Yang He

Two novel monoclonal antibodies to VWFA3 inhibit VWF-collagen and VWF-platelet interactions.

Summary.u2002 Background:u2002The interaction of collagen‐von Willebrand factor (VWF)‐GPIb is essential for platelet adhesion, especially under high shear conditions. VWF, which acts as a bridge between platelets and exposed subendothelium, interacts with collagen through its A3 domain, which is a new target for the antithrombotic agent. Objective:u2002To develop functional blockers that specifically inhibit VWF‐dependent adhesion of platelets to collagen under high shear stress. Methods:u2002To develop murine antihuman VWF A3 monoclonal antibodies (mAbs) by standard hybridoma technology, and characterize their abilities to block interactions between VWF A3 and collagen as well as platelet function. Results:u2002Thirty anti‐VWF‐A3 mAbs were obtained. Among them, two mAbs, designated as SZ‐123 and SZ‐125, were found to inhibit VWF‐collagen type III interaction. SZ‐123 and SZ‐125 inhibited the binding of purified human VWF (1.5 or 3u2003μgu2003mL−1) to human placenta collagen type III (IC50u2003=u20030.07u2003±u20030.02 and 0.15u2003± 0.03u2003μgu2003mL−1, respectively) or to calf skin collagen type III (IC50u2003=u20030.48u2003±u20030.06 and 0.51u2003±u20030.07u2003μgu2003mL−1, respectively) coated on plates. Under flow shear condition (1000u2003s−1), SZ‐123 and SZ‐125 inhibited platelet adhesion on human placenta collagen‐ or calf skin collagen‐coated surfaces. Both mAbs also inhibited platelet aggregation induced by ristocetin, botrocetin or bovine plasma. Conclusions:u2002SZ‐123 and SZ‐125 inhibited VWF‐collagen and VWF‐platelet interactions.

N-Glycosylation Is Required for Matriptase-2 Autoactivation and Ectodomain Shedding

Background: Matriptase-2 is a hepatic membrane serine protease that regulates hepcidin expression and iron metabolism. Results: Mutations at specific N-glycosylation sites prevented matriptase-2 activation and ectodomain shedding. Conclusion: N-Glycans play an important role in regulating matriptase-2 activity. Significance: The results provide new insights into the mechanism underlying matriptase-2 expression, zymogen activation, and ectodomain shedding. Matriptase-2 is a hepatic membrane serine protease that regulates iron homeostasis. Defects in matriptase-2 cause iron deficiency anemia. In cells, matriptase-2 is synthesized as a zymogen. To date, how matriptase-2 expression and activation are regulated remains poorly understood. Here we expressed human matriptase-2 in HEK293 and hepatic BEL-7402, SMMC-7721, and QGY-7703 cells. By labeling cell surface proteins and Western analysis, we examined matriptase-2 cell surface expression, zymogen activation, and ectodomain shedding. Our results show that matriptase-2 was activated on the cell surface but not intracellularly. Activated matriptase-2 underwent ectodomain shedding, producing soluble fragments in the conditioned medium. By testing inactive mutants, R576A and S762A, we found that matriptase-2 activation and shedding were mediated by its own catalytic activity and that the one-chain form of matriptase-2 had little activity in ectodomain shedding. We made additional matriptase-2 mutants, N136Q, N184Q, N216Q, N338Q, N433Q, N453Q, and N518Q, in which each of the predicted N-glycosylation sites was mutated. All of these mutants were expressed on the cell surface. However, mutants N216Q, N453Q, and N518Q, but not the other mutants, had impaired zymogen activation and ectodomain shedding. Our results indicate that N-glycans at specific sites are critical for matriptase-2 activation. Together, these data provide new insights into the cell surface expression, zymogen activation, and ectodomain shedding of matriptase-2.

Detection of autoantibodies against platelet glycoproteins in patients with immune thrombocytopenic purpura by flow cytometric immunobead array

BACKGROUNDnThe goal of this study is to develop a flow cytometric immunobead array (FCIA) assay to detect platelet autoantibodies commonly present in bleeding patients with immune thrombocytopenic purpura (ITP).nnnMETHODSnPolystyrene microbeads coated with antibodies against human platelet glycoproteins (GPs) IX (SZ1), Ib (SZ2), IIIa (SZ21), IIb (SZ22), and P-selectin (SZ51) were incubated with platelet lysate from 50 ITP patients and 86 controls. The platelet antigen-autoantibody complexes were detected by flow cytometry using an FITC-labeled antibody. The results were compared with that of a monoclonal antibody immobilization of platelet antigen (MAIPA) assay.nnnRESULTSnBy FCIA, platelet autoantibodies against GPIb, GPIIb, GPIIIa, GPIX and P-selectin were detected in ITP patients. Mean fluorescent intensity values with antibodies SZ1, SZ2, SZ21, SZ22 and SZ51 were all higher in ITP patients than controls (p values<0.01). In ROC analysis, values of the area under the curve were 0.89, 0.82, 0.93, 0.94 and 0.95, respectively. In ITP diagnosis, the FCIA assay with these five antibodies had better sensitivity and accuracy than the MAIPA assay (96% vs. 44% in sensitivity; 80.9% vs. 64.7% in accuracy, p<0.01).nnnCONCLUSIONnFCIA assays with multiple antibodies against platelet GPs may be used to improve the diagnosis of ITP in hospitals.

Efficacy of immunomodulatory therapy with all-trans retinoid acid in adult patients with chronic immune thrombocytopenia

INTRODUCTIONnImmune thrombocytopenia (ITP) is a common hematologic disorder characterized by isolated thrombocytopenia. In adults, ITP is more likely to be chronic, requiring individualised treatment and management. Corticosteroids and splenectomy are the most common therapy for ITP. However, these routine approaches failed in these patients with chronic ITP. The aim of this study was to evaluate the efficacy of immunomodulatory therapy with all-trans retinoid acid (ATRA) in adult patients with chronic ITP.nnnMATERIALS AND METHODSnATRA therapy was applied in a total of 35 patients with chronic ITP who failed with standard dose corticosteroids and/or splenectomy. The response ratio and the change of the T cell subsets including Th1, Th2, Th17 and Treg, were evaluated.nnnRESULTSnComplete response and overall response were observed in 10 (28.6%) and 19 patients (54.3%), respectively. Compared with the control group, a significant decreased level of Treg cells, IL-10 and Foxp3 expression were found in ITP patients. ATRA therapy could significantly increase the percentage of Treg cell, IL-10 level and Foxp3 expression.nnnCONCLUSIONSnOur findings indicate that ATRA therapy could induce significant changes of Treg cells to induce response in patients with chronic ITP.

An improved flow cytometric immunobead array to detect autoantibodies in plasma from patients with immune thrombocytopenic purpura.

BACKGROUNDnAutoantibodies against platelet glycoproteins (GPs) play an important role in immune thrombocytopenic purpura (ITP). This study was to develop an improved flow cytometric immunobead array (FCIA) assay to detect platelet autoantibodies in ITP patient plasma.nnnMETHODSnPlasma samples were isolated from 71 ITP patients and 136 non-ITP controls and incubated with platelets from normal individuals. After washing, platelets were lysed and the platelet lysates were incubated with polystyrene microbeads coupled with monoclonal antibodies against human GPs IX (SZ1), Ib (SZ2), IIIa (SZ21), IIb (SZ22), and P-selectin (SZ51). Platelet GP-autoantibody complexes were detected by flow cytometry using a FITC-labeled secondary antibody.nnnRESULTnAutoantibodies against platelet GPIb, GPIIb, GPIIIa, GPIX and P-selectin were detected in plasma from ITP patients, as indicated by high mean fluorescent intensity values when microbeads with antibodies SZ1, SZ2, SZ21, SZ22, and SZ51 were used. In ROC analysis, values of the area under the curve were 0.74, 0.83, 0.80, 0.79 and 0.87, respectively. Compared with the previously reported assays, this new FCIA eliminated the need of isolating platelets from ITP patients without compromising assay sensitivity and accuracy in predicting ITP.nnnCONCLUSIONnThis simplified FICA assay may be more suitable for ITP diagnosis in clinical laboratory settings.

An Uncoordinated-5 Homolog B Receptor Monoclonal Antibody Regulates A375 Melanoma Cell Migration

Uncoordinated-5 homolog B receptor (UNC5B) was first found to mediate neural chemorepulsive effects by binding to its ligand netrin-1 in the nervous system. Newer evidence indicated that UNC5B also has functions outside the nervous system. In this study, we report on the generation of a monoclonal antibody specific to the outer-membrane immunoglobulin-like domains of UNC5B using the hybridoma technique. Western blot, immunofluorescence, and flow cytometry analyses showed that the antibody specifically bound to UNC5B protein. Interestingly, the antibody blocked the Netrin-1-induced inhibitory effect on the mobility of melanoma A375 cells by wound healing assay and transwell migration assay, whereas it had no effects on cell proliferation measured by CCK-8 assay. Thus, the functional antibody may provide a useful tool for the study of UNC5B expression profiles and functions outside the nervous system.

A novel monoclonal antibody targeting a novel epitope of VE‑cadherin inhibits vasculogenic mimicry of lung cancer cells

Vascular endothelial cadherin (VE‑cadherin) was first found in vascular endothelial cells to maintain normal vascular structures and regulate endothelial cell permeability by homology adhesion. New evidence indicates that certain invasive tumor cells also express VE‑cadherin, which is involved in vasculogenic mimicry to provide a blood supply required for tumor growth and metastasis. EC1 and EC3 domains of VE‑cadherin were reported to be important for intercellular homology adhesion. In the present study, a monoclonal antibody specific to the outer-membrane immunoglobulin-like domains of VE‑cadherin was generated and the binding epitope was identified as peptide LDREVVPWYNLTVEA in the EC4 domain. This antibody inhibited proliferation and capillary-like structure formation of lung cancer Glc‑82 cells in 3D Matrigel culture inxa0vitro. This effect was mediated by the inhibition of AKT phosphorylation. Our results suggested that the EC4 domain participates in VE‑cadherin clustering and the antibody targeting the EC4 domain of VE‑cadherin may be a promising anti‑vasculogenic mimicry agent for cancer treatment.

Altered circulating T follicular helper cells in patients with chronic immune thrombocytopenia

The present study aimed to illuminate the role of circulating T follicular helper (TFH) cells in patients diagnosed with chronic immune thrombocytopenia (cITP). Fifty-four patients with cITP and 30 age-matched healthy control subjects were enrolled in the present study. TFH cell frequencies, expression of CD4+ TFH cell-associated cytokines, including interleukin (IL)-2, IL-4, IL-10 and IL-21 and associated regulatory mRNA expression levels including Bcl-6, c-Maf, Blimp-1 and PD-1 pre- and post-treatment with intravenous immunoglobulin and corticosteroids, were detected by flow cytometry, ELISA and reverse transcription-quantitative polymerase chain reaction, respectively. TFH cell frequencies of patients were significantly higher compared with healthy controls pre-treatment (P<0.05). Following treatment, significantly decreased percentages of TFH cells were present in cITP responders (P<0.05). Correlation analysis revealed that the number of TFH cells was negatively correlated with the platelet count in the peripheral blood. Furthermore, analysis of inflammatory cytokines indicated significant differences in serum interleukin (IL)-21 and IL-10 between pretreated patients and healthy controls (P<0.05). Additionally, transcription factor B-cell lymphoma (Bcl)-6, c-Maf and programmed death-ligand (PD)-1 mRNA expression levels were significantly different between cITP patients prior to treatment and the healthy controls (P<0.05). However, the expression levels of Bcl-6, C-Maf and PD-1 mRNA were significantly changed post-treatment (P<0.05). These data demonstrated that circulating TFH cells and CD4+ TFH cell-associated cytokines may serve a role in cITP. The findings suggest that the overactivation of TFH cells may contribute to the immunopathogenesis of cITP, thus blocking the pathway of TFH cells may be reasonable for therapeutic intervention.

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients

BackgroundPlatelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation.MethodsCapture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs.ResultsThe coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%.ConclusionsA quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.