shed light on the mechanisms that induce the ectopic expression of the GAD65 and PNMA/Ta2 proteins in LES muscle tissue. It remains impossible to firmly state the sequence of events, e.g. whether the expression of ectopic antigens is due to tissue destruction or the presence of autoantibodies. Whether serum autoantibodies react with autoantigens of the LES muscle tissue of the patients with achalasia also remains unknown.IMPACTThis information yields new insights into pathophysiological mechanisms of achalasia for better disease understanding and possible design of novel therapeutic strategies.LAY SUMMARYMost of the achalasia sera have anti-GAD65 and anti-PNMA2. Esophageal tissues of achalasia patients have ectopic GAD65 and PNMA2. This information yields new insights into pathophysiological mechanisms of achalasia for better disease understanding.