•The majority of PNP patients develop autoantibodies against A2ML1.•The biological function of A2ML1 in the epidermis remains incompletely understood.•Current diagnostic methods in PNP are inadequate ...to detect anti-A2ML1 antibodies.•Here we developed sensitive and quantitative assays to detect anti-A2ML1 antibodies.•Our approach can be applied for the characterization of antibodies against various antigens.
Paraneoplastic pemphigus (PNP) is a devastating autoimmune multiorgan syndrome associated with autoantibodies against several autoantigens, including the alpha-2-macroglobulin-like-1 (A2ML1). A2ML1 is recognized by up to 70 % of PNP sera. The currently recommended techniques for serological diagnosis of PNP are inadequate to detect anti-A2ML1 antibodies.
To develop novel assays which allow to easily and reliably detect anti-A2ML1 autoantibodies in PNP sera.
We produced full-length A2ML1 in fusion with enhanced green fluorescent protein (EGFP-A2ML1) in transfected human embryonic kidney 293 T cells. The recombinant protein was used as fluorescent ligand for immunoprecipitation studies. We further developed an enzyme-linked immunosorbent assay (ELISA) by immobilizing EGFP-A2ML1 on 96-well plates.
A2ML1-positive PNP sera were able to immunoprecipitate EGFP-A2ML1. Direct measurement of fluorescence in immunoprecipitates correlates with the relative levels of anti-A2ML1 antibodies in the PNP sera. By the novel ELISA, based on the determined best cut-off value, 61 % of the tested 36 PNP sera were A2ML1 positive with a specificity of 88.9 % and a sensitivity of 95 %. The 20 tested normal sera (NHS) were negative, while 2 (10 %) of 20 pemphigus vulgaris and 3 (15 %) of 20 bullous pemphigoid sera showed borderline values.
Our novel immunoassays enable rapid stratification of PNP patients. The novel green fluorescent protein-based ELISA utilizing an active eukaryotic A2ML1 is highly sensitive and reliable and, hence, is useful for a better understanding of the immunological background of PNP. This approach may be easily applied for the rapid detection of antibodies to various other antigens.
T helper 9 (T
9) cells promote allergic tissue inflammation and express the type 2 cytokines, IL-9 and IL-13, as well as the transcription factor, PPAR-γ. However, the functional role of PPAR-γ in ...human T
9 cells remains unknown. Here, we demonstrate that PPAR-γ drives activation-induced glycolysis, which, in turn, promotes the expression of IL-9, but not IL-13, in an mTORC1-dependent manner. In vitro and ex vivo experiments show that the PPAR-γ-mTORC1-IL-9 pathway is active in T
9 cells in human skin inflammation. Additionally, we find dynamic regulation of tissue glucose levels in acute allergic skin inflammation, suggesting that in situ glucose availability is linked to distinct immunological functions in vivo. Furthermore, paracrine IL-9 induces expression of the lactate transporter, MCT1, in T
cells and promotes their aerobic glycolysis and proliferative capacity. Altogether, our findings uncover a hitherto unknown relationship between PPAR-γ-dependent glucose metabolism and pathogenic effector functions in human T
9 cells.
Paraneoplastic pemphigus (PNP) is a rare and severe variant of pemphigus associated with an underlying malignancy and poor therapeutic outcome. PNP is characterized by the presence of autoantibodies ...against a broad spectrum of desmosomal components, additionally more than 50% of patients also have antibodies against the extracellular protease inhibitor alpha 2 macroglobulin-like 1 (A2ML1). A2ML1 expression in the epidermis is restricted to the granular layer and its biological function is still unknown.The detection of anti-A2ML1 antibodies is presently based on immunoprecipitation of differentiated human keratinocyte extracts followed by polyacrylamide gel autoradiography or immunoblotting. Faster and more reliable diagnostic tools are thus needed. Our method relies on the expression of full-length A2ML1 in fusion with enhanced green fluorescent protein (EGFP). The secreted EGFP-A2ML1 and EGFP, as negative control, were expressed in transfected human embryonic kidney 293T cells and immunoprecipitated from the culture medium with PNP A2ML1-negative or -positive sera. The measurement of fluorescence in immunoprecipitates correlates with the results obtained with current methods. Moreover, we developed an ELISA test by immobilizing EGFP-A2ML1 and EGFP on 96-well plates. With this method and the presently set cutoff value, 42 % of the tested PNP sera (n=36) were positive. Among them, 25 % were strongly positive while none of the sera of pemphigus vulgaris (n=20) and bullous pemphigoid patients (n=20) were positive.In summary, we established efficient methods to test and quantify the reactivity of human sera against A2ML1. Similar assays may be applied for the detection of autoantibodies against other target antigens. Fluorescence immunoprecipitation is suitable for the analysis of few sera while ELISA is optimal for the screening of a large numbers of samples. Quantitative results will help to better define the phenotype(s) associated with anti-A2ML1 autoantibodies giving additional clues to the biological function of A2ML1.
Snakin-1 (SN1), a cysteine-rich peptide with broad-spectrum antimicrobial activity in vitro, was evaluated for its ability to confer resistance to pathogens in transgenic potatoes. Genetic variants ...of this gene were cloned from wild and cultivated Solanum species. Nucleotide sequences revealed highly evolutionary conservation with 91-98% identity values. Potato plants (S. tuberosum subsp. tuberosum cv. Kennebec) were transformed via Agrobacterium tumefaciens with a construct encoding the S. chacoense SN1 gene under the regulation of the ubiquitous CaMV 35S promoter. Transgenic lines were molecularly characterized and challenged with either Rhizoctonia solani or Erwinia carotovora to analyse whether constitutive in vivo overexpression of the SN1 gene may lead to disease resistance. Only transgenic lines that accumulated high levels of SN1 mRNA exhibited significant symptom reductions of R. solani infection such as stem cankers and damping-off. Furthermore, these overexpressing lines showed significantly higher survival rates throughout the fungal resistance bioassays. In addition, the same lines showed significant protection against E. carotovora measured as: a reduction of lesion areas (from 46.5 to 88.1% with respect to the wild-type), number of fallen leaves and thickened or necrotic stems. Enhanced resistance to these two important potato pathogens suggests in vivo antifungal and antibacterial activity of SN1 and thus its possible biotechnological application.
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