scholarly journals Molecular identification of T cells that respond in a primary bulk culture to a peptide derived from a platelet glycoprotein implicated in neonatal alloimmune thrombocytopenia.

1996 ◽  
Vol 98 (8) ◽  
pp. 1802-1808 ◽  
Author(s):  
K Maslanka ◽  
M Yassai ◽  
J Gorski
Keyword(s):  
T Cells ◽  
Molecular Identification ◽  
Platelet Glycoprotein ◽  
Alloimmune Thrombocytopenia ◽  
Bulk Culture

scholarly journals A novel murine model of fetal and neonatal alloimmune thrombocytopenia: response to intravenous IgG therapy

Blood ◽  
2006 ◽  
Vol 107 (7) ◽  
pp. 2976-2983 ◽  
Author(s):  
Heyu Ni ◽  
Pingguo Chen ◽  
Christopher M. Spring ◽  
Ebrahim Sayeh ◽  
John W. Semple ◽  
...  
Keyword(s):  
High Titer ◽  
Maternal Antibodies ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Wild Type Male ◽  
Glycoprotein Iiia ◽  
Life Threatening ◽  
Β3 Integrin ◽  
Pathogenic Antibodies ◽  
Alloimmune Thrombocytopenia

AbstractFetal and neonatal alloimmune thrombo cytopenia (FNAITP) is a life-threatening bleeding disorder caused by maternal antibodies directed against fetal platelet antigens. The immunoreactive epitopes in FNAITP are primarily located in the extracellular regions of the platelet glycoprotein IIIa (β3 integrin). Here we have established a novel animal model of FNAITP using β3 integrin–deficient (β3-/-) mice. We demonstrated first that these mice are immunoresponsive to β3 integrin; β3-/- mice transfused with wild-type platelets generated specific anti–β3 antibodies which were able to induce thrombocytopenia in wild-type mice. Subsequently, β3-/- female mice (both naive and immunized) were bred with wild-type male mice to recapitulate the features of FNAITP. The titer of generated maternal antibodies correlated with the severity of FNAITP. High titer maternal anti–β3 anti-bodies caused severe fetal thrombocytopenia, intracranial hemorrhage, and even miscarriage. Furthermore, maternal administration of intravenous immunoglobulin G (IgG) ameliorated FNAITP and down-regulated pathogenic antibodies in both the maternal and fetal circulations.

scholarly journals A new low-frequency alloantigen (Khab) located on platelet glycoprotein IIIa as a cause of maternal sensitization leading to neonatal alloimmune thrombocytopenia

Transfusion ◽  
2014 ◽  
Vol 55 (6pt2) ◽  
pp. 1584-1585 ◽  
Author(s):  
Mia J. Sullivan ◽  
Julie Peterson ◽  
Janice G. McFarland ◽  
Daniel Bougie ◽  
Richard H. Aster ◽  
...  
Keyword(s):  
Low Frequency ◽  
Platelet Glycoprotein ◽  
Glycoprotein Iiia ◽  
Alloimmune Thrombocytopenia

scholarly journals Cell-mediated suppression of megakaryocytopoiesis in acquired amegakaryocytic thrombocytopenic purpura

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 619-626 ◽  
Author(s):  
AM Gewirtz ◽  
MK Sacchetti ◽  
R Bien ◽  
WE Barry
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Lymphocytes ◽  
Colony Formation ◽  
Progenitor Cell ◽  
Mononuclear Cells ◽  
Platelet Glycoprotein ◽  
Severe Thrombocytopenia ◽  
Thrombocytopenic Purpura ◽  
Marrow Cells

Abstract Acquired amegakaryocytic thrombocytopenic purpura (AATP) is a disorder of hematopoiesis characterized by severe thrombocytopenia due to a selective reduction or total absence of megakaryocytes in an otherwise normal-appearing bone marrow. Although the development of autoantibodies directed against cells in the megakaryocyte progenitor cell pool has been implicated in the pathogenesis of this disorder, cell-mediated suppression of megakaryocytopoiesis has not been described. Accordingly, we report two cases of AATP in which in vitro suppression of megakaryocyte colony formation by autologous ancillary marrow cells was demonstrable. Light-density bone marrow mononuclear cells (MNCs) obtained from both patients were either plated directly into plasma clot cultures, or after first being depleted by adherent monocytes (M phi) or T lymphocytes using standard methodologies. In some experiments, the depleted ancillary marrow cells were recovered for autologous co-culture studies with the MNCs from which they had been depleted. Megakaryocyte colony formation was detected in the cultures using an indirect immunofluorescence assay with a rabbit anti- human platelet glycoprotein antiserum. Removal of M phi (n = 6), or T lymphocytes (n = 4) from normal marrow MNCs had no apparent effect on colony formation. In contrast, depleting T lymphocytes from the MNCs of patient 1 significantly augmented megakaryocyte colony formation; a similar effect was observed after depleting M phi from the MNCs of patient 2. This observed augmentation in colony formation could be abrogated by autologous co-culture with the putative suppressor cell at effector cell/target cell ratios of 1:10 in the case of T lymphocytes or 1:5 in the case of M phi. Neither suppression nor stimulation of megakaryocyte colony formation was observed after culturing normal MNCs with autologous T cells (n = 4) or M phi (n = 3) at similar or greater ratios. We also observed inhibition of megakaryocyte colony formation after culturing normal MNCs in the presence of tissue culture medium conditioned by the M phi of patient 2. This effect was shown to be specific for megakaryocytes since this same conditioned medium had no significant effect on BFU-E and CFU-E-derived colony formation by autologous marrow mononuclear cells. These results suggest that: both T cells and M phi are capable of exerting a regulatory effect on the proliferation of human megakaryocyte progenitor cells (CFU-Meg); in the case of M phi, a soluble factor elaborated by these cells may be responsible for suppressing CFU-Meg growth; and aberrant ancillary cell- megakaryocyte progenitor cell interactions may lead to clinically significant disease.

scholarly journals Single point mutation in human glycoprotein IIIa is associated with a new platelet-specific alloantigen (Mo) involved in neonatal alloimmune thrombocytopenia

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 70-76 ◽  
Author(s):  
RW Kuijpers ◽  
S Simsek ◽  
NM Faber ◽  
R Goldschmeding ◽  
RK van Wermerkerken ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Single Point ◽  
Blot Hybridization ◽  
Platelet Glycoprotein ◽  
Single Point Mutation ◽  
Polymorphism Analysis ◽  
Coding Region ◽  
Dot Blot ◽  
Glycoprotein Iiia ◽  
Alloimmune Thrombocytopenia

Abstract Here we describe a new platelet-specific alloantigen that was identified in a case of neonatal alloimmune thrombocytopenia. This antigen has provisionally been called “Mo.” By studying the Mo family, it was shown to be inherited in an autosomal dominant manner. Immunoprecipitation and Western blot analysis showed that the antigen resides on platelet glycoprotein IIIa (GP IIIa). Genomic analysis, performed by applying polymerase chain reaction and sequencing, showed a C-->G substitution of base pair 1267 of the coding region of the DNA for GP IIIa, resulting in a substitution of Proline407 by Alanine407. That this substitution is associated with the antigen could be demonstrated by restriction fragment length polymorphism analysis of cDNA, prepared from platelet RNA, and of genomic DNA. It was confirmed by dot-blot hybridization with allele-specific oligonucleotides. All family members, also those being Mo antigen-positive, were healthy. None of them appeared to suffer from increased tendency of bleeding or thrombosis. Thus, the Mo mutation does not lead to significant platelet dysfunction in vivo with heterozygous carriers. One of 450 random healthy blood donors who were tested was positive for the Mo antigen. Typing was performed by the classical serologic methods as well as by DNA analysis.

Platelets: New Understanding of Platelet Glycoproteins and Their Role in Disease

Hematology ◽  
2000 ◽  
Vol 2000 (1) ◽  
pp. 222-240 ◽  
Author(s):  
James B. Bussel ◽  
Thomas J. Kunicki ◽  
Alan D. Michelson
Keyword(s):  
Point Of View ◽  
Platelet Glycoprotein ◽  
Clinical Implications ◽  
New Developments ◽  
Advantages And Disadvantages ◽  
Current State ◽  
Platelet Antigen ◽  
Low Platelet Count ◽  
Case Presentations ◽  
Alloimmune Thrombocytopenia

Abstract This review covers new developments and their clinical implications in three areas: platelet antigen polymorphisms, inhibition of platelet glycoprotein IIb-IIIa, and autoimmune thrombocytopenia (ITP). In Section I, Dr. Kunicki reviews platelet polymorphisms and their clinical implications. A current tabulation of the numerous platelet antigens, both those that are platelet specific and not platelet specific, are summarized. The immunogenic clinical implications of these polymorphisms are considered, including fetal and neonatal alloimmune thrombocytopenia, post transfusion purpura, and refractoriness to platelet transfusion. The functional relationship to hemostasis and thrombosis is also discussed, in particular whether one haplotype of the PIA1/PIA2 (HPA-1a/1b) polymorphism predisposes to myocardial infarction. Finally, novel investigations of polymorphisms will be considered, including hormonal induction of certain polymorphisms. In Section II, Dr. Michelson reviews the newest generation of platelet inhibitors, those blocking glycoprotein IIB/IIIA, from the point of view of the hematologist who might be consulted about a patient receiving this form of treatment. The current use of available IIb-IIIa inhibitors and those in trial and the accepted and possible future indications for their use are addressed. The mechanism of action and actual and theoretical advantages and disadvantages of each inhibitor are explored. Scenarios that prompt consultation with a hematologist are presented, including management of bleeding, thrombocytopenia, and management of the patient requiring emergency surgery. In Section III, Dr. Bussel reviews controversies in ITP, looking at both the current state of the art and the potential for the future. Case presentations are used to illustrate the issues in both children and adults. Three primary areas are addressed: 1) the diagnosis of ITP, 2) when and for which patient to recommend splenectomy, and 3) the management of the refractory splenectomized patient who still has a low platelet count and bleeding symptoms.

Isolation and Characterization of CD4+ T Cells Specific for the HPA 1a Epitope Associated with Neonatal Alloimmune Thrombocytopenia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2091-2091
Author(s):  
Maria T. Ahlen ◽  
Mette K. Killie ◽  
Bjorn Skogen ◽  
Anne Husebekk ◽  
Tor B. Stuge
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cell Detection ◽  
Severe Thrombocytopenia ◽  
Isolation And Characterization ◽  
T Cell Lines ◽  
Alloimmune Thrombocytopenia

Abstract Neonatal alloimmune thrombocytopenia (NAIT) can cause severe complications such as intrauterine death or intracranial hemorrhage (ICH) in the newborn, and is caused by the transfer of platelet-depleting antibodies from the mother to the fetus during pregnancy. These antibodies react with allogeneic epitopes, most commonly human platelet antigen (HPA) 1a, when present on fetal platelets. Although these responses are thought to be a result of a T cell-dependent immune response, HPA 1a specific T cells have not yet been isolated. To examine whether HPA 1a specific T cells could be detected and isolated, we collected PBMC post delivery from an HPA 1a negative mother who gave birth to an HPA 1a positive neonate suffering from severe thrombocytopenia (platelet count <50×109/L). The cells were stimulated with HPA 1a peptides (20aa) in long term cultures supplemented with IL-7 and IL-2, and subsequently, IL-15. After 4 weeks in culture these cells were labeled with CFSE dye and restimulated with HPA 1a or control peptides. After additional 2 weeks in culture supplemented with IL-2 and IL-15, specific proliferative responses were detectable by CFSE dye dilution by flow cytometry. The cells were cloned by fluorescent-activated cell sorting (FACS) and expanded in numbers with anti-CD3 stimulation in the presence of irradiated allogeneic PBMC and IL-2. The resulting clonal T cell lines were characterized in proliferation assays, ELISPOT assays and phenotyped by flow cytometry. All clones were CD3+, CD4+ and CD19−, and the majority of the clones proliferated and secreted cytokines in response to stimulation with HPA 1a peptides, but not control peptides. In ELISPOT assays, peptide-pulsed antigen-presenting cells were required for T cell detection. These clonal HPA 1a specific CD4+ T cell lines represent formal evidence of the existence of HPA 1a specific T cell responses related to NAIT and will serve as important tools for further characterization of maternal immune responses associated with NAIT.

Cell-Based Immunotherapy with Different Subsets of Tolerogenic Dendritic Cell in Idiopathic Thrombocytopenic Purpura.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3408-3408
Author(s):  
Xiao-Lin Zhang ◽  
Jun Peng ◽  
Shu-Qian Xu ◽  
Xin-Guang Liu ◽  
Yuan Yu ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Idiopathic Thrombocytopenic Purpura ◽  
Peripheral Tolerance ◽  
Platelet Glycoprotein ◽  
Autoantibody Production ◽  
Autoreactive T Cells ◽  
Thrombocytopenic Purpura ◽  
Autoreactive T Cell ◽  
Tolerogenic Dcs

Abstract There is growing evidence that tolerogenic dendritic cells (DCs) play an important role in maintaining peripheral tolerance through the induction of anergic or regulatory T cells. However, in humans, little is known about the ability of tolerogenic DCs to induce tolerance to autoantigens in autoimmune patients. Idiopathic thrombocytopenic purpura (ITP) is an immune-mediated disease in which platelets are destroyed by antiplatelet autoantibodies. Here, we explored in vitro the ability of four subsets of tolerogenic DCs (i.e., immature DCs (imDCs), IL-10-modulated DCs (IL-10-DCs), vasoactive intestinal peptide-modulated DCs (VIP-DCs) or plasmacytoid DCs (pDCs)) derived from patients with ITP, to induce an anergic state or regulatory T cells in autologous platelet glycoprotein (GP)-specific T cells. GPIIb/IIIa-reactive T cells were preincubated with GPIIb/IIIa-loaded imDCs, IL- 10-DCs, pDCs or VIP-DCs, and then rechallenged with autologous mature DCs (mDCs) in the presence of GPIIb/IIIa. Only when T cells were cultured with GPIIb/IIIa-loaded VIP-DCs in primary incubation, inhibited proliferation of GPIIb/IIIa-reactive T cells could be observed at rechallenge with GPIIb/IIIa-loaded mDCs. The anergic state of VIPDC- primed GPIIb/IIIa-reactive T cells could be reversed when rechallenged with GPIIb/ IIIa-loaded mDCs in the presence of a high concentration of exogenous IL-2. Meanwhile, GPIIb/IIIa-reactive T cells were also cultured with VIP-DCs loaded with tetanus toxoid (TT). In contrast to T cells pretreated with GPIIb/IIIa-loaded VIP-DCs, GPIIb/IIIareactive T cells pretreated with TT-loaded VIP-DCs proliferated when rechallenged with GPIIb/IIIa-loaded mDCs, which demonstrated that the induced anergy of autoreactive T cells is antigen specific. Additionally, functional analysis showed that VIP-DC-modulated T cells could not suppress the proliferation of newly induced GPIIb/IIIa-reactive T cells when cocultured with GPIIb/IIIa-loaded mDCs. These results indicated that VIP-DCs could induce autoreactive T cells anergic but not functionally suppressive. Moreover, we found that coculture of VIP-DCs with autologous PBMCs resulted in reduced production of anti-GPIIb/IIIa antibodies, suggesting that GPIIb/IIIa-reactive T cells lost their helper function for inducing autoantibody production by B cells. In contrast, reduced antibody production could not be found when autologous PBMCs were cocultured with imDCs, IL-10-DCs or pDCs. In conclusion, our studies revealed the therapeutic potential of VIPDCs, compared with imDCs, IL-10-DCs or pDCs, to induce autoreactive T-cell anergy to GP antigens, which would in turn facilitate the reestablishment of autoantigen-specific tolerance in patients with ITP.

scholarly journals New platelet glycoprotein polymorphisms causing maternal immunization and neonatal alloimmune thrombocytopenia

Transfusion ◽  
2011 ◽  
Vol 52 (5) ◽  
pp. 1117-1124 ◽  
Author(s):  
Julie A. Peterson ◽  
Shannon M. Pechauer ◽  
Maria L. Gitter ◽  
Adam Kanack ◽  
Brian R. Curtis ◽  
...  
Keyword(s):  
Platelet Glycoprotein ◽  
Maternal Immunization ◽  
Alloimmune Thrombocytopenia

SELECTIVE INHIBITION OF HUMAN MEGAKARYOCYTOPOIESIS IN VITRO BY HIGHLY PURIFIED PLATELET FACTOR 4

1987 ◽  
Author(s):  
A Gewirtz ◽  
W Y Xu ◽  
B Rucinski ◽  
S Niewiarowski
Keyword(s):  
T Cells ◽  
T Cell ◽  
Colony Formation ◽  
Solid Phase ◽  
Specific Inhibitor ◽  
Platelet Glycoprotein ◽  
Lymphocyte Function ◽  
Erythroid Progenitor ◽  
Platelet Factor

Platelet (plt) factor 4 (PF4) is an alpha granule protein which can modulate T lymphocyte function. T cells may help regulate megakaryocytopoiesis. Therefore, we hypothesized that T cell-PF4 interactions might play a role in autoregulating marrow megakaryocyte (MEG) production. To test this idea, we studied MEG colony formation in plasma clot cultures containing human serum derived solely from pit poor normal AB plasma, enriched hematopoietic progenitor cells (HPC), autologous T cells, and exogenous PF4. Highly purified PF4 (single band on SDS gel) was prepared from outdated human pits by a combination of heparin-agarose, Sephacryl G-200, and Sephadex G-50 column chromatography. HPC were prepared by depleting normal light density marrow mononuclear cells of adherent monocytes, and T cells. T cells were further fractionated into helper (Leu 3+) and suppressor (Leu 2+) subtypes by solid phase immunoabsorption ("panning"). MEG colonies were enumerated by indirect immunofluorescence with an anti-human platelet glycoprotein antiserum. HPC(5×105/ml) were co-cultured with Leu 3+, or Leu 2+ T cells at target;T cell ratios of 2:1 (n=3; n=4 respectively) and l:l(n=4; n=4 respectively) in the presence of 2.5 μg/ml PF4. Under these growth conditions, MEG colony formation was unchanged (p>0.5) when compared to colonies formed by HPC in the absence of PF4. When the above experiments were repeated (n=2-3/condition) at a higher PF4 concentration [25 μg/ml], MEG colony formation was markedly (>60%) inhibited. To determine if PF4 directly inhibited MEG or erythroid progenitor cell growth (CFU-Meg; CFU-E) in vitro, HPC were cloned in PF4 (25μg/ml) without added T cells. Mean ± SEM of MEG and CFU-E derived colonies formed without vs. with PF4 was as follows:These results suggest that: 1) PF4 may be a non-T cell dependent, lineage specific inhibitor of CFU-MEG, and 2) PF4 may play a role in autoregulating human megakaryocytopoiesis.

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