Triosephosphate isomerase, carbonic anhydrase, and creatinine kinase-brain isoform are possible antigen targets in patients with achalasia.

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.

Anti-retinal antibodies in patients with macular telangiectasia type 2.

PURPOSE: Macular telangiectasia type 2 (MacTel-2) is a retinal disease that can cause loss of central vision. To gain better understanding of the etiology and pathogenesis of MacTel-2, we investigated antigens that prompt the generation of retinal autoantibodies in the serum of patients with MacTel-2. METHODS: We screened for the presence of retinal autoantibodies in 45 serum samples collected from patients with MacTel-2 and 58 serum samples from healthy control subjects by Western blot. We then isolated and identified three retinal proteins that are putative targets of three of the most frequently detected autoantibodies in the serum of patients with MacTel-2 using chromatographic fractionation and liquid chromatography coupled to tandem mass spectrometry. We also validated the retinal location of the three antigens by immunohistochemisty using MacTel-2 sera as primary antibodies and commercial antibodies. RESULTS: Retinal autoantibodies were detected in a significantly higher proportion of patients with MacTel-2 than in controls (31 of 45 [69%] vs. 9 of 58 [16%], P < 0.0001). The three antigens that were targeted by the most frequently detected MacTel-2 autoantibodies were identified as glycogen debranching enzyme (hereafter AGL, named for the gene symbol AGL), retinol-binding protein 3 (RBP3), and creatine kinase type B (CK-B); autoantibodies against these antigens were found in four, eleven, and nine MacTel-2 serum samples, respectively. CONCLUSIONS: We found that most patients with MacTel-2 possess retinal autoantibodies, the most prevalent of which were directed against AGL, RBP3, and CK-B. The localization of retinal proteins bound by AGL, RBP3, and CK-B autoantibodies is consistent with their putative physiological functions. These findings provide potentially novel mechanisms for the etiology and pathogenesis of MacTel-2.

Alpha actinin is specifically recognized by Multiple Sclerosis autoantibodies isolated using an N-glucosylated peptide epitope.

Sophisticated approaches have recently led to the identification of novel autoantigens associated with Multiple Sclerosis (MuS), e.g. neurofascin, contactin, CNPase, and other T-cell receptor membrane anchored proteins. These putative antigens, although differing from the conventional myelin derivatives, are conceptually based on an animal model of experimental autoimmune encephalomyelitis. In this report we describe the identification of putative antigens based on their recognition by autoantibodies isolated from MuS patient serum. In a previous work from this laboratory we have shown that a peptide probe, named CSF114(Glc), specifically identifies serum autoantibodies in a subset of MuS patients, representing ∼30% of the patient population. The autoantibodies, purified from MuS patients' sera (six), through CSF114(Glc) affinity chromatography, detected three immunoreactive protein bands present in the rat brain. Proteomic analysis of the immunoreactive bands, involving MALDI and MS/MS techniques, revealed the presence of four proteins distinguishable by their mass: alpha fodrin, alpha actinin 1, creatine kinase, and CNPase. The immunoreactive profile of these rat brain proteins was compared with that of commercially available standard proteins by challenging against either CSF114(Glc) purified MuS autoantibodies, or monoclonal antibodies. Further discrimination among the rat brain proteins was provided by the following procedure: whereas monoclonal antibodies recognized all rat brain proteins, isolated MuS specific antibodies recognize only alpha actinin 1 as a putative antigen. In fact, alpha actinin 1 displayed a robust immunoreactive response against all MuS patients' sera examined, whereas the other three bands were not consistently detectable. Thus, alpha actinin 1, a cytoskeleton protein implicated in inflammatory/degenerative autoimmune diseases (lupus nephritis and autoimmune hepatitis) might be regarded as a novel MuS autoantigen, perhaps a prototypic biomarker for the inflammatory/degenerative process typical of the disease.