Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX–specific quinine-dependent antibodies

Blood ◽  
2000 ◽  
Vol 95 (6) ◽  
pp. 1988-1992 ◽  
Author(s):  
Janette K. Burgess ◽  
Jose A. Lopez ◽  
Leonie E. Gaudry ◽  
Beng H. Chong
Keyword(s):  
Flow Cytometric Analysis ◽  
Antibody Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Drug Induced ◽  
Complementary Dna ◽  
Glycoprotein Complex ◽  
Flow Cytometric ◽  
Antibody Binding Site ◽  
Close Proximity ◽  
Drug Dependent

Abstract The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because (1) it immunoprecipitated drug-dependently the former but not the latter glycoprotein complex and (2) the MAIPA assay showed strong rifampicin-dependent antibody binding when anti-GPIb-IX monoclonal antibodies (mAbs) (AK2 and FMC25) but not anti-GPIIb-IIIa mAbs (AP2, SZ21, and SZ22) were used to capture the antigen. The antibody binding site was further localized to the GPIX subunit of the GPIb-IX complex because flow cytometric analysis revealed drug-dependent antibody binding to L cells transfected with human GPIbβ and GPIX complementary DNA (L βIX cells) but not with human GPIb and GPIbβ complementary DNA (L β cells). Finally, in the MAIPA assay, the rifampicin-dependent antibody almost completely cross-blocked the binding of the anti-GPIX mAb (SZ1) to platelets. Similar cross-blocking of SZ1binding to platelets by the quinine-dependent antibodies was also observed. This finding not only confirms that the epitope of the rifampicin-dependent antibody is on GPIX but it is also identical to or located in close proximity to that of the quinine-dependent antibody and SZ1. Further characterization of the epitopes of these antibodies may have important implications for a general understanding of the mechanism of drug-induced thrombocytopenia.

Fibrinogen Receptor Antagonist-Induced Thrombocytopenia in Chimpanzee and Rhesus Monkey Associated With Preexisting Drug-Dependent Antibodies to Platelet Glycoprotein IIb/IIIa

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 587-599 ◽  
Author(s):  
Bohumil Bednar ◽  
Jacquelynn J. Cook ◽  
Marie A. Holahan ◽  
Michael E. Cunningham ◽  
Patricia A. Jumes ◽  
...  
Keyword(s):  
Platelet Count ◽  
Rhesus Monkey ◽  
Human Subjects ◽  
Active Form ◽  
Flow Cytometric Analysis ◽  
Platelet Glycoprotein ◽  
Fibrinogen Receptor ◽  
Flow Cytometric ◽  
Drug Dependent ◽  
Potent Antagonist

Abstract Most clinical trials with fibrinogen receptor antagonists (FRAs) have been associated with thrombocytopenia. This report describes the occurrence of thrombocytopenia in one chimpanzee and one rhesus monkey upon administration of potent FRAs. Chimpanzee A-264 experienced profound thrombocytopenia on two occasions immediately upon intravenous administration of two different potent FRAs, L-738,167 and L-739,758. However, an equally efficacious antiaggregatory dose of another potent antagonist, L-734,217, caused no change in platelet count. These compounds did not affect platelet count in five other chimpanzees or numerous other nonhuman primates. Flow cytometric analysis showed drug-dependent antibodies (DDAbs) in the plasma of chimpanzee A-264 that bound to platelets of chimpanzees, humans, and all other primates tested only in the presence of the compounds that induced thrombocytopenia. Rhesus monkey 94-R021 experienced thrombocytopenia upon administration of a different antagonist, L-767,679, and several prodrugs that are converted into the active form, L-767,679, in the blood. More than 20 other FRAs, including those that induced thrombocytopenia in chimpanzee A-264, had no effect on platelet count in this monkey. Flow cytometric measurements again identified DDAbs that reacted with platelets of all primates tested and required the presence of L-767,679. Screening for DDAbs in the plasma of 1,032 human subjects with L-738,167 and L-739,758 demonstrated that the incidence of these preexisting antibodies in this population was 0.8% ± 0.6% and 1.1% ± 0.6%, respectively.

scholarly journals Flow cytometric analysis of drug-Induced basophil histamine release

2015 ◽  
Vol 90 (3) ◽  
pp. 285-288 ◽  
Author(s):  
N. Cop ◽  
A. P. Uyttebroek ◽  
V. Sabato ◽  
C. H. Bridts ◽  
L. S. De Clerck ◽  
...  
Keyword(s):  
Histamine Release ◽  
Flow Cytometric Analysis ◽  
Drug Induced ◽  
Flow Cytometric

scholarly journals Quinine-dependent, platelet-reactive monoclonals mimic antibodies found in patients with quinine-induced immune thrombocytopenia

Blood ◽  
2009 ◽  
Vol 113 (5) ◽  
pp. 1105-1111 ◽  
Author(s):  
Daniel W. Bougie ◽  
Jessica Birenbaum ◽  
Mark Rasmussen ◽  
Mortimer Poncz ◽  
Richard H. Aster
Keyword(s):  
Molecular Basis ◽  
Immune Thrombocytopenia ◽  
Antibody Binding ◽  
Membrane Glycoproteins ◽  
Binding Studies ◽  
Platelet Destruction ◽  
Drug Induced ◽  
N Terminus ◽  
Sequencing Studies ◽  
Drug Dependent

Abstract Drug-induced immune thrombocytopenia (DITP) is caused by drug-dependent antibodies (DDAbs) that are nonreactive in themselves but bind tightly to specific platelet membrane glycoproteins (GP) when soluble drug is present at pharmacologic concentrations. This reaction takes place without covalent linkage of drug to the target, indicating that drug does not function as a classical hapten to promote antibody binding. Studies to define other mechanism(s) responsible for this interaction have been frustrated by the polyclonal nature of human DDAbs and limited quantities of antibody usually available. We produced 2 monoclonal antibodies (mAbs), 314.1 and 314.3, from a mouse immunized with purified human GPIIb/IIIa and quinine that recognize the N terminus of the GPIIb β propeller domain only when soluble quinine is present. Both monoclonals closely mimic the behavior of antibodies from patients with quinine-induced immune thrombo-cytopenia in their reactions at various concentrations of quinine and quinine congeners. Sequencing studies showed that the 2 mAbs are closely related structurally and that mAb 314.3 probably evolved from mAb 314.1 in the course of the immune response. These monoclonal reagents are the first of their kind and should facilitate studies to define the molecular basis for drug-dependent antibody binding and platelet destruction in DITP.

scholarly journals Generation of iPSC-Derived Human Megakaryocytes for Flow Cytometric Detection of Platelet-Specific Alloantibodies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 740-740
Author(s):  
Nanyan Zhang ◽  
Peter Newman
Keyword(s):  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Hla Class I ◽  
Blood Type ◽  
Class I ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Transfusion Therapy ◽  
Flow Cytometric ◽  
Simple Flow

Abstract Antibodies that form against human platelet alloantigens (HPAs) are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia, posttransfusion purpura, and multitransfusion platelet refractoriness. Some of HPAs are relatively rare in the population, and difficult to obtain for purposes of transfusion therapy and diagnostic testing. In addition, HPA alloantisera often contain antibodies against human leucocyte antigen (HLA) class I, thereby limiting antibody detection to glycoprotein (GP)-specific assays such as the modified antigen capture enzyme-linked immunosorbent assay (MACE) and the monoclonal immobilization of platelet antigen (MAIPA), which are tedious and require solubilization of platelet GPs that may cause the loss of epitopes. In this study we aimed to generate gene-edited, HPA-specific, megakaryocytes (MKs) derived from human induced pluripotent stem cells (iPSCs) that could be used for simple flow cytometric detection of specific HPA alloantibodies present in patient sera. The HPA-3a/HPA-3b alloantigen system, also known as Baka/Bakb, is caused by a single T13809G nucleotide substitution in the ITGA2B gene, resulting in an Ile874Ser amino acid polymorphism near the C terminus of the integrin αIIb subunit (GPIIb). Here we targeted HPA-3 system because alloantibodies targeting HPA-3 are often hard to detect with current detection methods, in part due to the requirement for cell type-specific glycosylation. To prevent interference of anti-A or anti-B antibodies in patient sera, a blood type O iPSC line (OT1-1) was generated from human peripheral blood mononuclear cells derived from a healthy donor using integration-free episomal vectors. The gene for β2 microglobulin (B2M) was first ablated from the OT1-1 iPS cell line using CRISPR/Cas9 to prevent binding of HLA class I alloantibodies. The resulting B2M knockout (B2MKO) cells were then additionally gene edited to convert the endogenous HPA-3a alloantigenic epitope present on B2MKO OT1-1 cells to HPA-3b. Two different guide RNAs targeting sequences that flank exon 26 of the ITGA2B gene were designed such that the entire exon harboring the HPA-3 polymorphic site was removed. A plasmid harboring a template replacing exon 26 with the G13809 mutation, flanked by 600 bp homology arms, was cotransfected into the B2MKO OT1-1 iPSCs together with the two CRISPR/Cas9 guide RNA constructs. iPSC clones containing the desired targeted T13809G mutation were identified by a diagnostic MfeI digestion specific for the G13089-bearing HPA-3b allele. Sequence analysis confirmed conversion of T13089 to G in these HPA-3b clones. Flow cytometric analysis showed the HPA-3a iPSCs, when differentiated into CD41+/CD42b+ MKs, specifically reacted with HPA-3a, but not HPA-3b, patient sera, while the HPA-3b iPSC-derived MKs lost reactivity with HPA-3a patient serum, and gained the reactivity with HPA-3b patient sera. Taken together, we have established genetically modified iPSC-derived MKs expressing specific HPAs that are suitable for simple flow cytometry-based detection of HPA alloantibodies in patient sera, with low non-specific background binding. This system provides intact antigens on the cell surface with carbohydrate moieties that likely mimic those found on human platelets, thus facilitating the detection of HPA alloantibodies that are normally hard to detect with current methods. Application of this strategy to genetically edit this and other clinically-important HPAs holds great potential for producing Designer Platelets for diagnostic, investigative and ultimately therapeutic use. Disclosures No relevant conflicts of interest to declare.

HNA−1a, −1b, −2a, −3a and −4a Frequencies in Brazilian Persons.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3837-3837 ◽  
Author(s):  
Angela M. Norcia ◽  
Elisa Y. Kimura ◽  
Akemi K. Chiba ◽  
Elyse Moritz ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
African Americans ◽  
Blood Donors ◽  
Mononuclear Cells ◽  
Flow Cytometric Analysis ◽  
Fluorescein Isothiocyanate ◽  
Blue Staining ◽  
Drug Induced ◽  
Flow Cytometric ◽  
The Gift

Abstract Granulocyte reactive antibodies have been found to cause clinical disorders such as transfusion related acute lung injury (TRALI), febrile transfusion reactions, alloimmune neonatal neutropenia, immune neutropenia after bone marrow transplantation, refractoriness to granulocyte transfusion, drug-induced neutropenia and autoimmune neutropenia. Using the granulocyte immunofluorescence test (GIFT) by microscopic and flow cytometric analysis, the frequencies of the neutrophil antigens HNA−1a, −1b, −2a, −3a and −4a were determined among 100 random Brazilian blood donors from the Blood Center of Universidade Federal de Sao Paulo, SP, Brazil. Granulocytes were separated from mononuclear cells and red cells by sedimentation with 5% dextran, followed by centrifugation on Ficoll-paque (d = 1.077), and then incubated with anti-sera (anti-HNA−1a, −1b, −2a, −3a, and −4a obtained from American Red Cross, North Central Blood Services, St. Paul, MN) conjugated with fluorescein isothiocyanate (FITC) labeled F(ab’)2 fragments of anti-human IgG. Only cell suspensions containing ≥95% neutrophils with viability ≥90% according to the trypan-blue staining were analyzed. The frequencies of HNA−1a, −1b and −2a were 65%, 83% and 94%, respectively, and for such alloantigens exact same results were observed using either the GIFT performed by microscopy or by flow cytometry. The frequency of HNA-3a was 86% by the microscopic GIFT, and 95% by the flow cytometry analysis; while the frequency of HNA−4a was 93% by the microscopic GIFT, and 94% by the flow cytometric technique. These results indicate that: GIFT by flow cytometry is more sensitive than the GIFT by microscopy to detect HNA−3a; the phenotypic frequencies found for neutrophil antigens HNA−1a and −1b among Brazilian blood donors are quite similar to those reported among African Americans, but different from those reported for Japanese and Chinese individuals; the phenotype frequencies of the neutrophil antigens HNA−2a, −3a, and −4a in Brazilians are quite similar to those found among Caucasians (Table). (These studies were funded by FAPESP, SP, Brazil - 05/55237–9). Asians Brazilians Antigen African Americans Chineses Hindus Japaneses Caucasians M FC M, microscopy; FC, flow cytometry; nd, not done HNA-1a 46 – 68 90 44 88 52 – 54 65 65 HNA-1b 78 – 84 52 83 51 – 64 87 – 89 83 83 HNA-2a nd 99 nd 89 87 – 97 94 94 HNA-3a nd nd nd nd 99 86 95 HNA-4a nd nd nd nd 96 93 94

scholarly journals Effects of Radix Adenophorae and Cyclosporine A on an OVA-Induced Murine Model of Asthma by Suppressing to T Cells Activity, Eosinophilia, and Bronchial Hyperresponsiveness

2008 ◽  
Vol 2008 ◽  
pp. 1-11 ◽  
Author(s):  
Seong-Soo Roh ◽  
Seung-Hyung Kim ◽  
Young-Cheol Lee ◽  
Young-Bae Seo
Keyword(s):  
T Cells ◽  
Lung Tissue ◽  
Cyclosporine A ◽  
Murine Model ◽  
Flow Cytometric Analysis ◽  
Bronchial Hyperresponsiveness ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inhibitory Effects ◽  
Lung Weight ◽  
Flow Cytometric

The present study is performed to investigate the inhibitory effects of Radix Adenophorae extract (RAE) on ovalbumin-induced asthma murine model. To study the anti-inflammatory and antiasthmatic effects of RAE, we examined the development of pulmonary eosinophilic inflammation and inhibitory effects of T cells in murine by RAE and cyclosporine A (CsA). We examined determination of airway hyperresponsiveness, flow cytometric analysis (FACS), enzyme-linked immunosorbent assay (ELISA), quantitative real time (PCR), hematoxylin-eosin staining, and Masson trichrome staining in lung tissue, lung weight, total cells, and eosinophil numbers in lung tissue. We demonstrated how RAE suppressed development on inflammation and decreased airway damage.

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3540-3546 ◽  
Author(s):  
Jeffrey T. Billheimer ◽  
Ira B. Dicker ◽  
Richard Wynn ◽  
Jodi D. Bradley ◽  
Debra A. Cromley ◽  
...  
Keyword(s):  
Chemical Structure ◽  
Blood Donors ◽  
Solid Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Drug Induced ◽  
Immune Mediated ◽  
Antagonist Binding ◽  
Orally Bioavailable ◽  
Drug Dependent ◽  
High Level

Glycoprotein (GP) IIb/IIIa antagonists are effective therapeutic agents, but elicit thrombocytopenia with a frequency that approaches 2%. Here, we provide evidence that thrombocytopenia in humans treated with the GP IIb/IIIa antagonist roxifiban is immune mediated. Two patients underwent conversion to a highly positive drug-dependent antibody (DDAB) status temporally associated with thrombocytopenia. Despite the continued presence of DDABs, the fall in platelet count was reversed by discontinuation of drug treatment, pointing to the exquisite drug dependency of the immune response. DDABs appear to bind to neoepitopes in GP IIb/IIIa elicited on antagonist binding. This information was used to develop an enzyme-linked immunosorbent assay (ELISA) for DDAB using solid-phase GP IIb/IIIa. A high level of specificity is indicated by the observation that DDAB binding is dependent on the chemical structure of the GP IIb/IIIa antagonist and that only 2% to 5% of human blood donors and 5% of chimpanzees present with pre-existing DDABs. Furthermore, none of 108 nonthrombocytopenic patients from the phase II roxifiban study showed an increase in antibody titer. Absorption of thrombocytopenia plasma with platelets reduced the DDAB ELISA signal, indicating that the test detects physiologically relevant antibodies. Screening patients for pre-existing or increasing DDAB titer during treatment with GP IIb/IIIa antagonists may reduce the incidence of drug-induced thrombocytopenia.

scholarly journals Aggravated brain injury after neonatal hypoxic ischemia in microglia depleted male mice

2019 ◽  
Author(s):  
Shunichiro Tsuji ◽  
Elena Di Martino ◽  
Takeo Mukai ◽  
Shoko Tsuji ◽  
Takashi Murakami ◽  
...  
Keyword(s):  
Neuronal Damage ◽  
Quantitative Polymerase Chain Reaction ◽  
Flow Cytometric Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Disease States ◽  
Flow Cytometric ◽  
Cytokine Analysis ◽  
Hypoxic Ischemia ◽  
Significant Difference ◽  
Polymerase Chain

Abstract Background: Neuroinflammation plays important roles in neonatal hypoxic–ischemic encephalopathy (HIE). Microglia are largely responsible for the injury-induced inflammatory response, but they also play beneficial roles in both normal and disease states. Nevertheless, the effects of microglial depletion in neonatal HIE remain unclear. Methods: Tamoxifen was administered to Cx3cr1 CreER/+ Rosa26 DTA/+ (microglia-depleted model) and Cx3cr1 CreER/+ Rosa26 DTA/- (control) mice at P8 and P9 in order to assess the effect of microglial depletion. The density of microglia was quantified using Iba-1 staining and the proportion of resident microglia after the hypoxic–ischemic (HI) insult was also analyzed using flow cytometric analysis. The HI insult employed the Rice-Vannucci procedure at P10. Infarct size and apoptotic cells were analyzed at P13. Cytokine analysis was performed by quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) at P13. Results: Tamoxifen administration induced over 99% microglial depletion at P10 in DTA + mice. Microglial depletion over 97% persisted at P13 following the HI insult. Male DTA + mice exhibited significantly larger infarct volumes than did male DTA - mice, but there was no significant difference in females. The density of TUNEL + cells in DTA + mice was significantly higher than that in DTA - mice in the caudoputamen, cerebral cortex, and thalamus in males. Females showed significantly greater numbers of TUNEL + cells in the hippocampus and thalamus in DTA + mice compared to DTA - mice. ELISA revealed that IL-10 and TGF-β levels were significantly lower in both male and female DTA + mice compared to DTA - mice, both under normal conditions and, more pronounced, after HI. Conclusion: We established a model of microglial depletion that aggravated neuronal damage and apoptosis after the HI insult, an effect most predominant in males.

Sign in / Sign up

Export Citation Format

Share Document