Irreversible electroporation holds great potential for cell-specific lysis due to the size-dependent susceptibility of cells to externally imposed electric fields. Previous attempts at selective cell lysis lead to significant overlap between affected populations and struggle with inconsistent biological outcome. We propose that charge transfer at the electrode-liquid interface is responsible by inducing multifactorial effects originating from both the electric field and electrochemical reactions. A promising remedy is the coating of electrodes with a high-k dielectric layer. The resulting capacitive coupling restores the selective potential of electric field mediated lysis in a microfluidic setup. Initial experiments show the consistent depletion of erythrocytes from whole blood while leaving leukocytes intact. The same is true for the reproducible and selective depletion of Jurkat and MCF-7 cells in a mixture with leukocytes. Unexpectedly, the observed order of lysis cannot be correlated with cell size. This implies that the cellular response to capacitive coupling features a selective characteristic that is different from conventional lysis configurations..
Birch pollen allergy is among the most prevalent pollen allergies in Northern and Central Europe. This IgE-mediated disease can be treated with allergen immunotherapy (AIT), which typically gives rise to IgG antibodies inducing tolerance. Although the main mechanisms of allergen immunotherapy (AIT) are known, questions regarding possible Fc-mediated effects of IgG antibodies remain unanswered. This can mainly be attributed to the unavailability of appropriate tools, i.e., well-characterised recombinant antibodies (rAbs). We hereby aimed at providing human rAbs of several classes for mechanistic studies and as possible candidates for passive immunotherapy. We engineered IgE, IgG1, and IgG4 sharing the same variable region against the major birch pollen allergen Bet v 1 using Polymerase Incomplete Primer Extension (PIPE) cloning. We tested IgE functionality and IgG blocking capabilities using appropriate model cell lines. In vitro studies showed IgE engagement with FcεRI and CD23 and Bet v 1-dependent degranulation. Overall, we hereby present fully functional, human IgE, IgG1, and IgG4 sharing the same variable region against Bet v 1 and showcase possible applications in first mechanistic studies. Furthermore, our IgG antibodies might be useful candidates for passive immunotherapy of birch pollen allergy.
To the Editor, Cow's milk (CM) allergy (CMA) is defined as an immune-mediated adverse response to CM proteins. 2% to 3% of children are suffering from CMA, but many develop natural tolerance after 3-4 years. 1,S01 Food allergen immunotherapy (FA-AIT) applying increasing antigen doses (oral immunotherapy, OIT) can contribute to improvement of CMA, S02 with variable clinical efficacy, 2,S03 but immunologically often resulting in decreased specific IgE levels and increased specific IgG4 levels S04,S05 . IgG4 is (a)anti-inflammatory as it does not activate the complement system; (b) bi-specific due to fab-arm exchange and, thus, has less crosslinking capacity than IgE, but has (c) blocking capacity. 3 The interplay of IgE and IgG4 may hence be decisive for the immune balance in CMA.Antibodies of different isotypes with the same variable region could be essential in molecular studies of the mechanism underlying CMA and might shed light onto the function of specific IgG4 antibodies during the acquisition of tolerance.Polymerase Incomplete Prime Extension (PIPE) cloning is a cutting-edge technique to engineer antibodies against various targets and facilitates simple exchange of the constant regions to increase the range of subtypes of antibodies. [4][5][6] In this study, we combined variable regions from the high-affinity IgE antibody "D1" 7 (KD = 1.4 × 10 −9 M; k on = 1.4 × 10 6 , k off = 1.54 × 10 −3 ) S06 specific for the major CM allergen beta-lactoglobulin (BLG), with the constant domains of IgE or IgG4 antibodies (Figure 1A). Correct antibody assembly was confirmed by SDS-PAGE in comparison with commercialThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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