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.

Anti-phosphatidylserine/prothrombin antibodies in patients with polyarteritis nodosa.

INTRODUCTION: Anti-phospatidylserine/prothrombin (aPS/PT) antibodies have been described in cutaneous Polyarteritis Nodosa (PAN) in association with specific manifestations. OBJECTIVES: To determine aPS/PT antibodies in patients with PAN and its correlation with clinical manifestations. METHODS: Cross-sectional comparative study including PAN patients and 20 controls (10 Microscopic Polyangiitis [MPA] and 10 Behçet's disease [BD]). Clinical and demographic variables, treatment, serologic markers, prognosis, activity and damage indexes were evaluated. aPS/PT, anti-cardiolipin (aCL), anti-beta 2 glycoprotein 1 (anti-B2GP1) antibodies, and lupus anticoagulant (LA) were determined. RESULTS: Fourteen patients with PAN were included, 11 (79%) women, with disease duration of 207 months, and mostly inactive disease. Only one patient with PAN and one with BD were positive for aPS/PT IgG. LA was the most frequent antibody identified. One patient with MPA and one with BD were positive for aCL IgM; one with MPA for anti-B2GP1 IgG, and one with PAN for anti-B2GP1 IgM. CONCLUSIONS: aPS/PT antibodies are not frequent in patients with longstanding inactive PAN.

Cytokines secretion from human mesenchymal stem cells induced by bovine bone matrix.

BACKGROUND: Bovine bone matrix is a natural material that has been used in the treatment of bone lesions. In this study, bovine bone matrix Nukbone® (NKB) was investigated due its osteoconductive and osteoinductive properties. This biomaterial induces CBFA-1 activation and osteogenic differentiation, although the cytokines involved in these processes is still unknown. OBJECTIVE: The aim of this work was to determine the influence of NKB on the pro-osteoblastic and anti-osteoblastic cytokines secretion from human mesenchymal stem cells (hMSCs). METHODS: The hMSCs were cultured onto NKB and cytokines IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ and TNF-α were analized at 0-14 days by immunoassay. In addition, hemocompatibility of NKB and characterization of hMSCs were evaluated. RESULTS: NKB induces an increase on pro-osteoblastic cytokine secretion IL-4 and a decrease on anti-osteoblastic cytokine IL-6 secretion, at days 7 and 14 of cell culture. Interestingly, there was no statistical difference between secretion profiles of others cytokines analized. CONCLUSIONS: The up-regulation of IL-4 and down-regulation of IL-6, and the secretion profiles of other cytokines examined in this work, are findings that will contribute to the understanding of the role of NKB, and similar biomaterials, in bone homeostasis and in the osteoblastic differentiation of hMSCs.

Alternative complement pathway activation in thrombotic microangiopathy associated with lupus nephritis.

INTRODUCTION/OBJECTIVE: Thrombotic microangiopathy (TMA) in systemic lupus erythematosus is a rare manifestation associated with activation of the complement system. This study aimed to compare plasma and urine complement activation products between patients with active lupus nephritis (aLN) and those with acute TMA plus concomitant active LN (aTMA+aLN). METHODS: Plasma and urine samples were obtained from 20 patients with aTMA+aLN, 20 patients with aLN matched by the histological activity index, 5 patients with chronic TMA, 20 patients with inactive LN, and 10 kidney donors. Complement fragments C3a, C4a, C4d, Ba, C5a, C5bC9, and factor H were determined by ELISA; and kidney C4d deposition was detected by immunohistochemistry. Patients were followed for > 12 months and complement activation products re-measured after treatment in 10 aTMA+aLN patients. RESULTS: Both aTMA+aLN and aLN groups had increased circulating C3a, Ba, and C5bC9; and decreased circulating C3, C4, C4a, C4d, and factor H. Urinary C3a, C5a, Ba, and C5bC9 were higher in patients with aTMA+aLN than in aLN. After treatment, levels of circulating C3, C4, and factor H increased; while levels of urinary C3a, C5a, Ba, and C5bC9 decreased in patients with aTMA+aLN. These changes were observed at each aTMA episode in two patients studied during repeated TMA episodes. There was no difference in C4d deposition in glomerular capillaries, tubular basement membrane, peritubular capillaries, and arterioles between patients with aLN and those aTMA+aLN. CONCLUSIONS: Circulating and urine complement activation products suggest that thrombotic microangiopathy associated with LN is mediated through activation of the alternative complement pathway. Key Points • Immune-complex kidney disease in systemic lupus erythematosus (SLE) is associated with activation of the classical, lectin, and alternative complement pathways • Indirect evidence from measurement of circulating and urinary complement pathway activation products suggests that renal acute thrombotic microangiopathy in SLE is mediated by activation of the alternative complement pathway • C4d kidney immunohistochemistry may be positive in both immune complex nephritis and thrombotic microangiopathy. Therefore, it is not a specific marker of renal thrombotic microangiopathy in SLE.

The Transcription Factor SCX is a Potential Serum Biomarker of Fibrotic Diseases.

Fibrosing diseases are causes of morbidity and mortality around the world, and they are characterized by excessive extracellular matrix (ECM) accumulation. The bHLH transcription factor scleraxis (SCX) regulates the synthesis of ECM proteins in heart fibrosis. SCX expression was evaluated in lung fibroblasts and tissue derived from fibrotic disease patients and healthy controls. We also measured SCX in sera from 57 healthy controls, and 56 Idiopathic Pulmonary Fibrosis (IPF), 40 Hypersensitivity Pneumonitis (HP), and 100 Systemic Sclerosis (SSc) patients. We report high SCX expression in fibroblasts and tissue from IPF patients versus controls. High SCX-serum levels were observed in IPF (0.663 ± 0.559 ng/mL, p < 0.01) and SSc (0.611 ± 0.296 ng/mL, p < 0.001), versus controls (0.351 ± 0.207 ng/mL) and HP (0.323 ± 0.323 ng/mL). Serum levels of the SCX heterodimerization partner, TCF3, did not associate with fibrotic illness. IPF patients with severely affected respiratory capacities and late-stage SSc patients presenting anti-topoisomerase I antibodies and interstitial lung disease showed the highest SCX-serum levels. SCX gain-of-function induced the expression of alpha-smooth muscle actin (α-SMA/ACTA2) in fibroblasts when co-overexpressed with TCF3. As late and severe stages of the fibrotic processes correlated with high circulating SCX, we postulate it as a candidate biomarker of fibrosis and a potential therapeutic target.

Anti-Phosphatidylserine/Prothrombin antibodies in patients with Polyarteritis Nodosa. / Anticuerpos antifosfatidilserina/protrombina en pacientes con poliarteritis nodosa.

INTRODUCTION: Anti-phosphatidylserine/prothrombin (aPS/PT) antibodies have been described in cutaneous Polyarteritis Nodosa (PAN) in association with specific manifestations. OBJECTIVES: To determine aPS/PT antibodies in patients with PAN and its correlation with clinical manifestations. METHODS: Cross-sectional comparative study including PAN patients and 20 controls (10 Microscopic Polyangiitis [MPA] and 10 Behçet's disease [BD]). Clinical and demographic variables, treatment, serological markers, prognosis, activity and damage indexes were evaluated. aPS/PT, anti-cardiolipin (aCL), anti-beta 2 glycoprotein 1 (anti-B2GP1) antibodies, and lupus anticoagulant (LA) were determined. RESULTS: Fourteen patients with PAN were included, 11 (79%) women, with disease duration of 207 months, and mostly inactive disease. Only one patient with PAN and one with BD were positive for aPS/PT IgG. LA was the most frequent antibody identified. One patient with MPA and one with BD were positive for aCL IgM; one with MPA for anti-B2GP1 IgG, and one with PAN for anti-B2GP1 IgM. CONCLUSIONS: aPS/PT antibodies are not frequent in patients with longstanding inactive PAN.

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.

Inhibition of HIV-1 envelope-dependent membrane fusion by serum antilymphocyte autoantibodies is associated with low plasma viral load.

The HIV-1 envelope protein (Env) mediates the membrane fusion process allowing virus entry to target cells and the efficiency to induce membrane fusion is an important determinant of HIV-1 pathogenicity. In addition to virus receptors, other adhesion/signaling molecules on infected and target cells and virus particles can enhance fusion. The presence of antilymphocyte autoantibodies (ALA) in HIV patients' serum suggests that they may contribute to the inhibition of Env-mediated membrane fusion. Here, sera from 38 HIV-1 infected treatment-naïve men and 30 healthy donors were analyzed for the presence of IgG and IgM able to bind to CD4-negative Jurkat cells. The use of CD4-negative cells precluded the binding of virus-antibody immune complexes, and allowed detection of ALA different from anti-CD4 antibodies. IgG and IgM antibodies binding to Jurkat CD4-negative cells was detected in 74% and 84% of HIV-positive sera, respectively. Then, the activity of sera on fusion of CD4+ with HIV Env+ Jurkat cells was determined before and after their adsorption on CD4-negative Jurkat cells to remove ALA. Sera inhibited fusion at variable extents, and inhibitory activity decreased in 58% of serum samples after adsorption, indicating that ALA contributed to fusion inhibition in these sera (herein called fusion inhibitory ALA). The contribution of ALA to fusion inhibition in individual sera was highly variable, with an average of 33%. IgG purified from a pool of HIV+ sera inhibited fusion of primary CD4 T lymphocytes with Jurkat Env+, and adsorption of IgG on CD4-negative Jurkat cells diminished the fusion inhibitory activity. Thus, the inhibitory activity of sera was related to IgG ALA. Our observations suggest that fusion inhibitory ALA other than anti-CD4 antibodies may contribute significantly to the inhibition of Env-mediated cell-cell fusion. Fusion inhibitory ALA, but not total ALA levels, associated with low plasma viral loads, suggesting that specific ALA may participate in virus containment by inhibiting virus-cell fusion in a significant fraction of HIV-infected patients.

Myocardial fibrosis detected by magnetic resonance imaging, elevated U-CRP and higher mRSS are predictors of cardiovascular complications in systemic sclerosis (SSc) patients.

INTRODUCTION: In previous studies we showed that prevalence of myocardial fibrosis as assessed by late enhancement on cardiac MRI in SSc patients is 45% and is associated to diffuse disease (dcSSc) and lower left ventricle ejection fraction; microvascular damage defined as decreased perfusion on cardiac MRI after adenosine infusion, was also very frequent (79%). Our aim was to identify baseline characteristics associated to the development of cardiovascular outcomes (heart failure, coronary artery disease, arrhythmias, vasculopathy, elevated systolic pulmonary artery pressure and death) in SSc patients with previously documented myocardial fibrosis and microvascular damage. PATIENTS AND METHODS: We included 62 SSc patients who participated in the study of prevalence of myocardial fibrosis (2008-2010) and in our local SSc cohort. We performed baseline clinical evaluation, cardiac MRI, coronary CT angiography, transthoracic echocardiogram, and yearly clinical and cardiovascular evaluation that included Medsger's severity scale items, electrocardiogram, echocardiogram, chest X-ray or HRCT and spirometry; we registered presence and severity of internal organ involvement and cardiovascular outcomes. Ordinal variables were analyzed using Chi square test and Fisher test when appropriate, numeric variables were compared using Student's t-test or Mann Whitney U when appropriate, logistic regression and Cox proportional hazard ratio were used to perform multivariable analysis. RESULTS: We obtained follow-up information from 62 patients (29 dcSSc, 33 lcSSc), mean follow-up was 43.5 months. Multivariable analysis showed that elevated basal ultrasensitive CRP was associated to mortality (p = 0.004, OR: 11.9, 95% CI 2.1-65.7) and recurrent digital tip ischemic ulcers (p = 0.001, OR 26.8, 95% CI 3,9-181.3) on follow-up. Myocardial fibrosis, particularly in the middle segments (p = 0.01, OR: 11.49, 95% CI 1.6-83), and older age (p = 0.02, OR: 1.11, 95% CI 1.01-1.22) were associated to heart failure on follow-up. Higher maximum mRSS was associated to coronary artery disease (p = 0.02, OR: 1.2, 95% CI 1.02-1.38), while insertion point fibrosis (p = 0.001, OR: 12.5 95% CI 2.7-56.6) was associated to recurrent digital tip ischemic ulcers. CONCLUSIONS: This study shows that myocardial fibrosis, elevated ultrasensitive CRP, and higher maximum mRSS are independent predictors of cardiovascular outcomes in SSc patients. Future studies should focus on early preventive and therapeutic strategies for this group of patients.

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.

Anti-RNP/Sm antibodies in patients with systemic lupus erythematosus and its role in thrombosis: a case-control study.

OBJECTIVE: To validate the association of thrombotic events with positive lupus anticoagulant (LA) and co-presence of anti-RNP/Sm, as well as the diagnostic accuracy of this combination of antibodies for thrombosis. METHODS: Case-control study of patients with systemic lupus erythematosus (SLE) who presented thrombosis after SLE diagnosis and controls with SLE without thrombosis. Comorbidities, traditional risk factors, clinical variables, and treatment were evaluated. Antiphospholipid (aPL) and anti-RNP/Sm antibodies were determined. RESULTS: Sixty-three cases and 63 controls were studied, 88% women, median age of 40 years, and disease duration of 135 months at study inclusion. No differences were found between groups regarding age, comorbidities, or clinical characteristics at SLE diagnosis. Patients with thrombosis were more frequently positive for anti-RNP/Sm (p = 0.001), IgG aCL (p = 0.02), IgG anti-B2GPI (p = 0.02), IgM anti-B2GPI (p = 0.02), LA (p < 0.001), the combination of anti-RNP/Sm + LA (p < 0.001), and aPL triple marker (p = 0.002), compared to controls. The combination of anti-RNP/Sm + LA, SLEDAI-2 K, and prednisone dose was associated with thrombosis (p < 0.05). The combination of anti-RNP/Sm + LA showed 56% sensitivity, 79% specificity, 73% positive predictive value, 64% negative predictive value, positive likelihood ratio (LR) 2.69, and negative LR 0.56 for predicting thrombosis. No difference was found in the comparison of area under the curve between LA alone and the combination of anti-RNP/Sm + LA (p = 0.73). CONCLUSION: Thrombosis was associated with disease activity, dose of prednisone, and the combination of anti-RNP/Sm antibodies and LA. This combination of antibodies could be useful in the identification of SLE patients at risk of thrombosis.

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.

Cellular immune response to ß2-glycoprotein-I valine/leucine247 phenotypes in Mexican patients with primary antiphospholipid syndrome.

Homozygote genotype V247 of the ß2-glycoprotein-I (ß2GP-I) gene has been associated with anti-ß2GP-I and thrombosis in patients with primary anti-phospholipid syndrome APS (PAPS). However, the cellular immune response to ß2GP-I247 has been little studied. OBJECTIVE: To evaluate the immune cellular proliferation in response to native and non-native ß2GP-I247 valine/leucine phenotype from Mexican patients with PAPS. METHODS: We studied 10 patients with PAPS and 10 healthy control subjects (HC). The polymorphism at position 247 of the ß2GP-I gene was determined by PCR-RFLP and the corresponding ß2GP-I protein was subsequently purified from normal human plasma by affinity chromatography. PBMC purified from patients and controls were stimulated with ß2GP-I under native and in non native (reduced) conditions. We also determined the anti-ß2GP-I production in vitro by B cell clones (EBV) generated in cocultures experiments. Differential Scanning Calorimetry (DSC) was studied to determine the structural differences between the ß2GP-I247 valine/leucine isoforms. Cytokine profile (IL-2, IL-4, IL-6, TNFα, INFγ) was evaluated in culture supernatants. RESULTS: PAPS and healthy control PBMCs had a higher proliferative response when stimulated with ß2GP-I under reduced cultures conditions compared to non-denatured conditions. PBMCs response from PAPS patients was higher. We observed more cell proliferation in response to ß2GP-I247 valine/leucine or valine isoforms in non-native conditions. In contrast, this response was not significant against ß2GP-I247 leucine. These findings were T CD4+-dependent. Similar results were obtained with B cell clones derived from PAPS patients, which showed more pronounced proliferation in non native conditions and higher against ß2GP-I247 valine. No differences were found in anti-ß2GP-I production, but high levels of IL-6 in vitro were identified. The structural analysis of both ß2GP-I247 isoforms by DSC showed a major conformational change due to a single mutation in the ß2GP-I variants. CONCLUSIONS: PAPS PBMCs had a higher cellular response against ß2GP-I247 in non-native culture conditions preferentially to the ß2GP-I247 valine phenotype. This effect is T CD4+ dependent and appears to be driven by tertiary structural changes adopted by ß2GP-I247 polymorphism.

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.

Venous and Arterial Thrombotic Events in Systemic Lupus Erythematosus.

OBJECTIVE: The incidence of thrombosis in patients with systemic lupus erythematosus (SLE) is 25 to 50-fold higher than in the general population; we aimed to define the characteristics of venous thrombotic events (VTE) and arterial thrombotic events (ATE) to identify the patients at highest risk. METHODS: The study included 219 patients with recent-onset SLE. At baseline, standardized medical history and laboratory tests were done. Followup visits occurred quarterly, and information about damage accrual, comorbidities, and cardiovascular risk factors was updated annually. Main outcome was development of TE after SLE diagnosis. RESULTS: Thirty-five patients (16%) developed TE (27 VTE, 8 ATE) during 5.21 years of followup; incidence rate 31/1000 patient-years. Most events (57%) developed within the first year of diagnosis, and 69% were not associated with lupus anticoagulant (LAC), determined with 1 method. VTE developed earlier than ATE (2.0 vs 57.5 mos, p = 0.02). In the multivariate analysis, variables preceding VTE included cutaneous vasculitis, nephrotic syndrome, dose of prednisone, and LAC in combination with anti-RNP/Sm antibodies (p < 0.03). Patients with ATE were older (median age 44 vs 29 yrs, p = 0.04), smokers, and had hypertension, diabetes mellitus, dyslipidemia, at least 2 traditional risk factors, nephrotic syndrome, chronic damage, and a higher cumulative dose of prednisone (p < 0.05). LAC in combination with anti-RNP/Sm antibodies was associated with VTE and improved the accuracy for predicting it. CONCLUSION: Our study suggests that in SLE, VTE and ATE have different risk factors. Understanding these differences is helpful for identifying patients at highest risk. The use of LAC plus anti-RNP/Sm for predicting VTE deserves further study.

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.

Predicting Sjögren's syndrome in patients with recent-onset SLE.

OBJECTIVE: To determine the prevalence of SS in a cohort of recent-onset SLE patients and evaluate the clinical and immunological variables that may identify SLE patients prone to develop SS. METHODS: A total of 103 patients participating in a prospective cohort of recent-onset SLE were assessed for fulfilment of the American European Consensus Group criteria for SS using a three-phase approach: screening (European questionnaire, Schirmer-I test and wafer test), confirmation (fluorescein staining test, non-stimulated whole-salivary flow and anti-Ro/La antibodies) and lip biopsy. Anti-Ro/SSA and anti-La/SSB antibodies and RF were measured at entry into the cohort and at SS assessment. RESULTS: Ninety-three females and 10 males were included. Mean age at lupus diagnosis was 25.9 ± 8.9 years, and lupus duration at SS assessment was 30.9 ± 9.1 years. SS was diagnosed in 19 (18.5%) patients, all female, and the patients were older at SLE diagnosis than patients without SS (30.8 ± 9.3 vs 24 ± 8.8 years, P = 0.004). Anti-Ro/SSA antibody was more common in SLE-SS patients (84% vs 55%, P = 0.02, LR + 1.53, 95% CI 1.14, 2.04). In the multivariate analysis, age ≥25 years and anti-Ro/SSA antibodies at SLE diagnosis were identified as predictors of SLE-SS, while the absence of anti-Ro/SSA, anti-La/SSB and RF seems to be protective (LR- 0.14, 95% CI 0.02, 0.95). CONCLUSION: The overlap of SLE and SS occurs in almost one-fifth of SLE patients and presents early during its evolution. SLE onset at age ≥25 years plus the presence of anti-Ro/SSA antibody at diagnosis are useful predictors, while the absence of anti-Ro/SSA, anti-La/SSB and RF identifies patients at lowest risk.