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.

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.

Cytotoxicity and apoptosis induced by alfalfa (Medicago sativa) leaf extracts in sensitive and multidrug-resistant tumor cells.

Alfalfa (Medicago sativa) has been used to cure a wide variety of ailments. However, only a few studies have reported its anticancer effects. In this study, extracts were obtained from alfalfa leaves and their cytotoxic effects were assessed on several sensitive and multidrug-resistant tumor cells lines. Using the mouse leukaemia P388 cell line and its doxorubicin-resistant counterpart (P388/DOX), we showed that the inhibition of cell growth induced by alfalfa leaf extracts was mediated through the induction of apoptosis, as evidenced by DNA fragmentation analysis. The execution of programmed cell death was achieved via the activation of caspase-3, leading to PARP cleavage. Fractionation of toluene extract (To-1), the most active extract obtained from crude extract, led to the identification of 3 terpene derivatives and 5 flavonoids. Among them, (-)-medicarpin, (-)-melilotocarpan E, millepurpan, tricin, and chrysoeriol showed cytotoxic effects in P388 as well as P388/DOX cells. These results demonstrate that alfalfa leaf extract may have interesting potential in cancer chemoprevention and therapy.

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.

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.

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*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*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*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-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.

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.

Resveratrol induces cell-cycle disruption and apoptosis in chemoresistant B16 melanoma.

Resveratrol, a naturally occurring polyphenol, has been shown to possess chemopreventive activities. In this study, we show that resveratrol (0-500 microM) inhibits the growth of a doxorubicin-resistant B16 melanoma cell subline (B16/DOX) (IC(50) = 25 microM after 72 h, P < 0.05). This was accomplished by imposing an artificial checkpoint at the G(1)-S phase transition, as demonstrated by cell-cycle analysis and down-regulation of cyclin D1/cdk4 and increased of p53 expression level. The G(1)-phase arrest of cell cycle in resveratrol-treated (10-100 microM) B16/DOX cells was followed by the induction of apoptosis, which was revealed by pyknotic nuclei and fragmented DNA. Resveratrol also potentiated at subtoxic dose (25 microM for 24 h) doxorubicin cytotoxicity in the chemoresistant B16 melanoma (P < 0.01). When administered to mice, resveratrol (12.5 mg/kg) reduced the growth of an established B16/DOX melanoma and prolonged survival (32% compared to untreated mice). All these data support a potential use of resveratrol alone or in combination with other chemotherapeutic agents in the management of chemoresistant tumors.

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.

Enhanced immune response induced by BSA loaded in hydroxyethylstarch microparticles.

Microparticles and nanoparticles represent promising carriers for the in vivo delivery of peptides, proteins or deoxyribonucleic acid (DNA). In this study, new hydroxyethylstarch (HES) microparticles were obtained by interfacial cross-linking with terephtaloyl chloride. These microparticles exhibit the characteristics required to improve antigen release and presentation to antigen presentating cells compared to free antigens. The adjuvant activity of HES microparticles as vaccine carrier was investigated in mice using bovine serum albumin (BSA) as model antigen. We showed HES microparticles were phagocyted by peritoneal mononuclear cells. The immunization with BSA-microparticles induced antibody synthesis that was predominantly immunoglobulin G1 (IgG1). Aluminium hydroxide remained more efficient to induce IgG synthesis. The analysis of the cytokine profile from spleen cells revealed that BSA-microparticles induced the secretion of both interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). However, the immune responses induced by BSA-microparticles were qualitatively and quantitatively affected by the route of injection. Taken together, these results demonstrate that HES microparticles induce a mixed T helper 1/T helper 2 response against BSA and may be a suitable delivery and presentation system in the field of vaccine development.

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.