Nematode Galectin Inhibits Basophilic Leukaemia RBL-2H3 Cells Apoptosis in IgE-Mediated Activation.

Mast cells are essential immune cells involved in the host's defence against gastrointestinal nematodes. To evade the immune response, parasitic nematodes produce a variety of molecules. Galectin 1, produced by Teladorsagia circumcincta (Tci-gal-1), reduces mast cell degranulation and selectively regulates mediator production and release in an IgE-dependent manner. To uncover the activity of Tci-gal-1, we have examined the effect of the protein on gene expression, protein production, and apoptosis in activated basophilic leukaemia RBL-2H3 cells. Rat RBL-2H3 cells were activated with anti-DNP IgE and DNP-HSA, and then treated with Tci-gal-1. Microarray analysis was used to examine gene expression. The levels of several apoptosis-related molecules and cytokines were determined using antibody arrays and ELISA. Early and late apoptosis was evaluated cytometrically. Degranulation of cells was determined by a ß-hexosaminidase release assay. Treatment of activated RBL-2H3 cells with Tci-gal-1 resulted in inhibited apoptosis and decreased degranulation, although we did not detect significant changes in gene expression. The production of pro-apoptotic molecules, receptor for advanced glycation end products (RAGE) and Fas ligand (FasL), and the cytokines IL-9, IL-10, IL-13, TNF-α, and IL-2 was strongly inhibited. Tci-gal-1 modulates apoptosis, degranulation, and production of cytokines by activated RBL-2H3 cells without detectable influence on gene transcription. This parasite protein is crucial for modulation of the protective immune response and the inhibition of chronic inflammation driven by mast cell activity.

Shiikuwasha leaf and peel extracts inhibit allergic reactions by suppressing degranulation in RBL-2H3 rat basophilic leukemia cells and immunoglobulin production in mouse spleen lymphocytes.

This study aims to investigate the antiallergic effects of Shiikuwasha (Citrus depressa Hayata) leaf and peel extracts by examining the regulation of degranulation and inflammatory cytokine production from rat basophilic leukemia (RBL-2H3) cells and antigen-specific antibody production in sensitized mouse spleen lymphocytes. In vivo antiallergic activity was evaluated using the passive cutaneous anaphylaxis (PCA) reaction model. Extracts of Shiikuwasha leaves and peel were prepared using 80% methanol and dissolved in dimethylsulfoxide. The dinitrophenyl-human serum albumin-induced ß-hexosaminidase levels in immunoglobulin (Ig) E-sensitized RBL-2H3 cells were assessed using enzymatic assays. Cytokine production was measured by enzyme-linked immunosorbent assay. Antibody production capacity was evaluated using lymphocytes isolated from spleens of type I allergy model mice. Lymphocytes were cultured for 72 h with Shiikuwasha extracts, and ovalbumin-specific IgE, IgG1, and IgG2a levels were measured. Shiikuwasha leaf and peel extract significantly reduced ß-hexosaminidase release and suppressed interleukin-4 and tumor necrosis factor-α production from RBL-2H3 cells. Ovalbumin-specific IgE and IgG1 production decreased in Shiikuwasha extract-treated lymphocytes. These extracts also significantly suppressed the PCA reaction. Shiikuwasha leaf and peel extract reduce degranulation in RBL-2H3 cells and antibody production in spleen-derived lymphocytes and therefore exhibit antiallergic effects.

Complex Karyotype Acute Basophilic Leukemia with Reactive Mast Cell Hyperplasia.

BACKGROUND: The goal of the study is to improve the understanding of complex karyotype acute basophilic leukemia with reactive mast cell hyperplasia. METHODS: A case of clinical and laboratory characteristics of complex karyotype acute basophilic leukemia with reactive mast cell hyperplasia and review of related literature of patient with acute basophilic leukemia were retrospectively analyzed. RESULTS: Laboratory tests: alanine aminotransferase 225 U/L, aspartate aminotransferase 129 U/L, total protein 3.0 g/L, albumin 25.0 g/L, globulin 17.8 g/L, total bilirubin 183.9 µmol/L, indirect bilirubin 65.6 µmol/L, D-dimer 13.02 mg/L, prothrombin time 19.30 S, white blood cell 37.30 x 109/L, red blood cells 2.06 x 1012/L, hemoglobin 71 g/L, platelets 13 x 109/L, and basophilic granulocyte cells 5.01 x 109/L. Bone marrow smear: a large number of abnormal primitive cells were scattered and distributed. The characteristics of these cells were as follows: the cell body size was different, most of them were round or quasi-round, the cytoplasm volume was medium, and the cytoplasm was stained gray blue. The cytoplasmic processes were visible, and basophilic particles were visible in part of the cytoplasm and/or on the nucleus. The nuclei were mostly round or almost round, with distortion and folding. The chromatin was detailed, the nucleoli varied in number, and some nucleoli were deeply stained. The cells showed double and multiple nuclei, scattered and occasionally small clumps. Bone marrow biopsy: acute myeloid leukemia, not excluding basophilic leukemia. Bone marrow molecular biology: All 29 myeloid leukemia genes, including RARA and BCR-ABL fusion genes, were negative. Flow cytometry results showed abnormal cell population which accounted for 21.98% of nuclear cells. The phenotypes are CD33++, CD38+, CD13+, CD123+, CD7+, CD36+, CD203c+, CD81+, part of CD34+, part of HLA-DR+, CD4 weak+, weak CD117+, the TDT-, cCD3-, cCD79a-, MPO-, CD11c-, CD25-, CD2-. In addition, mast cells accounted for 1.15% of nuclear cells, expressing CD203c, but not CD25 and CD2. Karyotype analysis results were 46, XY, -7, psudic (9; 19) (p24; p13.3), add (15) (p12), add (21) (q22), +r, +1 - 2 mar [cp20]. CONCLUSIONS: Acute basophilic leukemia (ABL) is a rare malignant hematologic tumor. Bone marrow smears, biopsies, flow cytometry, and cytogenetic tests play an important role in the diagnosis and differentiation of complex karyotype acute basophilic leukemia with reactive mast cell hyperplasia.

Monoblastic leukemia (M5a) with chronic basophilic leukemia in a cat.

A cat was presented with depression and anorexia. The complete blood cell count (CBC) revealed non-regenerative anemia (PCV, 8.5%), marked thrombocytopenia (2,400/µl), and leukocytosis (32,090/µl). In the peripheral blood, proliferation of blast cells (85%; 27,276/µl) and basophils (7.7%; 2,460/µl) was observed. Bone marrow aspirate showed hyperplasia with 8.8% blasts and 90.2% basophils of all nucleated cells. The blast cells were negative for myeloperoxidase staining and positive for alpha-naphthol butyrate esterase staining, indicating the agranular blasts are monoblasts. Thus, acute monoblastic leukemia (M5a) with chronic basophilic leukemia was diagnosed. Basophils accounted for more than 40% of the bone marrow, and we diagnosed secondary basophilic leukemia. Secondary basophilic leukemia should be included in the differential list when abnormal basophil increases are observed in feline bone marrow.

Next-generation sequencing in two cases of de novo acute basophilic leukaemia.

Acute basophilic leukaemia (ABL) is a rare subtype of acute myeloid leukaemia (AML); therefore, few data are available about its biology. Herein, we analysed two ABL patients using flow cytometry and next-generation sequencing (NGS). Two cell populations were detected by flow cytometry in both patients. In Case no. 1, blasts (CD34+ , CD203c- , CD117+ , CD123dim+ ) and basophils (CD34- , CD203c+ , CD117± , CD123+ ) were identified, both of which were found by NGS to harbour the 17p deletion and have loss of heterozygosity of TP53. In Case no. 2, blasts (CD33+ , CD34+ , CD123- ) and basophils (CD33+ , CD34+ , CD123+ ) were identified. NGS detected NPM1 mutations in either blasts or basophils, and TET2 in both. These data suggest an overlap of the mutational landscape of ABL and AML, including TP53 and TET2 mutations. Moreover, additional mutations or epigenetic factors may contribute for the differentiation into basophilic blasts.

Anti-Allergic Potential of Cinnamaldehyde via the Inhibitory Effect of Histidine Decarboxylase (HDC) Producing Klebsiella pneumonia.

Allergy is an immunological disorder that develops in response to exposure to an allergen, and histamines mediate these effects via histidine decarboxylase (HDC) activity at the intracellular level. In the present study, we developed a 3D model of Klebsiella pneumoniae histidine decarboxylase (HDC) and analyzed the HDC inhibitory potential of cinnamaldehyde (CA) and subsequent anti-allergic potential using a bacterial and mammalian mast cell model. A computational and in vitro study using K. pneumonia revealed that CA binds to HDC nearby the pyridoxal-5'-phosphate (PLP) binding site and inhibited histamine synthesis in a bacterial model. Further study using a mammalian mast cell model also showed that CA decreased the levels of histamine in the stimulated RBL-2H3 cell line and attenuated the release of ß-hexoseaminidase and cell degranulation. In addition, CA treatment also significantly suppressed the levels of pro-inflammatory cytokines TNF-α and IL-6 and the nitric oxide (NO) level in the stimulated mast cells. A gene expression and Western blotting study revealed that CA significantly downregulated the expressions of MAPKp38/ERK and its downstream pro-allergic mediators that are involved in the signaling pathway in mast cell cytokine synthesis. This study further confirms that CA has the potential to attenuate mast cell activation by inhibiting HDC and modifying the process of allergic disorders.

Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies.

The majority of bioassays are cell-lethal and thus cannot be used for cell assay and selection prior to live-cell sorting. A quad microraft array-based platform was developed to perform semi-automated cell sampling, bioassay, and banking on ultra-small sample sizes. The system biopsies and collects colony fragments, quantifies intracellular protein levels via immunostaining, and then retrieves the living mother colonies based on the fragments' immunoassay outcome. To accomplish this, a magnetic, microwell-based plate was developed to mate directly above the microraft array and capture colony fragments with a one-to-one spatial correspondence to their mother colonies. Using the Signal Transducer and Activator of Transcription 3 (STAT3) model pathway in basophilic leukemia cells, the system was used to sort cells based on the amount of intracellular STAT3 protein phosphorylation (pSTAT3). Colonies were detected on quad arrays using bright field microscopy with 96 ± 20% accuracy (true-positive rate), 49 ± 3% of the colonies were identified as originating from a single cell, and the majority (95 ± 3%) of biopsied clonal fragments were successfully collected into the microwell plate for immunostaining. After assay, biopsied fragments were matched back to their mother colonies and mother colonies with fragments possessing the greatest and least pSTAT3/STAT3 were resampled for expansion and downstream biological assays for pSTAT3/STAT3 and immune granule exocytosis. This approach has the potential to enable colony screening and sorting based on assays not compatible with cell viability, greatly expanding the cell selection criteria available to identify cells with unique phenotypes for subsequent biomedical research.

Development of Fluorescently Labeled SSEA-3, SSEA-4, and Globo-H Glycosphingolipids for Elucidating Molecular Interactions in the Cell Membrane.

Glycosphingolipids (GSLs), such as the globo-series GSLs stage-specific embryonic antigen 3 (SSEA-3), SSEA-4, and Globo-H, are specifically expressed on pluripotent stem cells and cancer cells, and are known to be associated with various biological processes such as cell recognition, cell adhesion, and signal transduction. However, the behavior and biological roles of these GSLs are still unclear. In our previous study, we observed the interactions between the lipid raft and GSLs in real-time using single-molecule imaging, where we successfully synthesized various fluorescent analogs of GSLs (e.g., GM1 and GM3). Here, we have developed fluorescent analogs of SSEA-3, SSEA-4, and Globo-H using chemical synthesis. The biophysical properties of these analogs as raft markers were examined by partitioning giant plasma membrane vesicles from RBL-2H3 cells into detergent-resistant membrane fractions and liquid-ordered/liquid-disordered phases. The results indicated that the analogs were equivalent to native-type GSLs. The analogs could be used to observe the behavior of globo-series GSLs for detailing the structure and biological roles of lipid rafts and GSL-enriched nanodomains during cell differentiation and cell malignancy.

Preventative and Therapeutic Effects of Low-dose Ionizing Radiation on the Allergic Response of Rat Basophilic Leukemia Cells.

The prevalence of allergies has increased over the last four decades. In allergic reactions, mast cells induce a hypersensitive immune response to a substance that is normally harmless. Ionizing radiation has different biological effects depending on the dose and dose rate. In this study, we investigated whether low-dose irradiation before (preventative effect) or after (therapeutic effect) an antigen-antibody reaction has an anti-allergic effect. To test this, we activated rat basophilic leukemia (RBL-2H3) mast cells with anti-2,4-dinitrophenyl IgE (antibody) and 2,4-dinitrophenyl human serum albumin, which served as an antigen. To test for both the potential of a preventative effect and a therapeutic effect, we irradiated mast cells both before and after mast cell activation, and we measured mediator release and signaling pathway activity. Low-dose ionizing radiation suppressed mediator release from RBL-2H3 mast cells activated by the antigen-antibody reaction regardless of when the mast cells were irradiated. These results were due to the suppression of FcεRI expression. Therefore, we suggest that low-dose ionizing radiation has a preventative and therapeutic effect in allergic reactions via the FcεRI-mediated RBL-2H3 mast cell activation system.

Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines.

Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.

Cytoskeletal actin patterns shape mast cell activation.

Activation of immune cells relies on a dynamic actin cytoskeleton. Despite detailed knowledge of molecular actin assembly, the exact processes governing actin organization during activation remain elusive. Using advanced microscopy, we here show that Rat Basophilic Leukemia (RBL) cells, a model mast cell line, employ an orchestrated series of reorganization events within the cortical actin network during activation. In response to IgE antigen-stimulation of FCε receptors (FCεR) at the RBL cell surface, we observed symmetry breaking of the F-actin network and subsequent rapid disassembly of the actin cortex. This was followed by a reassembly process that may be driven by the coordinated transformation of distinct nanoscale F-actin architectures, reminiscent of self-organizing actin patterns. Actin patterns co-localized with zones of Arp2/3 nucleation, while network reassembly was accompanied by myosin-II activity. Strikingly, cortical actin disassembly coincided with zones of granule secretion, suggesting that cytoskeletal actin patterns contribute to orchestrate RBL cell activation.

In vitro cyto-toxic assessment of heavy metals and their binary mixtures on mast cell-like, rat basophilic leukemia (RBL-2H3) cells.

We investigated the cytotoxicity and mechanisms of cell death induced by salts of Cadmium (Cd2+), Lead (Pb2+), Arsenic (AsO43-) and Chromium (Cr+6) on RBL-2H3 cells (a model mast cell line). In addition, cyto-toxic effect on cell viability was assessed to reveal their nature of interaction in binary mixture. The individual cytotoxic characteristics of these metals on RBL-2H3 cell viability showed a concentration-dependent reduction of cell viability. We observed that concentration-dependent cytotoxic potency on RBL-2H3 cells of these metals range in the following order Cd2+>Cr+6>As O43- > Pb2+ with LC50 values of 0.11 µM, 93.58 µM, 397.9 µM and 485.3 µM respectively. Additive effects were observed with Pb2+ + Cd2+, Pb2+ + AsO43-, Pb2+ + Cr+6 and AsO43- + Cr+6. The study revealed that Pb2+, Cd2+, AsO43- and Cr+6 could induce significant (P < 0.01) cell death by apoptosis in RBL-2H3. Highly significant necrotic cell death was observed with Pb2+ and Cr+6 (P < 0.01) than Cd2+ and AsO43- (P < 0.05). Overall, it can be deduced that several cell death executing pathways may be concomitantly activated on exposure to heavy metals and the predominance of one over others might depend on the type of heavy metal, concentration and the metabolic state of the cell. Eventually, binary mixtures of some of these metals showed less cytotoxicity than would be expected from their individual actions and may depend on the co-exposure of the metal ions and their modes of action.

Antiallergic Activity of the Wild Mushrooms of Nepal and the Pure Compound Hispidin.

In the present study, ethanol extracts of 90 wild mushroom samples from Nepal, and the pure compound hispidin, were screened for their ability to inhibit ß-hexosaminidase release (BHR) from rat basophilic leukemia-2H3 cells. Simultaneously, the toxicity of the extracts toward the cells was also determined, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Samples belonging to the groups Hymenochaetales and Polyporales showed promising anti-allergic activity, with Phellinus adamantinus and Ganoderma lingzhi 3 allowing a mere 19.4% and 16.7% BHR, respectively, without any cell cytotoxicity. Moreover, the 50% inhibitory concentration (IC50) values for Inonotus clemensiae and P. adamantinus were determined to be 51.24 and 50.65 µg/mL, respectively; whereas hispidin, the major bioactive compound in I. clemensiae showed an IC50 value of 82.47 µg/mL. These findings are crucial in underscoring the medicinal value of the wild mushrooms of Nepal, as a source of strong antiallergic agents.

CD45 exclusion- and cross-linking-based receptor signaling together broaden FcεRI reactivity.

For many years, the high-affinity receptor for immunoglobulin E (IgE) FcεRI, which is expressed by mast cells and basophils, has been widely held to be the exemplar of cross-linking (that is, aggregation dependent) signaling receptors. We found, however, that FcεRI signaling could occur in the presence or absence of receptor cross-linking. Using both cell and cell-free systems, we showed that FcεRI signaling was stimulated by surface-associated monovalent ligands through the passive, size-dependent exclusion of the receptor-type tyrosine phosphatase CD45 from plasma membrane regions of FcεRI-ligand engagement. Similarly to the T cell receptor, FcεRI signaling could also be initiated in a ligand-independent manner. These data suggest that a simple mechanism of CD45 exclusion-based receptor triggering could function together with cross-linking-based FcεRI signaling, broadening mast cell and basophil reactivity by enabling these cells to respond to both multivalent and surface-presented monovalent antigens. These findings also strengthen the case that a size-dependent, phosphatase exclusion-based receptor triggering mechanism might serve generally to facilitate signaling by noncatalytic immune receptors.

Construction and identification of the recombinant hFcεRIα/RBL-2H3 cells.

IgE/FcεRI signal pathway plays a crucial role in triggering allergic reactions, and there is no cross-recognition between IgE and FcεRI in human and rats. In order to obtain the hFcεRIα/ RBL-2H3 cell line, total RNA was extracted from U937 cells, and the human FcεRIα gene was obtained by RT-PCR technology. Then the amplified product was digested and inserted into the pIRES2-EGFP vector. After the plasmid was transfected into the RBL-2H3 cells using lipofectamine, and the RBL-2H3 cell lines of stable expression were screened by G418. The transfection efficiency reached 60.45% with optimizing transfection parameters. The last the expression of hFcεRIα was detected by RT-PCR, western blotting and fluorescent microscopy. The present results demonstrated that the pIRES2-EGFP-hFcεRIα vector was constructed and a stable cell line of hFcεRIα/ RBL-2H3 cells was established successfully. This cell line is promising tools for further research on the pathogenesis and drug development of allergic diseases.