Effects of Sarcoptes scabiei Translationally Controlled Tumor Protein (TCTP) on Histamine Release and Degranulation of KU812 Cells.

Scabies is a common parasitic dermatological infection worldwide that is often neglected. Scabies mites stimulate host inflammatory symptoms via secreted and excreted proteins, which induce basophil and mast cell degranulation and host histamine release. However, the mechanism of degranulation and histamine release is unclear. Moreover, the Sarcoptes scabiei translationally controlled tumor protein (TCTP) is predicted as an excreted protein, which may be involved in host inflammatory response regulation. First, we evaluated S. scabiei TCTP gene (SsTCTP) transcription in larvae, nymphs, and adults by qRT-PCR, and SsTCTP transcription was highest in larvae, followed by nymphs. Second, we found that the S. scabiei TCTP recombinant protein (rSsTCTP) promoted mice histamine release in vivo by Evans blue Miles assay. Therefore, to further explore the possible role of S. scabiei TCTP in host inflammatory response regulation, we established a degranulation model of KU812 cells. The results of the degranulation model suggested that rSsTCTP could induce enhanced degranulation of KU812 cells and increase the secretion of histamine and the expression of IL-4, IL-6, and IL-13 in vitro. In conclusion, we speculate that scabies mites could stimulate host histamine release and Th2 response by excreting S. scabiei TCTP.

Basophils as a potential therapeutic target in cancer.

Basophils, which are considered as redundant relatives of mast cells and the rarest granulocytes in peripheral circulation, have been neglected by researchers in the past decades. Previous studies have revealed their vital roles in allergic diseases and parasitic infections. Intriguingly, recent studies even reported that basophils might be associated with cancer development, as activated basophils synthesize and release a variety of cytokines and chemokines in response to cancers. However, it is still subject to debate whether basophils function as tumor-protecting or tumor-promoting components; the answer may depend on the tumor biology and the microenvironment. Herein, we reviewed the role of basophils in cancers, and highlighted some potential and promising therapeutic strategies.

Basophils balance healing after myocardial infarction via IL-4/IL-13.

The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent remodeling. Here, we show that basophil granulocytes infiltrated infarcted murine hearts, with a peak occurring between days 3 and 7. Antibody-mediated and genetic depletion of basophils deteriorated cardiac function and resulted in enhanced scar thinning after MI. Mechanistically, we found that basophil depletion was associated with a shift from reparative Ly6Clo macrophages toward increased numbers of inflammatory Ly6Chi monocytes in the infarcted myocardium. Restoration of basophils in basophil-deficient mice by adoptive transfer reversed this proinflammatory phenotype. Cellular alterations in the absence of basophils were accompanied by lower cardiac levels of IL-4 and IL-13, two major cytokines secreted by basophils. Mice with basophil-specific IL-4/IL-13 deficiency exhibited a similarly altered myeloid response with an increased fraction of Ly6Chi monocytes and aggravated cardiac function after MI. In contrast, IL-4 induction in basophils via administration of the glycoprotein IPSE/α-1 led to improved post-MI healing. These results in mice were corroborated by the finding that initially low counts of blood basophils in patients with acute MI were associated with a worse cardiac outcome after 1 year, characterized by a larger scar size. In conclusion, we show that basophils promoted tissue repair after MI by increasing cardiac IL-4 and IL-13 levels.

E-cadherin is regulated by GATA-2 and marks the early commitment of mouse hematopoietic progenitors to the basophil and mast cell fates.

E-cadherin is a calcium-dependent cell-cell adhesion molecule extensively studied for its involvement in tissue formation, epithelial cell behavior, and suppression of cancer. However, E-cadherin expression in the hematopoietic system has not been fully elucidated. Combining single-cell RNA-sequencing analyses and immunophenotyping, we revealed that progenitors expressing high levels of E-cadherin and contained within the granulocyte-monocyte progenitors (GMPs) fraction have an enriched capacity to differentiate into basophils and mast cells. We detected E-cadherin expression on committed progenitors before the expression of other reported markers of these lineages. We named such progenitors pro-BMPs (pro-basophil and mast cell progenitors). Using RNA sequencing, we observed transcriptional priming of pro-BMPs to the basophil and mast cell lineages. We also showed that GATA-2 directly regulates E-cadherin expression in the basophil and mast cell lineages, thus providing a mechanistic connection between the expression of this cell surface marker and the basophil and mast cell fate specification.

The role of basophils in acquired protective immunity to tick infestation.

Ticks are blood-feeding ectoparasites that transmit a variety of pathogens to host animals and humans, causing severe infectious diseases such as Lyme disease. In a certain combination of animal and tick species, tick infestation elicits acquired immunity against ticks in the host, which can reduce the ability of ticks to feed on blood and to transmit pathogens in the following tick infestations. Therefore, our understanding of the cellular and molecular mechanisms of acquired tick resistance (ATR) can advance the development of anti-tick vaccines to prevent tick infestation and tick-borne diseases. Basophils are a minor population of white blood cells circulating in the bloodstream and are rarely observed in peripheral tissues under steady-state conditions. Basophils have been reported to accumulate at tick-feeding sites during re-infestation in cattle, rabbits, guinea pigs and mice. Selective ablation of basophils resulted in a loss of ATR in guinea pigs and mice, illuminating the essential role of basophils in the manifestation of ATR. In this review, we discuss the recent advance in the elucidation of the cellular and molecular mechanisms underlying basophil recruitment to the tick-feeding site and basophil-mediated ATR.

Author's reply: Basophil activation test. Tool for the diagnosis of interstitial nephritis / Réplica a la carta al Editor: Test de activación de basófilos. Herramienta para el diagnóstico de nefritis intersticial

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Mast Cell and Basophil Cell Lines: A Compendium.

Mast cells and basophils play a crucial role during type I hypersensitivity reactions. However, despite efforts to elucidate their role in the pathogenesis of allergy and inflammation, our understanding of MC and basophil biology is still relatively scarce. The practical difficulty in obtaining a sufficient number of purified primary cells from biological samples has slowed down the process of reaching a full understanding of the physiological role of these functionally similar cell types. The establishment of several immortalized cell lines has been a useful tool to establish and perform sophisticated laboratory protocols that are impractical using primary cells. Continuous cell lines have been extensively used to investigate allergen/IgE-mediated cell activation, to elucidate the degranulation dynamics, to investigate structural and functional properties of the high-affinity receptor (FcεRI), and to test cell-stabilizing compounds. In this chapter, we review the most widely used and better-characterized MC and basophil cell lines, highlighting their advantages and drawbacks. It must be pointed out, however, that while cell lines represent a useful in vitro tool due to their easy manipulability and reduced culture costs, they often show aberrant characteristics which are not fully representative of primary cell physiology; results obtained with such cells therefore must be interpreted with due care.

Use of Engineered Nanoparticles (ENPs) for the Study of High-Affinity IgE FcεRI Receptor Engagement and Rat Basophilic Leukemia (RBL) Cell Degranulation.

Degranulation of mast cells and basophils occurs after the cross-linking of FcεRI receptor-bound IgE by multivalent allergens, resulting in the release of a range of de novo synthesized and preformed mediators of the allergic response. ß-Hexosaminidase release is usually measured as a simple readout for degranulation. Furthermore, the rat basophilic leukemia (RBL)-2H3 cell line is commonly used for measuring degranulation, monitoring ß-hexosaminidase release. Here, we describe surface-engineered and modified nanoparticles with specific ligands in order to study the signaling and cellular responses of the RBL-2H3 cell line.

Basophil Activation Techniques: Staining of Exteriorized Granule Matrix.

The basis of traditional flow cytometry allergy diagnosis is measurement of the expression of basophilic surface activation and/or degranulation markers. Basophils, upon encounter with a specific allergen that cross-links surface FcRI-bound IgE antibodies, not only secrete and release quantifiable bioactive mediators but also upregulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies. Here, we describe a novel technique that relies upon the staining of exteriorized anionic proteoglycans from a basophil granule matrix by cationic fluorescent avidin probes.

Filling the Antibody Pipeline in Allergy: PIPE Cloning of IgE, IgG1 and IgG4 against the Major Birch Pollen Allergen Bet v 1.

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.

Constructing and deconstructing GATA2-regulated cell fate programs to establish developmental trajectories.

Stem and progenitor cell fate transitions constitute key decision points in organismal development that enable access to a developmental path or actively preclude others. Using the hematopoietic system, we analyzed the relative importance of cell fate-promoting mechanisms versus negating fate-suppressing mechanisms to engineer progenitor cells with multilineage differentiation potential. Deletion of the murine Gata2-77 enhancer, with a human equivalent that causes leukemia, downregulates the transcription factor GATA2 and blocks progenitor differentiation into erythrocytes, megakaryocytes, basophils, and granulocytes, but not macrophages. Using multiomics and single-cell analyses, we demonstrated that the enhancer orchestrates a balance between pro- and anti-fate circuitry in single cells. By increasing GATA2 expression, the enhancer instigates a fate-promoting mechanism while abrogating an innate immunity-linked, fate-suppressing mechanism. During embryogenesis, the suppressing mechanism dominated in enhancer mutant progenitors, thus yielding progenitors with a predominant monocytic differentiation potential. Coordinating fate-promoting and -suppressing circuits therefore averts deconstruction of a multifate system into a monopotent system and maintains critical progenitor heterogeneity and functionality.

Changes in passively-sensitized basophil activation to αS1-casein after oral immunotherapy.

INTRODUCTION: Immune response to cow's milk allergen (CMA) has been analyzed mostly using crude milk antigen or a mixture of various caseins. This study aimed to assess the changes in the immunological response against αS1-casein during oral immunotherapy (OIT) and to investigate the mechanism of tolerance. METHODS: We have performed rush OIT to 39 patients with CMA and obtained the serum samples up to 3 years after OIT. Immunoglobulin E (IgE) and IgG4 antibodies specific to highly purified αS1-casein as well as passively-sensitized basophil activation were evaluated using the serial samples. Furthermore, we examined whether basophil activation led by the pre-OIT serum was suppressed by the post-OIT serum, or by the tolerant serum obtained from naturally outgrown patients. RESULTS: Specific IgE to αS1-casein was significantly reduced after OIT. Specific IgG4 (sIgG4) to αS1-casein was also detected in most of the pre-OIT sera, which was not significantly increased after OIT. Activation of passively-sensitized basophils to αS1-casein was significantly reduced after 2 years (14% ± 19%) and 3 years (19% ± 18%) post-OIT compared with pre-OIT (%CD63high basophils; 51% ± 27%). Furthermore, the addition of post-OIT or tolerant serum to pre-OIT serum significantly suppressed the basophil activation. This suppression was abrogated by washing the supernatant after passive sensitization, but not by depleting IgG antibodies from post-OIT or tolerant sera, nor by blocking FcγRIIb using an anti-FcγR antibody. CONCLUSIONS: αS1-casein-sIgG4 plays a minor role in tolerance mechanisms in cases of CMA; humoral factors other than antigen-sIgG4 may be involved.

Pathophysiologic mechanisms of itch in bullous pemphigoid.

BACKGROUND: One of the hallmarks of bullous pemphigoid (BP) is moderate to severe chronic itch. Managing this is difficult because little is known about the mechanisms of itch in BP. OBJECTIVE: We sought to elucidate the pathophysiologic mechanisms of itch in BP. METHODS: The expression of itch mediators in lesions of 24 patients with BP and 6 healthy individuals were examined through immunofluorescence staining. Furthermore, the expression of itch mediators and itch severity was correlated. RESULTS: Itch severity was correlated with eosinophils, substance P, neurokinin 1R, interleukin (IL) 31 receptor A, oncostatin M receptor-ß, IL-13, periostin, and basophils. There was also a trend between itch severity and IL-31 expression. Most of the cells expressing IL-31 or neurokinin 1R were identified as eosinophils. Intraepidermal nerve fiber density was decreased. Other itch mediators, including mast cells, IL-4, thymic stromal lymphopoietin, transient receptor potential vanilloid 1 and ankyrin 1, and protease activated receptor 2 were not significantly correlated with itch severity. LIMITATIONS: The relatively small sample size, the examination of protein expression exclusively through immunofluorescent analysis, and lack of functional assays in patients are the limitations. CONCLUSIONS: Multiple factors are involved in BP-associated itch, including eosinophils, substance P, neurokinin 1R, IL-31, IL-31 receptor A, oncostatin M receptor-ß, IL-13, periostin, and basophils. They could be useful therapeutic targets.

Clinical utility of the basophil activation test in the diagnosis of sweat allergy.

BACKGROUND: Many patients with atopic dermatitis and cholinergic urticaria display an immediate-type allergy to autologous sweat. Although the histamine release test (HRT) using semi-purified sweat antigen (QR) was available for the detection of immediate sweat allergy, the existence of HRT low responders could not be disregarded. Furthermore, it has not been established whether the results of the HRT are consistent with the autologous sweat skin test (ASwST). We aimed to compare the HRT and basophil activation test (BAT) for the diagnosis of immediate sweat allergy. METHODS: The HRT and BAT were performed on 47 subjects (35 ASwST positive, 12 negative) whose symptoms had worsened on sweating. For the BAT, blood was incubated with QR or crude sweat and CD203c upregulation was assessed. A commercial HRT was performed and histamine release induced by QR was quantified. RESULTS: When excluding non-responders for anti-IgE antibody, the BAT using QR and the HRT had a sensitivity of 100% and 44% and specificity of 75% and 100%, respectively. The BAT and HRT had a positive predictive value of 91.3% and 100% and negative predictive value of 100% and 30%, respectively. The BAT detected 0% non-responders, whereas the HRT identified 22.5%. When using crude sweat for the BAT, the false-positives observed when using QR were not detected. CONCLUSIONS: The BAT using QR displayed a higher sensitivity and negative predictive value and a lower number of non-responders compared with the HRT. Furthermore, the BAT using crude sweat can also be an alternative tool for the ASwST.

Pru p 3-Glycodendropeptides Based on Mannoses Promote Changes in the Immunological Properties of Dendritic and T-Cells from LTP-Allergic Patients.

SCOPE: Glycodendropeptides (GDPs) functionalized with mannose can enhance allergen interaction with dendritic cells (DCs) via C-type lectin receptors (CLRs), modulating the immune response. They can present multiple peptides and have potential applications for diagnosis and treatment of food allergy (FA). The immune response induced by GDPs with mannose and Pru p 3 peptides (mono/tetravalent) with ester (D1 ManPrup3/D4 ManPrup3) or ether linkers (D1 Man-O- Prup3/D4 Man-O- Prup3) in lipid-transfer-protein-allergic patients and tolerant controls is analyzed. METHODS AND RESULTS: The immunological response induced by GDPs is studied by assessing monocyte-derived-DC maturation, lymphocyte proliferation, cytokine production, and basophil response by flow cytometry. Dn ManPrup3 was recognized by DCs via CLRs inducing DC maturation in all subjects. However, CCR7 expression is significantly upregulated in allergic patients compared to tolerant controls. These changes correlate with lymphocyte proliferation and specific production of Th2/Th1 cytokines in allergic patients. Moreover, D1 ManPrup3 does not induce basophil activation. CONCLUSION: Dn ManPrup3 induces changes in DC maturation and lymphocyte proliferation, indicating specific recognition via CLRs. Prup3-GDPs are recognized by immune cells, inducing a specific immune response and modulating the immunological response in FA patients. The specific geometry of D1 ManPrup3 in particular makes it a potential candidate for specific immunotherapy development.

Interaction of peripheral nerves and mast cells, eosinophils, and basophils in the development of pruritus.

Mast cells, eosinophils and basophils are central effector immune cells in allergic skin inflammation including atopic dermatitis (AD). Recent studies revealed that the bidirectional interaction between these three immune cell types (mast cells, eosinophils and basophils) and the nervous system is involved in the pathogenesis of neurogenic inflammation, pain and pruritus. Emerging evidence shows that these cells are the main source of pruritogens such as histamine, neuropeptides and cytokines, which are potential new therapeutic targets for drug development in chronic pruritus. For instance, many Th2 cytokines including interleukin (IL)-4, 13 and 31 have been recognized as some of the most promising targets for the treatment of chronic pruritus in AD. In this review, we highlight the link between these three immune cell subsets and peripheral nerves, with emphasis on the development of chronic pruritus such as AD. We present cytokines and receptors of these three immune cells and peripheral nerves, and discuss the therapeutic potential of targeting these neuro-immunological processes.

Test de activación de basófilos. Herramienta para el diagnóstico de nefritis intersticial / Basophil activation test. Tool for the diagnosis of interstitial nephritis

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Selection and characterization of FcεRI phospho-ITAM specific antibodies.

Post-translational modifications, such as the phosphorylation of tyrosines, are often the initiation step for intracellular signaling cascades. Pan-reactive antibodies against modified amino acids (e.g., anti-phosphotyrosine), which are often used to assay these changes, require isolation of the specific protein prior to analysis and do not identify the specific residue that has been modified (in the case that multiple amino acids have been modified). Phosphorylation state-specific antibodies (PSSAs) developed to recognize post-translational modifications within a specific amino acid sequence can be used to study the timeline of modifications during a signal cascade. We used the FcεRI receptor as a model system to develop and characterize high-affinity PSSAs using phage and yeast display technologies. We selected three ß-subunit antibodies that recognized: 1) phosphorylation of tyrosines Y218 or Y224; 2) phosphorylation of the Y228 tyrosine; and 3) phosphorylation of all three tyrosines. We used these antibodies to study the receptor activation timeline of FcεR1 in rat basophilic leukemia cells (RBL-2H3) upon stimulation with DNP24-BSA. We also selected an antibody recognizing the N-terminal phosphorylation site of the γ-subunit (Y65) of the receptor and applied this antibody to evaluate receptor activation. Recognition patterns of these antibodies show different timelines for phosphorylation of tyrosines in both ß and γ subunits. Our methodology provides a strategy to select antibodies specific to post-translational modifications and provides new reagents to study mast cell activation by the high-affinity IgE receptor, FcεRI.