Characterisation of the novel HLA-DQA1*04:14N allele by next-generation sequencing.

HLA-DQA1*04:14N differs from HLA-DQA1*04:01:01:03 by a single nucleotide deletion in codon 113 in exon 3.

Molecular allergology: a clinical laboratory tool for precision diagnosis, stratification and follow-up of allergic patients.

Identification of the molecular culprits of allergic reactions leveraged molecular allergology applications in clinical laboratory medicine. Molecular allergology shifted the focus from complex, heterogeneous allergenic extracts, e.g. pollen, food, or insect venom, towards genetically and immunologically defined proteins available for in vitro diagnosis. Molecular allergology is a precision medicine approach for the diagnosis, stratification, therapeutic management, follow-up and prognostic evaluation of patients within a large range of allergic diseases. Exclusively available for in vitro diagnosis, molecular allergology is nonredundant with any of the current clinical tools for allergy investigation. As an example of a major application, discrimination of genuine sensitization from allergen cross-reactivity at the molecular level allows the proper targeting of the culprit allergen and thus dramatically improves patient management. This review aims at introducing clinical laboratory specialists to molecular allergology, from the biochemical and genetic bases, through immunological concepts, to daily use in the diagnosis and management of allergic diseases.

Characterization of the novel HLA-B*15:648 allele by next-generation sequencing.

HLA-B*15:648 differs from HLA-B*15:02:01:01 by one nucleotide substitution in codon 77 in exon 2.

Characterization of the novel HLA-A*33:220 allele by next-generation sequencing.

HLA-A*33:220 differs from HLA-A*33:03:01:01 by one nucleotide substitution in codon 245 in exon 4.

Characterization of the novel HLA-A*01:383 allele by next-generation sequencing.

HLA-A*01:383 differs from HLA-A*01:01:01:01 by two nucleotide substitutions at positions 28 and 48 in exon 1.

Characterization of the novel HLA-B*53:64 allele by next-generation sequencing.

HLA-B*53:64 differs from HLA-B*53:01:01:01 by one nucleotide substitution at position 1617 in exon 4.

Characterization of the novel HLA-C*07:1001N allele by next-generation sequencing.

HLA-C*07:1001N differs from HLA-C*07:01:01:01 by a deletion of 17 nucleotides in exon 1.

Characterization of the novel HLA-C*03:04:94 allele by next-generation sequencing.

HLA-C*03:04:94 differs from HLA-C*03:04:01:01 by one nucleotide substitution at position 737 in exon 4.

Characterization of the novel HLA-C*16:184 allele by next-generation sequencing.

HLA-C*16:184 differs from HLA-C*16:02:01:01 by one nucleotide substitution at position 737 in exon 3.

Characterization of the novel HLA-C*05:255 allele by next-generation sequencing.

HLA-C*05:255 differs from HLA-C*05:01:01:02 by one nucleotide substitution at position 2013 in exon 5.

Characterization of the novel HLA-C*06:317 allele by next-generation sequencing.

HLA-C*06:317 differs from HLA-C*06:02:01:01 by one nucleotide substitution at position 921 in exon 3.

Characterization of the novel HLA-B*35:29:03 allele by next-generation sequencing.

HLA-B*35:29:03 differs from HLA-B*35:29:01 by one nucleotide substitution at position 374 in exon 2.

Absence of Anti-Glomerular Basement Membrane Antibodies in 200 Patients With Systemic Lupus Erythematosus With or Without Lupus Nephritis: Results of the GOODLUPUS Study.

Introduction: Anti-glomerular basement membrane (GBM) antibodies are pathogenic antibodies first detected in renal-limited anti-GBM disease and in Goodpasture disease, the latter characterized by rapidly progressive crescentic glomerulonephritis combined with intra-alveolar hemorrhage. Studies have suggested that anti-GBM antibody positivity may be of interest in lupus nephritis (LN). Moreover, severe anti-GBM vasculitis cases in patients with systemic lupus erythematosus (SLE) have been described in the literature, but few studies have assessed the incidence of anti-GBM antibodies in SLE patients. Objective: The main study objective was to determine if positive anti-GBM antibodies were present in the serum of SLE patients with or without proliferative renal damage and compared to a healthy control group. Methodology: This retrospective study was performed on SLE patients' sera from a Franco-German European biobank, developed between 2011 and 2014, from 17 hospital centers in the Haut-Rhin region. Patients were selected according to their renal involvement, and matched by age and gender. The serum from healthy voluntary blood donors was also tested. Anti-GBM were screened by fluorescence enzyme immunoassay (FEIA), and then by indirect immunofluorescence (IIF) in case of low reactivity detection (titer >6 U/ml). Results: The cohort was composed of 100 SLE patients with proliferative LN (27% with class III, 67% with class IV, and 6% with class V), compared to 100 SLE patients without LN and 100 controls. Patients were mostly Caucasian and met the ACR 1997 criteria and/or the SLICC 2012 criteria. Among the 300 tested sera, no significant levels of anti-GBM antibodies were detected (>10 U/ml) by the automated technique, three sera were found "ambivalent" (>7 U/ml): one in the SLE with LN group and two in the SLE without LN group. Subsequent IIF assays did not detect anti-GBM antibodies. Conclusion: Anti-GBM antibodies were not detected in the serum of Caucasian patients with SLE, even in case of renal involvement, a situation favoring the antigenic exposure of glomerular basement membranes. Our results reaffirm the central role of anti-GBM antibodies as a specific diagnostic biomarker for Goodpasture vasculitis and therefore confirm that anti-GBM antibody must not be carried out in patients with SLE (with or without LN) in the absence of disease-suggestive symptoms.

NET Formation in Bullous Pemphigoid Patients With Relapse Is Modulated by IL-17 and IL-23 Interplay.

Background: DNA extracellular traps (ETs), released by neutrophils (NETs), or eosinophils (EETs), play a pathogenic role in several autoimmune disorders. However, to date, NETs have never been investigated in bullous pemphigoid (BP) with respect to clinical and immunological activities, both at baseline and at time of relapse which have been characterized with specific IL-17 and IL-23 patterns. Objective: We sought to assess whether ETs were associated with BP as well as the relative contribution of IL-17 axis cytokines to NET induction. Methods: Skin biopsy specimens were obtained from 11 patients with BP. Immuno-detection of neutrophils and eosinophils combined to DNA staining allowed us to investigate the in-situ presence of NETs and EETs using confocal scanning microscopy. NETs release was evaluated ex vivo by stimulating polymorphonuclear cells from BP patients with BP biological fluids in presence of IL-17A and IL-23 or of glucocorticoids. Results: At baseline, ETs were observed in BP lesions at the site of dermal-epidermal cleavage. Despite an important infiltrate of eosinophils, ETs were essentially associated with neutrophils in situ and were not related to BP clinical activity at diagnosis. In situ observation of NETs was associated in 6 among 8 patients with serum capacity of NET induction. Notably both blister fluid and sera from BP patients at diagnosis and at time of relapse could induce NET formation ex vivo. In contrast, a longitudinal investigation showed a decrease of NET formation with time of treatment in patients undergoing remission. Mimicking relapse, complementation of sera from BP patients with ongoing remission with either IL-17A or IL-23 increased NET formation. Conversely, IL-17A inhibited NET formation induced by serum from BP patients with relapse supplemented or not with IL-23. Finally, glucocorticoids also inhibited NET formation ex vivo in BP. Conclusion: NET formation is an associated phenomenon with BP. Furthermore, we showed that IL-23 favored NET formation, whereas the effects of IL-17A are environment dependent. Indeed, IL-17A displayed a protective effect on NET formation when associated with IL-23, showing for the first-time differential effects of these two cytokines in BP.

Mucosal Involvement in Bullous Pemphigoid Is Mostly Associated with Disease Severity and to Absence of Anti-BP230 Autoantibody.

Bullous pemphigoid (BP) is the most common autoimmune bullous disease and typically affects the elderly. Binding of specific autoantibodies to BP180/230 hemidesmosomal components induces an inflammatory response leading to skin blister formation. Unusual manifestations of BP include additional mucous membrane involvement, without pathophysiological knowledge associated to the formation of these lesions. We here performed a prospective study on series of consecutive BP patients with (n = 77) and without (n = 18) mucosal involvements at baseline to further investigate why some BP patients display mucosal lesion and other not. Analysis of disease activity showed that BP patients with mucosal involvement displayed a higher total BP Disease Area Index (BPDAI) score (P = 0.008), but also higher skin and blister/erosion BPDAI scores (P = 0.02 and P = 0.001, respectively). By contrast, the erythema/urticaria BPDAI score was identical between the two groups of patients. The erythema/urticaria BPDAI score, but not the blister/erosion BPDAI score, was correlated with the serum concentration of anti-BP180 NC16A autoantibodies in patients with mucosal involvement. In multivariate analysis, the absence of anti-BP230 autoantibody was the only factor independently associated with mucosal involvement (OR 7.8; 95% CI, 3.1-19.6) (P < 0.0001). Analysis of the distribution of BP patients according to BPDAI scores revealed a shift toward higher blister/erosion BPDAI scores for BP patients with mucosal involvement. This study indicates that mucosal lesions are clinically mainly related to disease severity and immunologically to the absence of anti-BP230 antibodies.

Anti-Type VII Collagen Antibodies Are Identified in a Subpopulation of Bullous Pemphigoid Patients With Relapse.

Bullous pemphigoid (BP) is an autoimmune bullous skin disease characterized by anti-BP180 and anti-BP230 autoantibodies (AAbs). Mucous membrane involvement is an uncommon clinical feature of BP which may evoke epidermolysis bullosa acquisita, another skin autoimmune disease characterized by anti-type VII collagen AAbs. We therefore evaluated the presence of anti-type VII collagen AAbs in the serum of BP patients with and without mucosal lesions at time of diagnosis and under therapy. Anti-BP180, anti-BP230, and anti-type VII collagen AAbs were measured by ELISA in the serum of unselected patients fulfilling clinical and histo/immunopathological BP criteria at baseline (n = 71) and at time of relapse (n = 24). At baseline, anti-type VII collagen AAbs were detected in 2 out of 24 patients with BP presenting with mucosal involvement, but not in patients without mucosal lesions (n = 47). At the time of relapse, 10 out of 24 BP patients either displayed a significant induction or increase of concentrations of anti-type VII collagen AAbs (P < 0.01), independently of mucosal involvement. Those 10 relapsing BP patients were also characterized by a sustained high concentration of anti-BP180 AAb, whereas the serum anti-BP230 AAb concentrations did not vary in BP patients with relapse according to the presence of anti-type VII collagen AAbs. Thus, our study showed that anti-type VII collagen along with anti-BP180 AAbs detection stratified BP patients at time of relapse, illustrating a still dysregulated immune response that could reflect a potential epitope spreading mechanism in those BP patients.

Biomarkers related to bullous pemphigoid activity and outcome.

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease of the skin. Investigation of the BP-associated pathophysiological processes during the last decades showed that the generation of autoantibodies directed against the hemidesmosome proteins BP180 and BP230, a hallmark of the BP-associated autoimmune response, leads to the recruitment of inflammatory immune cells at the dermal-epidermal junction, and subsequently to the release of a large amount of inflammatory molecules involved in blister formation. Analysis in transversal and longitudinal studies of autoantibodies and inflammatory molecules production both at the time of diagnosis and under treatment was mainly performed within the serum but also in the blister fluid. Some autoimmune or inflammatory molecules expression was related to the presence of clinical signs, while others were mere bystanders. In this review, we focused on the autoimmune and inflammatory molecules that have been identified as potential biomarkers of BP development and outcome.

Eosinophil Cationic Protein (ECP), a predictive marker of bullous pemphigoid severity and outcome.

Bullous Pemphigoid (BP) is an inflammatory rare autoimmune bullous dermatosis, which outcome cannot be predicted through clinical investigations. Eosinophils are the main immune infiltrated cells in BP. However, the release of Major Basic Protein (MBP), Eosinophil Derived Neurotoxin (EDN), and Eosinophil Cationic Protein (ECP) upon eosinophil activation has still not been evaluated with respect to BP development. MBP, EDN and ECP were measured by ELISA in serum (n = 61) and blister fluid (n = 20) of patients with BP at baseline, and in serum after 2 months of treatment (n = 41). Eosinophil activation in BP patients was illustrated at baseline by significantly higher MBP, EDN and ECP serum concentrations as compared with control subjects (n = 20), but without distinction according to disease severity or outcome. EDN and ECP values were even higher in the blister fluids (P < 0.01 and P < 0.05, respectively), whereas MBP values were lower (P < 0.001). ECP serum concentration decreased after 60 days of treatment in BP patients with ongoing remission but not in patients who later relapsed (P < 0.05). A reduction of at least 12.8 ng/mL in ECP concentrations provided a positive predictive value for remission of 81%, showing that ECP serum variation could be a useful biomarker stratifying BP patients at risk of relapse.

Interpretation of serological tests in the diagnosis of celiac disease: Anti-deamidated gliadin peptide antibodies revisited.

Algorithms for celiac disease diagnosis provided by guidelines are based primarily on anti-tissue transglutaminase 2 (TG2) antibodies and/or anti-endomysium antibodies. The place of anti-deamidated gliadin peptide (DGP) antibodies is less well established. This study was designed to assess the clinical relevance of anti-DGP antibodies. Two thousand and twenty-six consecutive unselected patients systematically tested for anti-TG2, endomysium, gliadin, DGP antibodies and IgA dosage were investigated. The serological interpretation was assessed by analyzing the medical records of patients. From the 1984 newly investigated patients suspected of celiac disease, 10% had at least one celiac marker. Anti-TG2, anti-endomysium, anti-gliadin and anti-DGP antibodies were found in 1.1%, 0.6%, 6.8% and 4.1% of cases respectively, with different combinations. The diagnosis of celiac disease was retained in 0.45% of patients. When using the duodenal biopsies as a gold standard, analysis of the anti-DGP diagnosis performance showed that the specificity and the predictive positive value (PPV) were lower than that of the anti-TG2 assay. The combined detection of anti-TG2 and anti-DGP antibodies had a lower PPV than that of anti-TG2 and anti-endomysium antibodies (p = 0.04). When analyzing the contribution of anti-DGP antibodies as an additional marker to both anti-TG2 and anti-endomysium antibodies, the PPV of the three associated antibodies was shown to be significantly lower than the PPV of the both anti-TG2 and anti-endomysium antibodies (p = 0.04). As a conclusion, anti-DGP antibodies may not have the diagnosis value required as an additional screening test to anti-TG2 antibodies for identifying celiac disease patients in medical centers where anti-endomysium detection is available.

Medicarpin and millepurpan, two flavonoids isolated from Medicago sativa, induce apoptosis and overcome multidrug resistance in leukemia P388 cells.

BACKGROUND: High consumption of flavonoids has been associated with a decrease risk of cancer. Alfalfa (Medicago sativa) leaves have been widely used in traditional medicine and is currently used as a dietary supplement because of their high nutrient content. We previously reported the cytotoxic activity of alfalfa leaf extracts against several sensitive and multidrug resistant tumor cell lines. HYPOTHESIS/PURPOSE: We aimed to determine whether medicarpin and millepurpan, two isoflavonoids isolated from alfalfa leaves, may have pro-apoptotic effects against drug-sensitive (P388) and multidrug resistant P388 leukemia cells (P388/DOX). STUDY DESIGN/METHODS: Cells were incubated with medicarpin or millepurpan for the appropriate time. Cell viability was assessed by the MTT assay. DNA fragmentation was analyzed by agarose gel electrophoresis. Cell cycle analysis was realized by flow cytometry technics. Caspases 3 and 9 activities were measured using Promega caspACE assay kits. Proteins and genes expression were visualized respectively by western-blot using specific antibodies and RT-PCR assay. RESULTS: P-glycoprotein-expressing P388/DOX cells did not show resistance to medicarpin (IC50 ≈ 90 µM for P388 and P388/DOX cells) and millepurpan (IC50 = 54 µM and 69 µM for P388 and P388/DOX cells, respectively). Treatment with medicarpin or millepurpan triggered apoptosis in sensitive as well as multidrug resistant P388 cells. These effects were mediated through the mitochondrial pathway by modifying the balance pro/anti-apoptotic proteins. While 3 µM doxorubicin alone could not induce cell death in P388/DOX cells, concomitant treatment with doxorubicin and subtoxic concentration of medicarpin or millepurpan restored the pro-apoptotic cascade. Each compound increased sensitivity of P388/DOX cells to doxorubicin whereas they had no effect in sensitive P388 cells. Vinblastine cytotoxicity was also enhanced in P388/DOX cells (IC50 = 210 nM to 23 and 25 nM with medicarpin and millepurpan, respectively). This improved sensitivity was mediated by an increased uptake of doxorubicin in P388/DOX cells expressing P-gp. P-gp expression was not altered by exposure to medicarpin and millepurpan. CONCLUSION: These data indicate that medicarpin and millepurpan possess pro-apoptotic properties and potentiate the cytotoxicity of chemotherapy drugs in multidrug resistant P388 leukemia cells by modulating P-gp-mediated efflux of drugs. These flavonoids may be used as chemopreventive agents or as sensitizer to enhance cytotoxicity of chemotherapy drugs in multidrug resistant cancer cells.