CROCIN PROMOTES APOPTOSIS IN HUMAN EBV-TRANSFORMED B-LYMPHOCYTE VIA INTRINSIC PATHWAY

Background: As a major carotenoid in saffron, crocin demonstrates potent anti-cancer impacts. However, its anti-lymphoma effects remain vague, especially in the human EBV-associated B-cell lymphoproliferative disorders. This study examined crocin's apoptogenic potential and its underlying mechanism in CO 88BV59-1 cell line vs. normal human peripheral blood B cells. Methods: CO 88BV59-1 cells were treated with crocin alone or in combination with vincristine for up to 72 h. The cell viability was examined using a resazurin assay. Flow cytometry using annexin V and propidium iodide labeling was performed to detect apoptotic cells. Also, the expression levels of genes and proteins involved in apoptosis (CASP3, CASP8, CASP9, P53, Bax, and Bcl-2) were respectively determined via real-time PCR and Western blot analysis. Results: Crocin concentration-dependently reduced cell viability in CO 88BV59-1 cells with no significant toxicity toward normal B cells. Similar to vincristine, crocin significantly increased apoptosis in these cells during 72 h of incubation. Furthermore, the combination of crocin (80 μM) and vincristine (1 μM) enhanced apoptosis in CO 88BV59-1 cells. Therefore, this synergistic effect was detected in human EBV-transformed B-lymphocyte. CASP3, CASP9, P53, and Bax/Bcl-2 ratio expressions were significantly raised in CO 88BV59-1 cells, whereas CASP8 was unaltered. It was proposed that crocin promoted apoptosis in CO 88BV59-1 cells in a time- and concentration-dependent manner via the induction of the intrinsic pathway. Conclusion: The results suggest that crocin may serve as a good alternative/coadjuvant to vincristine in EBV-associated B-cell lymphoproliferative disorders. Keywords: Crocin, CO 88BV59-1 cells, EBV-associated B-cell lymphoproliferative disorders, apoptosis

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Monoclonal antibodies to the 140,000 mol wt glycoprotein of B lymphocyte membranes (CR2 receptor) initiates proliferation of B cells in vitro

Blood ◽

10.1182/blood.v66.4.824.bloodjournal664824 ◽

1985 ◽

Vol 66 (4) ◽

pp. 824-829

Author(s):

BS Wilson ◽

JL Platt ◽

NE Kay

Keyword(s):

Monoclonal Antibodies ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

B Lymphocyte ◽

Human Peripheral Blood ◽

Raji Cell ◽

Lymphoid Cells ◽

Humoral Immune

Several mouse monoclonal IgG antibodies (AB1, AB2, AB3, and AB5) were developed that reacted with a 140,000 mol wt glycoprotein on the surface of cultured RAJI B lymphoid cells. The antibodies reacted with purified normal human peripheral blood B cells and CLL Ig+ B cells and showed specific germinal center and mantle zone staining in tissue sections of secondary lymphoid organs. Immunodepletion studies using 125I surface-labeled Raji cell membrane antigens demonstrated that the antigen identified by AB5 is the same 140,000 mol wt glycoprotein detected by anti-B2 that has recently been shown to react with the C3d fragment or CR2 receptor. (Iida et al: J Exp Med 158:1021, 1983). Addition of the AB series and anti-B2 monoclonal antibodies to cultures of purified human peripheral blood B cells resulted in the uptake of 3H- thymidine at two to six times background control levels provided that irradiated autologous T cells were added to the culture. Stimulation was not evoked by other monoclonal antibodies to B cell surface molecules (ie, B1, BA-1, BA-2, and HLA-DR). Pepsin-generated F(ab')2 fragments of anti-CR2 antibodies were essentially as effective as the intact IgG molecule in stimulating B cells. Induction of B cell proliferation by antibody binding to CR2 suggests that the C3d receptor may have an integral role in regulation of humoral immune response.

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Artemisinin derivative SM934, influences the activation, proliferation, differentiation and antibody-secreting capacity of β-cells in systemic lupus erythematosus mice via inhibition of TLR7/9 signaling pathway

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v18i7.4 ◽

2021 ◽

Vol 18 (7) ◽

pp. 1391-1396

Author(s):

Yajuan Li ◽

Lixin Zhao ◽

Xuehui Yang ◽

Jing Chen ◽

Wenjing Xu ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

B Cells ◽

B Cell ◽

Lupus Erythematosus ◽

B Lymphocyte ◽

Underlying Mechanism ◽

Dependent Manner ◽

Artemisinin Derivative ◽

Systemic Lupus ◽

Dose Dependent

Purpose: To study the influence of artemisinin derivative, SM934 on activation, proliferation, differentiation and antibody-secreting capacity of B cells of systemic lupus erythematosus (SLE) mice, and the underlying mechanism. Methods: Female MRL/lpr mice (n = 60) were randomly assigned to four groups of 15 mice each: SLE, 2.5 mg/kg SM934; 5 mg/kg SM934, and 10 mg/kg SM934 groups. Serum levels of interleukins 6, 10, 17 and 21 (IL-6, IL-17, IL-10 and IL-21) were determined. The secretions of immunoglobulins G and M (IgG and IgM) by B cells were determined. The population of B lymphocyte subtypes was determined flow cytometrically. The expressions of Blimp-1 and Bcl-6, Toll-like receptors 7 and 9 (TLR7 and TLR9) mRNAs were determined. Results: SLE-induced upregulation of serum IL-10, IL-6, IL-17 and IL-21 was significantly and dosedependently reduced following a 2-month treatment with SM934 (p < 0.01). Treatment with SM934 significantly and dose-dependently accentuated B cell germinal center B cell populations, but significantly and dose-dependently decreased the populations of plasma and activated B cells (p < 0.01). The splenic levels of IgG and IgM were decreased in a dose-dependent fashion after 8 weeks of treatment (p < 0.01). Artemisinin derivative SM934 decreased the expression of Blimp-1, and upregulated the expression of Bcl-6, both in a dose-dependent manner (p < 0.01). Moreover, SM934 decreased the mRNA expressions of TLR7 and TLR9 in a dose-based manner (p < 0.01). Conclusion: Artemisinin derivative SM934 mitigates LSE syndromes by suppressing the TLR-induced B-cell stimulation and plasma cell generation

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Antitumor Effects of Nordihydroguaiaretic Acid (NDGA) in Bladder T24 Cancer Cells are Related to Increase in ROS Production and Mitochondrial Leak Respiration

Natural Product Communications ◽

10.1177/1934578x1801301128 ◽

2018 ◽

Vol 13 (11) ◽

pp. 1934578X1801301

Author(s):

Gustavo Ignacio Vázquez-Cervantesa ◽

Karla Villaseñor-Aguayoa ◽

Jacqueline Hernández-Damiána ◽

Omar Emiliano Aparicio-Trejoa ◽

Omar Noel Medina-Camposa ◽

...

Keyword(s):

Cell Viability ◽

Annexin V ◽

Nordihydroguaiaretic Acid ◽

Mitochondrial Respiratory Chain ◽

Cellular Respiration ◽

Dependent Manner ◽

Antitumor Effects ◽

Concentration Dependent Manner ◽

T24 Cells ◽

Iodide Solution

The aim of this study was to evaluate the effect of nordihydroguaiaretic acid (NDGA) on tumor bladder T24 cells. Bladder cancer T24 cells were cultured on Dulbecco's Modified Eagle Medium in presence of NDGA. Cell viability and apoptosis were evaluated after 24, 48 and 72 h by using fluorescein diacetate (FDA) and Alexa fluor 488 annexin-V/propidium iodide solution, respectively. To determine the mitochondrial effects of NDGA (0-24 h), reactive oxygen species (ROS) levels by dihydroethidium fluorescence, mitochondrial membrane potential (ΔΨm) by 5,5’,6,6'-tetrachloro-1,1’,3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) dual fluorescence and cellular respiration states by high resolution respirometry were evaluated. It was found that NDGA reduced T24 cell viability after 72 h of incubation in a concentration-dependent manner and apoptosis increased at 48 h. Furthermore, 20 μM NDGA increased ROS levels, decreased ΔΨm and promoted leak of respiration from mitochondrial respiratory chain in T24 cells which was associated to the death of tumor cells. Taken together these results suggested that antitumor effects of NDGA in T24 cells are related to its ability to induce mitochondrial alteration.

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Blimp-1-Dependent Repression of Pax-5 Is Required for Differentiation of B Cells to Immunoglobulin M-Secreting Plasma Cells

Molecular and Cellular Biology ◽

10.1128/mcb.22.13.4771-4780.2002 ◽

2002 ◽

Vol 22 (13) ◽

pp. 4771-4780 ◽

Cited By ~ 292

Author(s):

Kuo-I Lin ◽

Cristina Angelin-Duclos ◽

Tracy C. Kuo ◽

Kathryn Calame

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocyte ◽

Critical Role ◽

Cell Lineage ◽

Immunoglobulin M ◽

Dependent Manner ◽

Plasmacytic Differentiation

ABSTRACT B-cell lineage-specific activator protein (BSAP), encoded by the Pax-5 gene, is critical for B-cell lineage commitment and B-cell development but is not expressed in terminally differentiated B cells. We demonstrate a direct connection between BSAP and B-lymphocyte-induced maturation protein 1 (Blimp-1), a transcriptional repressor that is sufficient to drive plasmacytic differentiation. Blimp-1 binds a site on the Pax-5 promoter in vitro and in vivo and represses the Pax-5 promoter in a binding-site-dependent manner. By ectopically expressing Blimp-1 or a competitive inhibitor of Blimp-1, we show that Blimp-1 is both necessary and sufficient to repress Pax-5 during plasmacytic differentiation of primary splenic B cells. Blimp-1-dependent repression of Pax-5 is sufficient to regulate BSAP targets CD19 and J chain and is necessary but not sufficient to induce XBP-1. We further show that repression of Pax-5 is required for Blimp-1 to drive differentiation of splenocytes to immunoglobulin M-secreting cells. Thus, repression of Pax-5 plays a critical role in the Blimp-1-dependent program of plasmacytic differentiation.

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Anti-Leukemic Property of Sulphoraphane In Precursor B Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v116.21.3974.3974 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3974-3974

Author(s):

Koramit Suppipat ◽

Xiao Zhu ◽

Chun Shik Park ◽

H. Daniel Lacorazza

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Annexin V ◽

Dependent Manner ◽

Carcinogenic Activity ◽

Childhood All

Abstract Abstract 3974 Acute lymphoblastic leukemia (ALL) is the most common form of hematologic malignancy in children. In spite of significant advances achieved in the treatment of childhood ALL, one fifth of these patients still relapse after the standard treatment. Moreover, relapse ALL is the second most common cause of cancer-related deaths in children. The development of novel therapies is prevented by a limited understanding of the exact pathobiology. There are emerging evidences that the transcription factor KLF4 has a tumor suppressor property in ALL. Recently, a gene expression classifier study in pediatric precursor B-cell ALL (pre-B ALL) showed that KLF4 expression was significantly reduced in high risk ALL patients with positive MRD after induction. This finding suggests a possible role of this cell cycle inhibitor on the development, expansion and drug-resistant of leukemic cells. Several studies demonstrate that overexpression of KLF4 in normal B cells and BCR transformed B cells show increased apoptosis and reduced proliferation. Furthermore, we recently described that KLF4 inhibits proliferation of naïve lymphocytes by activating p21 (Yamada, et al, 2009). Sulphoraphane (SF; 4-methylsulfonylbutyl isothiocyanate) is a dietary compound derived from Cruciferae vegetables with anti-carcinogenic activity in colon cancer by upregulating KLF4 and p21 among other genes. Thus, we hypothesized that SF could also exhibit anti-leukemic activity in human-derived acute lymphoblastic leukemia cells via the activation of KLF4. The pre-B ALL cell lines (Nalm6, REH, RS-4, SUP-B15) and an EBV transformed B cell line were treated with different concentrations of SF (0-40 μM) for 24–48 hours. Then, cell number was estimated using an ATP-based viability method. Flow cytometric analysis of ANNEXIN-V/7-AAD binding as well as CFSE dilution was used to measure apoptosis and proliferation respectively. We found that SF induced cytotoxicity in Nalm-6, REH and RS-4 cell lines in a dose and time dependent manner. This cytotoxic effect was less pronounced in EBV-transformed B cell line. SF treatment led to increased ANNEXIN-V and 7-AAD positive cells (82% apoptotic cells in SF-treated versus 9% in DMSO control). Further, SF-treated cells displayed significantly less proliferation in comparison to DMSO controls thus suggesting that SF inhibits cellular proliferation. Preliminary data also suggest that SF-mediated apoptosis is caused by upregulation of KLF4. In conclusion, Sulphoraphane exhibits an anti-leukemic property by inducing apoptosis and abrogating proliferation in pre-B ALL cell lines. Thus, sulphoraphane could potentially be used as an adjunctive therapy in a subgroup of pre-B ALL patients who have decreased expression of KLF4. Disclosures: No relevant conflicts of interest to declare.

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Monoclonal antibodies to the 140,000 mol wt glycoprotein of B lymphocyte membranes (CR2 receptor) initiates proliferation of B cells in vitro

Blood ◽

10.1182/blood.v66.4.824.824 ◽

1985 ◽

Vol 66 (4) ◽

pp. 824-829 ◽

Cited By ~ 53

Author(s):

BS Wilson ◽

JL Platt ◽

NE Kay

Keyword(s):

Monoclonal Antibodies ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

B Lymphocyte ◽

Human Peripheral Blood ◽

Raji Cell ◽

Lymphoid Cells ◽

Humoral Immune

Abstract Several mouse monoclonal IgG antibodies (AB1, AB2, AB3, and AB5) were developed that reacted with a 140,000 mol wt glycoprotein on the surface of cultured RAJI B lymphoid cells. The antibodies reacted with purified normal human peripheral blood B cells and CLL Ig+ B cells and showed specific germinal center and mantle zone staining in tissue sections of secondary lymphoid organs. Immunodepletion studies using 125I surface-labeled Raji cell membrane antigens demonstrated that the antigen identified by AB5 is the same 140,000 mol wt glycoprotein detected by anti-B2 that has recently been shown to react with the C3d fragment or CR2 receptor. (Iida et al: J Exp Med 158:1021, 1983). Addition of the AB series and anti-B2 monoclonal antibodies to cultures of purified human peripheral blood B cells resulted in the uptake of 3H- thymidine at two to six times background control levels provided that irradiated autologous T cells were added to the culture. Stimulation was not evoked by other monoclonal antibodies to B cell surface molecules (ie, B1, BA-1, BA-2, and HLA-DR). Pepsin-generated F(ab')2 fragments of anti-CR2 antibodies were essentially as effective as the intact IgG molecule in stimulating B cells. Induction of B cell proliferation by antibody binding to CR2 suggests that the C3d receptor may have an integral role in regulation of humoral immune response.

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The Immunomodulatory Effects of Nidus Vespae on Human Peripheral Blood Immune CellsIn Vitro

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/705308 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Ming Zhu ◽

Yang Ling ◽

Qiufeng Qi ◽

Yaping Zhang ◽

Yanqing Bao ◽

...

Keyword(s):

B Cells ◽

Tumor Cell ◽

Cell Viability ◽

Immune Cells ◽

Peripheral Blood ◽

Human Peripheral Blood ◽

Cell Counting ◽

Dependent Manner ◽

Immunomodulatory Effects ◽

Gastric Cancer Cells

Nidus Vespae has been used in traditional Chinese medicine (TCM) to treat various cancers, but the underlying mechanisms were not yet clarified. This study was to investigate the effect of Nidus Vespae decoction (NVD) on tumor cell viability and immunoregulating functions of human peripheral blood immune cells. The effects on tumor cell viability, peripheral blood mononuclear cell (PBMC) proliferation activity, and the tumor cell phagocytosis of monocytes were evaluated by cell counting kit-8. Tumor-killing activity of cytotoxic T lymphocyte (CTL) was analyzed by51Cr releasing assay. IgG production of B cells and cytokine (TNF-αand IL-6) secretion of monocytes were determined by ELISA method. Data showed that NVD has no significant inhibiting effects on gastric cancer cells growth. Nevertheless, it could obviously promote PBMC proliferation in a time- and concentration-dependent manner. After treatment with NVD, the CTL cytotoxicity against SGC-7901 was significantly greater than control. The TNF-αand IL-6 secretion of monocytes and the IgG production of B cells also increased remarkably. Furthermore, NVD could significantly promote the phagocytosis of monocytes on tumor cells. These results suggest that NVD appears to have an immunoenhancing effect on immune cells, indicating that Nidus Vespae is worth exploring for immunomodulatory effects in tumor treatment.

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BI 836826, a Novel Fc-Engineered Antibody in Combination with Phosphoinositide-3-Kinase Inhibitor for Treatment of High Risk Chronic Lymphocytic Leukemia and Lymphoma

Blood ◽

10.1182/blood.v128.22.2767.2767 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2767-2767

Author(s):

Deborah M Stephens ◽

Kyle A. Beckwith ◽

Priscilla Do ◽

Carolyn Cheney ◽

Xiaokui Mo ◽

...

Keyword(s):

B Cells ◽

Cell Viability ◽

B Cell ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Single Agent ◽

Annexin V ◽

Lymphocytic Leukemia ◽

Advisory Committees

Abstract Background Targeting new antigens in chronic lymphocytic leukemia (CLL) and lymphoma may increase flexibility in the clinic and help circumvent resistance. The tetraspanin CD37 domain mediates transduction of survival and apoptotic signals (Lapalombella et al.,Cancer Cell, 2014), and has been clinically validated by recent trials of otlertuzumab (TRU-016) in CLL and Non-Hodgkin Lymphoma . Ligation of CD37 by this reagent simultaneously induced pro-apoptotic signaling and inhibited pro-survival signaling of phosphoinositide 3-kinase δ (PI3Kδ), which introduces a unique opportunity to use combination strategies employing activation of CD37 and inhibition of PI3Kδ. A new agent BI 836826 is an Fc-engineered anti-CD37 IgG1 that displays improved effector activities as well as crosslinker-independent direct cytotoxicity. We have evaluated the efficacy of BI 836826 combined with the PI3Kδ-selective inhibitor idelalisib in diffuse large B-cell lymphoma (DLBCL) cell lines and primary human CLL B-cells in the University and then by industry to validate the synergistic finding initially reported. Methods Cell viability assays usedCellTiterGlo to measure inhibition of antibody, isotype control, idelalisib or a combination of antibody and compound over 72h in culture. The cell viability of vehicle is measured at the time of dosing (T0) and after seventy-two hours (T72). A GI reading of 0% represents no growth inhibition, GI 100% represents complete growth inhibition, and a GI 200% represents complete death of all cells in the culture well. Annexin V-FITC and propidium iodide measure by flow cytometry was used to assess enhanced killing of primary CLL cells, with incubation of BI 836826 (0.1 µg/mL) and/or idelalisib (1 µM) at 37°C for 24 hours. Trastuzumab included as a non-specific IgG1 control. Data was reported as percentage of viable cells (Annexin V negative, PI negative) normalized to untreated control. Results DLBCL cell lines were variably sensitive to single agent BI 836826. In most of the cell lines tested, the cell viability was inhibited by 40%-50% with BI 836826 in the concentration range of 1-1000 ng/mL (Figure 1A). A synergistic effect was noted in several DLBCL cell lines when BI 836826 was combined with idelalisib. When the maximal effect of BI 836826 was greater than isotype control (GI% > 12, dotted line) and the effect of idelalisib showed a GI50 < 1uM, 3/5 cell lines showed synergy in combination (red dot, Figure 1B). A shift in the EC50of idelalisib can be seen with the addition of increasing amounts of BI 836826 (Figure 1C). In primary CLL B-cell cultures, 1 µM idelalisib displayed weak single agent activity following 24-hour incubation. The cytotoxicity of BI 836826 at 0.1 µg/mL was more variable, although treatment of samples from most CLL patients resulted in 20-50% B-cell death. The combination of these 2 agents resulted in enhanced cytotoxic activity (Figure 2A), and this effect was not attenuated by the presence of del(17)(p13.1), as there was no significant difference in cytotoxicity against these cells compared to those with lower risk cytogenetics (Figure 2B,C). Additionally, the combination was beneficial in CLL B-cells isolated from patients who were refractory to ibrutinib (Figure 2D). Conclusions This collaborative industry and academic endeavor with cross validation of initial mechanistic studies of synergy between CD37 and idelalisib demonstrates that addition of idelalisib to BI 836826 augments cytotoxicity against DLBCL cell lines and primary human CLL B-cells in an additive-to-synergistic manner. In addition, it maintains efficacy against CLL B-cells with del(17)(p13.1) and those from ibrutinib-refractory patients. Further exploration of this therapeutic strategy in clinical trials is strongly warranted. Disclosures Jones: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Grosmaire:Gilead: Employment. Jones:Gilead: Employment. DiPaolo:Gilead: Employment. Tannheimer:Gilead Sciences: Employment. Heider:4Boehringer Ingelheim RCV: Employment.

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Orelabrutinib, a Selective Bruton's Tyrosine Kinase (BTK) Inhibitor in the Treatment of Primary Immune Thrombocytopenia (ITP)

Blood ◽

10.1182/blood-2021-144691 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3172-3172

Author(s):

Tianshu Yu ◽

Lingjun Wang ◽

Xiaofei Ni ◽

Yu Hou ◽

Xinguang Liu ◽

...

Keyword(s):

Platelet Count ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Plasma Cells ◽

Annexin V ◽

Control Group ◽

Dependent Manner ◽

Murine Models ◽

Btk Inhibitor

Abstract Background: Primary immune thrombocytopenia (ITP) is the most common autoimmune hemorrhagic disorder characterized by decreased platelet count. increased risk of bleeding, and poor quality of life. Only about 70% patients response to first-line treatments, some patients are still refractory or relapsed after combined therapies, therefore it is necessary to explore new therapeutic targets. Bruton's tytosine kinase (BTK) is a non-receptor tyrosine kinase of Tec family, which is widely expressed in hematopoietic cells including B cells, monocytes/macrophages, and others. BTK participates in a variety of signaling pathways of innate and adaptive immunity, and plays an important role in cell survival and maturation. Platelet destruction mediated by anti-platelet glycoprotein antibodies is considered to be the main cause of ITP. B cells differentiate into plasma cells and produce autoantibodies due to the intolerance to autoantigens, which are important effectors in the pathogenesis of ITP. We speculated that inhibition of BTK may reduce platelet destruction by inhibiting B cell activation and autoantibody production. Orelabrutinib is a new generation of BTK inhibitor which has been used in hematological malignancies, this is the first study to explore the mechanisms of BTK inhibitor in the treatment of ITP. Methods: The concentrations of orelabrutinib were set at 1 nM, 10 nM, 100 nM and 1 μM in the in vitro study. Peripheral blood mononuclear cells (PBMCs) were isolated from active ITP patients and healthy controls and cultured for 72 hours, the apoptosis rate of PBMCs in each group was measured by Annexin V/PI double staining. CD19 + B cells of ITP patients were sorted by magnetic beads and stimulated with anti-human IgM to evaluate the activation of B-cell receptor (BCR) pathway and differentiation of plasma cells, respectively. Further, we transfused the splenocytes of immunized CD61-KO mice (C57BL/6) into the severe combined immunodeficient (SCID) mice to establish the active ITP murine models. Orelabrutinib was administered intragastrically at 10mg/kg, once a day. The control group was treated with 0.5% CMC at the same volume and frequency. Platelet count was measured weekly, the peripheral blood was collected and the B cell subsets in spleen were detected by flow cytometry at days 28 after splenocyte transfusion. Results: The proportion of early apoptotic cells (Annexin V +PI -) in PBMCs from both ITP patients and healthy controls was increased by orelabrutinib at 1μM,but there was no statistical difference. Orelabrutinib significantly inhibited the expression of CD69 in a dose-dependent manner at the concentrations of 10nM, 100nM and 1μM. Another early activation marker of BCR signaling pathway, CD86, was also found to be inhibited by orelabrutinib at 100nM and 1μM. The number of CD138 + plasma cells was decreased after treated with orelabrutinib at 10nM, 100nM and 1μM without dose-dependent manner. In the murine models, mice administered with orelabrutinib had significantly higher platelet count than the control mice at days 7, 14, 28 after splenocyte transfusion. The frequency of total B cells in peripheral blood leukocytes, the proportion of GL-7 + germinal center B cells and plasma cells in splenocytes were all determined to be lower in mice treated with orelabrutinib than the control group, though did not reach the statistical significance. Conculsion: Orelabrutinib could effectively suppress the activation and differentiation of B cells invitro and invivo, thus alleviate the thrombocytopenia in active ITP murine models. It could be the new treatment strategy for refractory/relapsed ITP patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Overexpression of Myo1e promotes albumin endocytosis by mouse glomerular podocytes mediated by Dynamin

PeerJ ◽

10.7717/peerj.8599 ◽

2020 ◽

Vol 8 ◽

pp. e8599

Author(s):

Huijun Shen ◽

Yu Bao ◽

Chunyue Feng ◽

Haidong Fu ◽

Jianhua Mao

Keyword(s):

Flow Cytometry ◽

Cell Viability ◽

Fluorescence Intensity ◽

Annexin V ◽

Underlying Mechanism ◽

Immunofluorescence Assay ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Foot Process ◽

Albumin Endocytosis

Background As a fundamental process internalizing molecules from the plasma membrane, endocytosis plays a crucial role in podocyte biology. Our previous study has identified that overexpression of Myole may enhance podocyte endocytosis. However, its potential mechanism has been not well understand. Thus, we aimed to analyze whether albumin endocytosis by mouse glomerular podocytes is dependent on Myo1e expression. Also, we aimed to elucidate whether the underlying mechanism is mediated by Dynamin. Methods Firstly, mouse podocyte cells (MPC5) were treated with different concentrations of FITC-bovine serum albumin (BSA). The fluorescence intensity and cell viability were detected by flow cytometry and MTT assays, respectively. Afterwards, the optimal concentration of FITC-BSA was determined. Secondly, MPC5 cells were treated with Myole overexpression or knockdown. Cell morphology was observed under microscope. Immunofluorescence assay was used to determine the expression of F-actin. The protein expression of nephrin and podocin was detected by western blot. Flow cytometry was used to detect MPC5 cell apoptosis with annexin V. Finally, MPC5 cells were treated with Myole overexpression and/or Dynasore (a GTPase inhibitor of Dynamin). The fluorescence intensity was detected using flow cytometry assay. Results MPC5 endocytosis BSA was elevated with a concentration-dependent manner. MTT results showed that MPC5 cell viability was inhibited with a concentration-dependent manner. Myo1e overexpression promoted podocyte endocytic FITC-BSA, which was contrary to its knockdown. Under microscope, after inhibition of Myo1e, podocyte foot process fusion was observed. Myo1e overexpression promoted the expression of cytoskeleton F-actin and podocyte-specific molecules (nephrin and podocin) in podocyte endocytic FITC-BSA. Furthermore, we found that Myo1e promoted the apoptosis of podocytes. Dynasore attenuated the increase in endocytosis of FITC-BSA induced by Myo1e overexpression, suggesting that podocytes might mediate albumin endocytosis via Myo1e-Dynamin-Albumin. Conclusion Our findings revealed that overexpression of Myo1e promotes albumin endocytosis in mouse glomerular podocyte endocytic albumin mediated by Dynamin.

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