Autoantigen characterization in the lower esophageal sphincter muscle of patients with achalasia.

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.

Esophagogastric junction outflow obstruction: Characterization of a new entity? Clinical, manometric, and neuroimmunological description.

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.

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.

The AIRE Ser196Ser synonymous variant is a risk factor for systemic lupus erythematosus.

The AIRE gene influences the expression of a wide array of self-antigens in the thymus, and is essential to the negative selection of self-reactive T cells and establishment of central tolerance. Single nucleotide variants (SNVs) such as rs878081C/T (Ser196Ser) and rs2075876G/T at this locus have been associated with susceptibility to rheumatoid arthritis, mainly in Asian populations, but its role in systemic lupus erythematosus (SLE) has not been documented. We performed a case-control association study with 379 SLE patients and 460 controls from central Mexico. In addition, we replicated our finding in another group of 179 SLE patients and 97 controls from the same region of Mexico. In the first group, we identified that the AIRE Ser196Ser synonymous variant was associated with SLE (OR 1.4, p = 0.009), meanwhile, in the second group we observed the following: OR 1.7, p = 0.024. No association was found between these AIRE SNVs and lupus nephritis. Our results suggest that AIRE is a risk factor for SLE in our population. This study is the first to document an association between AIRE and SLE susceptibility.

Prevalence and associations of anti-phosphatidylserine/prothrombin antibodies with clinical phenotypes in patients with primary antiphospholipid syndrome: aPS/PT antibodies in primary antiphospholipid syndrome.

OBJECTIVE: The clinical significance of anti-phosphatidylserine/prothrombin (aPS/PT) in antiphospholipid syndrome (APS) is still controversial. We assessed the prevalence of aPS/PT antibodies, their association with other anti-phospholipid antibodies (aPL) and with different APS clinical phenotypes. METHODS: We included 95 primary APS patients according to the Sydney classification criteria, and patients with thrombocytopenia and/or hemolytic anemia who also fulfilled the serological APS criteria. We tested aCL, anti-ß2GP-I and aPS/PT antibodies (both IgG and IgM isotypes) and lupus anticoagulant (LA). We used χ2 test, Spearman's correlation coefficient, Mann-Whitney U test and logistic regression. RESULTS: Seventy-seven percent of patients had thrombosis, 50% hematologic involvement and 25% obstetric events (non-exclusive groups). Twenty patients had only hematologic features. The prevalence of IgG and IgM aPS/PT antibodies was 61% and 60%, respectively. Patients with LA+ had a higher prevalence and higher titers of IgG and IgM aPS/PT antibodies. aPS/PT antibodies correlated with aPL antibodies including LA. IgG aPS/PT antibodies were associated with thrombosis (OR 8.6 [95% CI 2.13-33.8, p = 0.002]) and pure hematologic features (OR 0.2, CI 95% 0.05-0.97, p = 0.004). IgM anti-ß2GP-I antibodies conferred high risk for both hematologic (OR 7.9, 95% CI 1.88-34.61, p = 0.006) and thrombotic involvement (OR 7.4, 95% CI 1.76-31.12, p = 0.006). CONCLUSIONS: aPS/PT antibodies were highly prevalent and correlated with other aPL antibodies. IgG aPTS/PT conferred a high risk for thrombosis, but not for pure hematologic involvement. aPS/PT antibodies may be a useful serological tool in the diagnosis and phenotypic characterization of APS patients.

α-enolase is an antigenic target in primary Sjögren's syndrome.

OBJECTIVES: Although anti-cyclic citrullinated peptides antibodies are specific markers for rheumatoid arthritis (RA), they might be present in other diseases. Our aim was to assess the native or citrullinated antigens recognised by patients with primary Sjögren's syndrome (pSS) and to evaluate their association with clinical and serological features. METHODS: In an initial screening, we assessed the serum reactivity of 12 patients with pSS against native or in vitro citrullinated antigens of HEp-2 cells by immunoblotting. We identified a 47kDa band, which was preferentially recognised and corresponded to α-enolase. Thus, levels of IgA and IgG anti-native and citrullinated α-enolase antibodies were measured in 50 pSS patients, 20 RA patients and 20 healthy subjects (HS) by ELISA. RESULTS: We identified α-enolase as a predominant antigen recognised in pSS. These patients had higher levels of anti-citrullinated α-enolase IgG antibodies compared with RA or HS (p=0.003 and p<0.0001, respectively). Furthermore, there was an increase of IgG anti-citrullinated α-enolase vs IgG anti-non-citrullinated α-enolase antibodies in pSS patients (p=0.001), by contrast no difference was found in RA. The presence of IgA and IgG anti-non-citrullinated and anti-citrullinated α-enolase antibodies were not associated with any clinical manifestation whatsoever, including non-erosive arthritis among pSS, but an association of IgA anti-citrullinated α-enolase with anti-Ro/SSA antibodies was found. CONCLUSIONS: We characterised α-enolase as a dominant antigen in lysates of HEp- 2 cells in pSS. Nevertheless, their precise role in pSS remains to be elucidated.

Autoimmune comorbidity in achalasia patients.

BACKGROUND AND AIM: Idiopathic achalasia is a rare esophageal motor disorder. The disease state manifests local and systemic inflammation, and it appears that an autoimmune component and specific autoantibodies participate in the pathogenesis. The study aims to determine the prevalence of autoimmune and chronic inflammatory diseases in patients with achalasia and compare the results with those from patients with gastroesophageal reflux disease (GERD). METHODS: It was a cross-sectional and included 114 patients with idiopathic achalasia and 114 age-matched and sex-matched control patients with GERD. Data on the presence of autoimmune and inflammatory diseases, the time of presentation, and any family history of autoimmune disease were obtained from the hospital's medical records. RESULTS: Seventy three (64%) were female patients (mean age: 42.3 ± 15.5; median disease duration: 12 months). We identified the presence of autoimmune disease in 19 patients with achalasia (16.7%), hypothyroidism was the main diagnosis, and it was present in 52.6% of patients compared with 4.2% in controls. Thirteen of the 19 achalasia patients (68.4%) with autoimmune disease had history of familial autoimmunity. We identified 11 achalasia (9.6%) and 5 GERD patients (4.16%) with an inflammatory condition. Compared with the GERD, the achalasia group was 3.8 times more likely to have an autoimmune disease (95% CI: 1.47-9.83), 3.0 times more likely to have thyroidopathies (95% CI: 1.00-9.03), and 3.02 times more likely to suffer from any chronic inflammatory disease (95% CI: 1.65-6.20). CONCLUSIONS: The non-negligible number of patients with autoimmune diseases identified among the patients with idiopathic achalasia supports the hypothesis that achalasia has an autoimmune component.

Refractory ascites in systemic lupus erythematosus: further biological support of intraperitoneal steroid treatment as a suitable therapeutical option.

The objective of this report was to evaluate the ascitic fluid of a patient with refractory lupus ascites (proband) at different time points-pre- and post-intraperitoneal treatment with dexamethasone-using a multiparametric approach which included the presence of autoantibodies and pro- and anti-inflammatory cytokines and chemokines, and a proteomic analysis. As controls, we studied two additional patients also with lupus ascites (only at basal evaluation) and two patients with ascites due to alcoholic liver cirrhosis. High levels of anti-dsDNA and anti-nucleosomes autoantibodies were detected in the ascitic fluid of all lupus patients and remained elevated in the proband throughout the follow-up. All lupus patients have detectable ascitic high levels of IL-6, IL-8, IL-10, TNF-α, MCP-1, and IGF-1 which diminished gradually in the proband after intraperitoneal dexamethasone. In the proteomic analysis of the ascitic fluid, a marked increment of apolipoprotein A1 was observed and again, it diminished gradually after intraperitoneal treatment. Our findings further support the use of intraperitoneal steroids as an effective therapeutic option for refractory ascites in systemic lupus erythematosus.

The amount of citrullinated proteins in synovial tissue is related to serum anti-cyclic citrullinated peptide (anti-CCP) antibody levels.

The objective of this study was to determine the relationship between citrullinated proteins in synovial tissue with peripheral anti-citrullinated peptides autoantibodies (ACPA) and peptidylarginine deiminase (PADI) PADI2, PADI3, and PADI4 messenger RNA (mRNA) expressions in synovial tissue and fibroblast-like synoviocytes in rheumatoid arthritis (RA) patients. Eleven RA and 12 osteoarthritis (OA) patients who underwent knee replacement surgery were studied. We detected citrullinated proteins in synovial tissue homogenates by western blot and serum ACPA by ELISA to anti-cyclic citrullinated peptide (anti-CCP) antibodies, and PADI2, PADI3, and PADI4 mRNA expressions in synovial tissue and in fibroblast-like synoviocytes. Patients with high amount of citrullinated proteins in synovial tissue (3 out of 7) have high levels of anti-CCP in serum. However, in the remaining 4 patients, the amount of synovial citrullinated proteins was minimal and their sera showed low levels of anti-CCP antibodies. Furthermore, we observed an increase in PADI2 mRNA expression in RA synovial tissue compared with OA patients (p = 0.02). We detected PADI3 mRNA in the synovial tissue of RA patients, but not in the tissue of OA patients. Even though fibroblast-type synoviocytes in RA are not the main source of PADs in the synovial tissue, they express PADI2 mRNA moderately, PADI4 mRNA weakly, while there is no detectable expression of PADI3 mRNA. In conclusion, we found a variety of citrullinated proteins in the synovial tissue of RA patients and the amount of such proteins is related to serum concentration of anti-CCP antibodies. We identified the presence of PADI3 mRNA expression in synovial tissue and PADI2 and PADI4 mRNA expressions in fibroblast-like synoviocytes from patients with RA.

The role of ß2-glycoprotein I (ß2GPI) carbohydrate chains in the reactivity of anti-ß2GPI antibodies from patients with primary antiphospholipid syndrome and in the activation and differentiation of U937 cells.

Several studies have shown that conformational changes of ß(2)-glycoprotein I (ß(2)GPI) when bound to negatively charged components expose cryptic epitopes and subsequent binding of anti-ß(2)GPI from patients with antiphospholipid syndrome (APS). However, the role of the carbohydrate chains of ß(2)GPI in this anti-ß(2)GPI reactivity is poorly understood. We therefore studied the reactivity and inhibition of anti-ß(2)GPI antibodies from APS patients with native, partially glycosylated ß(2)GPI (pdß(2)GPI; without sialic acid) and completely deglycosylated ß(2)GPI (cdß(2)GPI). To determine the potential biologic importance of these glycoforms and their interaction with anti-ß(2)GPI in vitro, stimulation assays were performed with the U937 cell line. Circular dichroism (CD) and fluorescence analysis of the three ß(2)GPI forms were also studied. We found an increased reactivity of anti-ß(2)GPI against pdß(2)GPI and cdß(2)GPI compared to native ß(2)GPI. Both deglycosylated ß(2)GPI isoforms showed higher inhibition of the anti-ß(2)GPI reactivity than the native protein in soluble-phase. Likewise, the antibody/ß(2)GPI/glycoform complexes increased the synthesis of IL-6, IFNγ and TNFα and the expression of HLA-DR, CD14 and CD11c in U937 cells. CD and fluorescence studies of the glycoforms yielded considerable changes in the fluorescence signals. Our work suggests that the partial or complete removal of the carbohydrate chains uncover cryptic epitopes present in ß(2)GPI. The differentiation and increased synthesis of pro-inflammatory cytokines by U937 cells in vitro may have pathogenetic implications.

[Citrullinated proteins in rheumatoid arthritis]. / Proteínas citrulinadas en artritis reumatoide.

Rheumatoid arthritis is an autoimmune disease of multifactorial etiology characterized by inflammation of the joints and presence of autoantibodies directed against multiple autoantigens. Recently the study of the anti-citrullinated protein antibodies (ACP) has acquired great interest due to its high specificity and sensitivity for diagnosis, in addition to which it has shown to be a predictor of severity in patients with rheumatoid arthritis, suggesting an important participation in the pathogenesis of the disease.