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Biomedical subjects

John Freedman

Publications and source records attributed to John Freedman.

At least 19 recordsLinked to original sources
PubMed2003

Anti-D initially stimulates an Fc-dependent leukocyte oxidative burst and subsequently suppresses erythrophagocytosis via interleukin-1 receptor antagonist.

Previous results have demonstrated that anti-D therapy in children with chronic auto-immune thrombocytopenic purpura (AITP) induced a significant increase in several pro- and anti-inflammatory plasma cytokines within 2 hours of administration. To investigate the biologic basis of these early in vivo responses, we developed a flow cytometric assay to measure Fc-dependent responses of human peripheral leukocytes with fluorescently labeled and anti-D-opsonized red blood cells (RBCs). When anti-D-opsonized RBCs were incubated with peripheral blood leukocytes, the earliest detectible event observed was a significant oxidative burst in both monocytes (P <.05) and granulocytes (P <.0001), characterized by the production of hydrogen peroxide (H2O2), peroxynitrite (ONOO-), superoxide (O -2), and hydroxyl (OH) by 10 minutes which declined by 1 hour. By 2 hours, the opsonized RBCs were phagocytosed, particularly by granulocytes (P <.001), but the phagocytosis subsequently declined by 6 hours of incubation. The decline in phagocytosis was correlated with a significant production of interleukin-1 receptor antagonist (IL1ra) by both monocytes (P =.036) and granulocytes (P =.0002) within 4 hours. None of these events occurred if the RBCs were coated with anti-D F(ab)'2 fragments. When recombinant IL1ra was titrated into the assay, phagocytosis of the opsonized RBCs was significantly inhibited (P =.002). Taken together, these results suggest that at least one mechanism of action of anti-D is via the production of the anti-inflammatory cytokine IL1ra which can negatively regulate the ability of leukocytes to phagocytose opsonized cells.

Cytokines↗
PubMed2003

The relationship of von Willebrand factor binding to activated platelets from healthy neonates and adults.

von Willebrand Factor (VWF) is important in platelet adhesion and shear-dependent platelet activation. We performed flow cytometric analyses of VWF binding to and activation of platelets from healthy neonates, children, and adults. Platelets from cord blood (n = 38; gestational age: 36-42 wk; birth weight: 2.4-5.1 kg), neonatal venous blood (n = 19; d 2-3 of life), children (n = 15; age: 1.5-16.3 y), and adults (n = 22; age: 18-55 y) were studied. Binding of VWF was assessed using an antihuman VWF polyclonal antibody and a FITC-conjugated secondary antibody. Platelet activation was determined by the expression of CD62P, CD63, CD41, CD42b, activated GPIIb/IIIa (PAC-1), procoagulant surface (as reflected by annexin V binding), and microparticle formation. Although the mean percentage of VWF-positive platelets was not significantly higher in unstimulated platelets from 2- to 3-d-old neonates, their platelets were more activated than those from adults, and there was a positive correlation of VWF binding with platelet activation (CD62P: r = 0.74, p < 0.001; annexin V: r = 0.46, p < 0.05). In adults, after in vitro activation of platelets with thrombin and ADP, VWF binding to platelets increased and correlated significantly with CD62P expression (r = 0.71, p < 0.001). VWF binding to unstimulated neonatal platelets was, however, higher than that to in vitro-stimulated platelets from adults at the same level of expression of platelet activation markers. Further studies are required to assess the mechanism and significance of VWF binding to activated platelets in the neonatal period.

Adolescent↗
PubMed2003

IVIg-mediated amelioration of murine ITP via FcgammaRIIB is independent of SHIP1, SHP-1, and Btk activity.

It has been established that amelioration of murine immune thrombocytopenia purpura (ITP) by IVIg is dependent on the inhibitory receptor FcgammaRIIB. Co-cross-linking of the FcgammaRIIB with the B-cell receptor complex or with FcepsilonRI in mast cells results in cell inhibition, which is mediated by recruitment of the inositol phosphatase SHIP1 to the cytoplasmic tail of the FcgammaR. The FcgammaRIIB can also associate with protein tyrosine phosphatase SHP-1 as a potential secondary target of the receptor. Alternatively, homoaggregation of FcgammaRIIB can induce a proapoptotic state in B cells that is dependent on the presence of Bruton tyrosine kinase (Btk), a kinase also expressed in monocytes. We sought to determine if these signaling pathways may direct IVIg-mediated FcgammaRIIB-dependent regulation of in vivo monocyte function in a murine model of ITP in which IVIg functions in an FcgammaRIIB-dependent manner. We demonstrate that mice deficient in SHIP1, SHP-1, and Btk respond to the ameliorating effects of IVIg with the same kinetics as control mice. We conclude that IVIgmediated inhibitory pathways operating via monocyte FcgammaRIIB may involve a transmembrane signaling pathway different from that of B cells.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2003

Cooperation of TNF family members CD40 ligand, receptor activator of NF-kappa B ligand, and TNF-alpha in the activation of dendritic cells and the expansion of viral specific CD8+ T cell memory responses in HIV-1-infected and HIV-1-uninfected individuals.

Members of the TNF superfamily have been shown to be instrumental in enhancing cell-mediated immune responses, primarily through their interactions with dendritic cells (DCs). We systematically evaluated the ability of three TNF superfamily molecules, CD40 ligand (CD40L), receptor activator of NF-kappaB ligand (RANKL), and TNF-alpha, to expand ex vivo EBV-specific CTL responses in healthy human individuals and ex vivo HIV-1-specific CTL responses in HIV-1-infected individuals. In both groups of individuals, we found that all three TNF family molecules could expand CTL responses, albeit at differing degrees. CD40L treatment alone was better than RANKL or TNF-alpha alone to mature DCs and to expand CTL. In healthy volunteers, TNF-alpha or RANKL could cooperate with CD40L to maximize the ability of DCs to expand virus-specific CTL responses. In HIV-1 infection, cooperative effects between TNF-alpha or RANKL in combination with CD40L were variable. TNF-alpha and RANKL cooperated with CD40L via differing mechanisms, i.e., TNF-alpha enhanced IL-12 production, whereas RANKL enhanced survival of CD40L-stimulated DCs. These findings demonstrate that optimal maturation of DCs requires multiple signals by TNF superfamily members that include CD40L. In HIV-1 infection, DCs may only require CD40L to maximally expand CTL. Finally, CTL responses were higher in CD4(+) T cell-containing conditions even in the presence of TNF family molecules, suggesting that CD4(+) T cells can provide help to CD8(+) T cells independently of CD40L, RANKL, or TNF-alpha.

CD4-Positive T-Lymphocytes↗
PubMed2003

Prophylactic platelet transfusions: which dose is the best dose? A review of the literature.

Routine platelet transfusions for patients with acute leukemia were introduced in the early 1960s, and since then platelet use has increased steadily. Despite widespread use, good clinical evidence supporting prophylactic platelet transfusions is limited, and there are very few studies that have examined the dose for prophylactic platelet transfusions. Review of the platelet dose used in both early studies of routine platelet transfusions and more recent clinical trials of platelet transfusions shows wide variation in dosing, which is also reflected in clinical practice. As such, only limited recommendations for platelet dose have been forthcoming from consensus conferences or guidelines. The results from 3 recent clinical trials and a mathematical model examining the dose for prophylactic platelet transfusions suggest that lower dose transfusions may decrease the total number of platelets transfused; however, no definitive conclusions about the optimal platelet dose can be reached as these trials were not designed to evaluate bleeding outcomes or total platelet utilization. Future large clinical trials of platelet dose, which examine these critical outcomes, are required. Only with these results can the optimal platelet dose be determined.

Blood Donors↗
PubMed2003

Platelets in hemostasis and thrombosis: role of integrins and their ligands.

Platelet adhesion and aggregation at the site of vascular injury are two key events in hemostasis and thrombosis. The contribution of several platelet receptors and their ligands has been highlighted in these processes. In platelet adhesion, particularly at high shear stress, GP1b-von Willebrand factor (vWF) interaction may initiate this event, which is followed by firm platelet adhesion mediated by members of the integrin family, such as beta1 (alpha2beta1, alpha5beta1) and beta3 (alphaIIbbeta3) integrins. In platelet aggregation, although GP1b-vWF, P selectin-sulfatides, and other molecules, may play some roles, the process is mainly mediated by beta3 (alphaIIbbeta3) integrin and its ligands, such as fibrinogen and vWF. Recent studies with perfusion chambers and intravital microscopy have revised the dogma established with the static (low shear stress) conditions. It is intriguing that platelet adhesion and aggregation do still occur in mice lacking both vWF and fibrinogen, suggesting that other unexpected molecule(s) may also be important in hemostasis and thrombosis.

Animals↗
PubMed2003

Platelets and von Willebrand factor.

The interaction of platelets with von Willebrand factor (VWF) is crucial in the initiation of any hemostatic or thrombotic process. VWF enables the platelet, via its surface glycoprotein receptors, to adhere to exposed subendothelium and to respond to shear stress in the blood. Via VWF that is stored and released from platelet alpha-granules and from Weibel-Palade bodies of endothelial cells, the hemostatic system can respond locally to lesions in the vessel wall and can initiate the activation of other platelets. This review describes the molecular structure of VWF, its functions and its interactions with the platelet membrane glycoprotein receptors GP Ib-IX-V and GP IIb-IIIa. As well, the role of VWF in shear-induced platelet adhesion and aggregation is described, and mechanisms are discussed that control the size of VWF multimers and the responsiveness of platelets to multimeric VWF. Finally, the review discusses the role of locally released VWF from platelet alpha-granules and from Weibel-Palade bodies for platelet activation in neonates and adults.

Blood Platelets↗
PubMed2003

Platelet apoptosis in stored platelet concentrates and other models.

Apoptosis or programmed cell death was discovered in nucleate cells 30 years ago and has been well documented. In contrast, apoptosis in anucleate platelets has only a five-year research history and as yet but few publications related to it. In this review, we will present the data on platelet apoptosis in several models. These include in vitro models where platelet apoptosis was induced by calcium ionophores, natural platelet agonists, storage in capped tubes at 37 degrees C and storage at room temperature under standard blood banking conditions, and in vivo models where apoptosis was provoked by suppression of thrombopoiesis, malaria infection and injection of tumor necrosis factor or anti-platelet antibodies. Understanding of platelet apoptosis and its role in the platelet storage lesion is an exciting challenge; future research is likely to provide us with further insight into this field.

Animals↗
PubMed2003

Hemolink, an o-raffinose cross-linked haemoglobin-based oxygen carrier, does not affect activation and function of human platelets in whole blood in vitro.

Haemoglobin-based oxygen carriers (HBOCs) are anticipated to be safe and efficient alternatives to RBC transfusions. Haemoglobin (Hb) raffimer (Hemolink; Hemosol, Toronto, ON, Canada) is polymerized human Hb, cross-linked with o-raffinose. As administration of cell-free Hb may affect blood cells and tissues, this study was focused on evaluating effects of Hb raffimer on human platelets in whole blood in vitro. Citrated blood from healthy donors was incubated with Hb raffimer to achieve raffimer concentrations of 2-50 vol percentage (2-50 g/l). Platelet activation, phosphatidylserine exposure and microparticle generation were measured by flow cytometry. Aperture closure time on collagen/ADP- and collagen/epinephrine-coated membranes was determined by a platelet function analyser (PFA-100). We found that addition of Hb raffimer to blood samples up to 50 vol % did not affect human platelets as measured by various markers of platelet activation (CD42b, CD41, PAC-1, CD62, CD63), procoagulant activity (annexin V) and microparticle formation; differences between Hb raffimer- and lactated Ringer's-diluted blood were not significant. Similarly, no adverse effect of Hb raffimer on closure time was observed at concentrations up to 50 vol %, in comparison with Ringer's solution. These data indicate that exposure of human blood to high concentrations of Hb raffimer in vitro did not cause platelet activation nor affect platelet function.

Blood Platelets↗
PubMed2003

Cellular immune mechanisms in autoimmune thrombocytopenic purpura: An update.

Autoimmune thrombocytopenic purpura (AITP) is a bleeding disorder in which autoantibodies are directed against an individual's own platelets, leading to enhanced clearance through Fc receptor (R)-mediated phagocytosis by macrophages residing in the reticuloendothelial system (RES), particularly in the spleen. The production of IgG autoantibodies is critically dependent on cellular immune mechanisms particularly relating to T cells. We review the recent literature of the cell-mediated immunology of AITP focusing on platelet phenotype, genetics, T-cell reactivities, and cytokine profiles in patients with AITP. Understanding the interaction between these cell-mediated mechanisms is vital for developing antigen specific immunotherapies to treat this autoimmune disease.

Autoantibodies↗
PubMed2003

How should Canada fund the blood system? An evaluation of the chargeback proposal.

The Canadian blood system is a critical interface between public health and the delivery of patient care. The organization of the blood system plays a vital role in ensuring that its functions are effectively fulfilled. Previous structural problems in the blood system led to serious health consequences by contributing to the blood transmission of hepatitis C and HIV in the 1980s. To address these problems, the Canadian blood system has recently undergone considerable organizational reform. However, policy-makers, particularly in Ontario, are considering further structural reform specifically focusing on how the blood system is financed. This move for reform is partially motivated by the rising cost of blood products and the perception that the current system has failed to provide incentives for the efficient use of these products. The suggested payment mechanism, a "chargeback system," involves the provincial ministries of health funding hospitals so that hospitals can directly purchase blood products from the Canadian Blood Services. This approach would replace the current system in which the provinces directly fund the Canadian Blood Services, which in turn provides blood products free of charge to hospitals. Based on a review of documents and stakeholder interviews, we report the potential advantages and disadvantages of a change to a new system of funding and make recommendations on how provinces should proceed.

Administrative Personnel↗
PubMed2002

Monoclonal IgG can ameliorate immune thrombocytopenia in a murine model of ITP: an alternative to IVIG.

Intravenous immunoglobulin (IVIG) is used to treat immune thrombocytopenia resulting from a variety of autoimmune and nonautoimmune diseases such as idiopathic thrombocytopenic purpura (ITP), heparin-induced thrombocytopenia, and posttransfusion purpura. IVIG is a limited resource and although considered safe, may nevertheless carry some risk of transferring disease. Its high cost makes monoclonal antibodies, capable of mimicking the clinical effects of IVIG, highly desirable. We show here, using a murine model of ITP, that selected monoclonal antibodies can protect against thrombocytopenia. SCID mice were pretreated with 1 of 21 monoclonal antibodies before induction of thrombocytopenia by antiplatelet antibody. Four antibodies reacted with the CD24 antigen on erythrocytes. Two antibodies were of the IgM class, and although one IgM antibody caused a minimal degree of anemia (P <.05), neither antibody ameliorated immune thrombocytopenia. One of 2 anti-CD24 antibodies of the IgG class ameliorated immune thrombocytopenia and blocked reticuloendothelial system function at the same doses that protected against thrombocytopenia. Some antibodies reactive with other circulating cell types also protected against immune-mediated thrombocytopenia while no antibody without a distinct target antigen in the mice was protective. Protective monoclonal antibodies significantly prevented thrombocytopenia at down to a 1000-fold lower dose (200 microg/kg) as compared with standard IVIG treatment (2 g/kg). It is concluded that monoclonal IgG with specificity for a circulating cellular target antigen may provide an alternative therapeutic approach to treating immune thrombocytopenia.

Animals↗
PubMed2002

Gamma-globulins prepared from sera of multiparous women bind anti-HLA antibodies and inhibit an established in vivo human alloimmune response.

It has previously been shown that sera from multiparous women have increased levels of anti-idiotypic antibodies specific for anti-HLA molecules. gamma-Globulins prepared from these sera may be superior to commercial preparations of intravenous gamma-globulin (IVIg) for inhibiting HLA alloimmunization. To test this, F(ab')2 fragments prepared from either commercial IVIg or from the sera of men or multiparous women were coupled to CNBr-Sepharose and tested for their ability to bind F(ab')2 fragments derived from polyspecific anti-HLA sera. As determined by flow cytometry, compared with columns coated with F(ab')2 derived from commercial IVIg or sera from men, columns coated with F(ab')2 prepared from the sera of multiparous women bound significantly more anti-HLA. In addition, intact IgG molecules prepared from the sera of multiparous women significantly neutralized the reactivity of the anti-HLA F(ab')2 fragments. To determine whether the intact IgG molecules or their corresponding F(ab')2 fragments could affect in vivo alloimmunity, they were tested for their ability to inhibit an established IgG human alloimmune response in humanized severe combined immunodeficient (SCID) mice. Compared with commercial IVIg, when intact IgG or F(ab')2 fragments derived from multiparous women were administered to SCID mice making human anti-HLA antibodies, a significant reduction in anti-HLA reactivity was observed. The findings suggest that IgG molecules prepared from the sera of multiparous women have increased anti-idiotypic reactivity against anti-HLA antibodies, which can significantly inhibit an established human IgG alloimmune response in an Fc-independent manner.

Animals↗
PubMed2002

A rabbit model for monitoring in vivo viability of human platelet concentrates using flow cytometry.

BACKGROUND: Viability in vivo of novel platelet components cannot be readily determined in human transfusions. Elaboration of valid animal models may be useful for this purpose. STUDY DESIGN AND METHODS: Viability of platelet concentrates (PCs) WBC reduced before storage was determined by flow cytometry in rabbits whose reticuloendothelial system was inhibited by ethyl palmitate administration. PCs stored at 22 degrees C for 2 and 5 days (D2- and D5-PCs) or refrigerated PCs (3-6 days at 22 degrees C plus 1-4 days at 4 degrees C, RF-PCs) were transfused into rabbits. Five parameters of PC viability in vivo were calculated from human platelet survival curves: survival time, recovery 0.5 and 24 hours after transfusion (R0.5, R24), maximal recovery (Rmax), and total recovery for 0 to 24 hours (RSigma). RESULTS: No differences in viability of D2- and D5-PCs were found. In contrast, viability of RF-PCs was significantly lower than that of D2-PCs, as was revealed with diverse sensitivity by four parameters: RSigma > R24 > R0.5=survival time (p < 0.001, p < 0.01, and p < 0.05, respectively). CONCLUSION: The rabbit model elaborated is sufficiently sensitive to reveal differences in human platelet viability in vivo between conventional and cold-damaged PCs. It may be useful for comparing viability of different platelet components that cannot be readily tested in human transfusions.

Animals↗