![]() Although the adaptive and innate immune systems were reported to be responsible for peanut allergic reactions, the detailed molecular mechanisms of peanut allergic reactions largely remain unclear. Sometimes peanut allergy can lead to anaphylaxis, a life-threatening condition. Allergic reactions are characterized by various kind of symptoms, such as skin (rashes, angioedema), gastrointestinal (vomiting, diarrhea), respiratory (coughing, sneezing), and circulatory symptoms (cardiovascular collapse). Peanut allergy is one of the most common and serious types of food allergy, and its prevalence has been increasing. These techniques are being applied in several research areas, including characterization and/or monitoring of B-cell repertoires in various types of disease, such as food allergy as well as autoimmune diseases, infectious diseases, and cancer.įood allergy affects ~2–10% of children in the Unites States. Hence, a method to comprehensively analyze BCR repertoires including potential novel sequences is important. In our previous study, we identified several abnormally spliced or rearranged, and potentially novel T-cell receptor (TCR) transcripts by NGS. The recent advancement in next-generation sequencing (NGS) technologies has allowed us to generate millions of BCR cDNA sequence reads and characterize BCR repertoires comprehensively and quantitatively in a single experiment. Through clonal affinity selection for enhanced antigen binding, somatic hypermutation-mediated variation further increases diversity of the mature B-cell repertoire. Rearranged BCR genes obtain further diversity by helper T-cell-mediated somatic hypermutation induced by activation-induced cytosine deaminase. During the recombination, random deletion and/or non-templated insertion at the junction site occurred and this process significantly increases the BCR diversity within this rearranged protein, a complementarity-determining region 3 (CDR3) is considered to be critical for antigen recognition. BCR genes consist of multiple distinct gene segments corresponding to the variable (V), diversity (D, only for IGH genes), and joining (J) regions, and undergo site-specific V(D)J recombination. BCRs are a heterodimeric protein complex, composed of two identical heavy-chain (IGH) and two identical light-chain proteins. Healthy humans have ~3 × 10 9 B cells in their peripheral blood, and this population consists of a repertoire of distinct B cells expressing different BCRs to recognize a wide variety of antigens and play critical roles in an effective humoral immune response. Antibodies (immunoglobulin IG), the soluble form of B-cell receptors (BCRs), are essential in adaptive immunity. The adaptive immune system is a complex network of cells, including B and T cells, and multiple organs defending against pathogens. This newly developed BCR sequencing and analysis method can be applied to investigate B-cell repertoires in various research areas, including food allergies as well as autoimmune and infectious diseases. In the 17 peanut allergic subjects’ peripheral blood samples, we observed an oligoclonal enrichment of certain immunoglobulin heavy chain alpha (IGHA) and IGH gamma (IGHG) clones ( P = 0.034 and P = 0.027, respectively) in peanut allergic subjects after OIT. ![]() By our methods, we successfully identified all of variable (V), joining (J), and constant (C) regions, in an average of 79.1% of total reads and 99.6% of these VJC-mapped reads contained the C region corresponding to the isotypes that we aimed to analyze. Using this method and quantitative real-time PCR, we analyzed expression levels and repertoires of BCRs in a total of 17 peanut allergic subjects’ peripheral blood samples before and after receiving oral immunotherapy (OIT) or placebo. Here, we developed “Bcrip”, a novel approach to characterize BCR repertoire by sequencing millions of BCR cDNA using next-generation sequencer. To better understand immune responses, it is critically important to establish a quantitative and rapid method to analyze BCR repertoire comprehensively. B-cell receptors (BCRs) play a critical role in adaptive immunity as they generate highly diverse immunoglobulin repertoires to recognize a wide variety of antigens.
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