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Biomedical subjects

J Freedman

Publications and source records attributed to J Freedman.

At least 19 recordsLinked to original sources
PubMed1996

Differences in serum cytokine levels in acute and chronic autoimmune thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet T-cell reactivity.

Patients with both acute and chronic autoimmune thrombocytopenic purpura (AITP) have in vitro lymphocyte defects in the form of platelet-stimulated proliferation and cytokine secretion. A blinded study was performed to determine if these defects are related to serum cytokine levels and/or platelet antigen expression. Compared with controls, 53% of children with chronic AITP, but only 9% of those with acute AITP, had increased serum interleukin-2 (IL-2), interferon-gamma, and/or IL-10; however, none of the patients had detectible serum levels of IL-4 or IL-6, cytokine patterns suggesting and early CD4+ Th0 and Th1 cell activation. In children with chronic AITP, the levels of serum IL-2 correlated with in vitro platelet-stimulated IL-2 production. Few (17%) patients with AITP showed platelet activation, as measured by CD62 expression, or abnormal expression levels of platelet membrane glycoprotein (GP) IIbIIIa, but abnormal GPIb levels were observed in one-third of children with AITP. In contrast to normal controls and patients with nonimmune thrombocytopenia, a significant number of children with acute (80%), chronic (71%), or chronic-complex (55%) AITP and GPIb+ peripheral blood cells expressing HLA-DR. HLA-DR was variably coexpressed on distinct smaller and larger-sized GPIb+ cell populations with CD41, CD45, CD14, CD80, and/or glycophorin molecules. GPIb+ cells isolated from spleens of patients with chronic AITP had high expression (49% +/- 30%) of HLA-DR and splenic T cells had a high level of in vitro platelet-stimulated IL-2 secretion compared with controls. Platelet HLA-DR expression correlated inversely with platelet count, but not with therapy, serum cytokines, or in vitro lymphocyte antiplatelet reactivity. The results indicate that platelet HLA-DR expression is a common occurrence in patients with immune thrombocytopenia, whereas a large subpopulation of children with chronic AITP can be identified by increased serum cytokine levels and in vitro platelet-stimulated IL-2 secretion by lymphocytes, suggesting that differences exist in the immune pathogenesis of acute and chronic AITP, particularly at the level of platelet reactive T cells.

Acute Disease↗
PubMed1996

Plasmin accelerates platelet-dependent prothrombinase formation without activating the platelets.

Patients with acute myocardial infarction who undergo thrombolytic therapy may shortly thereafter present evidence for increased platelet activation and thrombin activity, and recurrent thrombosis. This study investigated whether plasmin activates platelets and prothrombin in recalcified platelet-rich plasma (RPRP) to cause (at least in part) these side-effects of thrombolytic therapy. Plasmin (0.1 and 1.0 CU/ml) addition to RPRP with microM r-tick anticoagulant peptide (the latter a factor Xa inhibitor which abrogates prothrombin activation by prothrombinase at the concentration used) resulted in no change in the concentration of prothrombin fragment 1 + 2, or in the expression of GMP-140, the resting and activated GP IIb-IIIa conformers, and GPIb on platelets. Thus, plasmin neither activates platelets nor prothrombin in RPRP. However, plasmin accelerated platelet activation and secretion, and prothrombin fragment 1 + 2 production in RPRP. When combined with 1 microM r-tick anticoagulant peptide and 1 or 10 mM alpha-thrombin to RPRP, plasmin also increased the number of GMP-140 molecules expressed/platelet without enhancing alpha-thrombin binding to the platelets. Additionally, plasmin accelerated prothrombin activation when it was added to washed platelets resuspended in factor V depleted plasma simultaneously with 10 mM CaCl2, 10 nM alpha-thrombin for 10 s (to activate platelets and platelet factor V), followed by 4 microM hirudin and 1 nM factor Xa. Thus, plasmin potentiates the platelet release reaction in response to alpha-thrombin (probably by increasing the availability of factor V on the platelets) to enhance prothrombin activation in RPRP. These actions of plasmin may contribute to the increased platelet activation and thrombotic side-effects that can occur after thrombolytic therapy.

Blood Platelets↗
PubMed1996

A survey of red cell use in 45 hospitals in central Ontario, Canada.

BACKGROUND: The purpose of this survey was to establish baseline information on blood component use in relation to patient diagnoses, procedures, and demographics and to identify patterns of blood use that may be used for blood program planning and transfusion audits. STUDY DESIGN AND METHODS: A cross-sectional survey of the transfusion of blood components in teaching and nonteaching hospitals in central Ontario between September 1991 and August 1992 was carried out. Coders of hospital medical records routinely record demographics, procedures, diagnoses, and other relevant information. A protocol was created by which medical records coders could add the components transfused to the discharge abstract for this study. Red cell use is reported here. RESULTS: Of the 61 hospitals invited to participate, from which 547,279 patients were discharged during the 12-month period of the study, 45 (74%) agreed to participate. Information was collected on 439,373 discharged patients. Of these, 26,611 (6.1%) received at least 1 unit of red cells. Of a total of 101,116 red cell units transfused, more than 74 percent were used in patients discharged with neoplasms, gastrointestinal diseases, circulatory system diseases, and trauma. High-transfusion-use procedures included operations and procedures on the digestive and cardiovascular systems, diagnostic and therapeutic procedures, musculoskeletal system, and hemic or lymphatic system procedures. CONCLUSION: This survey provides baseline blood transfusion information for a specific period that can help determine the need for hospital audits and maximum surgical blood-order schedule guideline reviews. This information is relevant to current recommendations to reduce patient's exposure to blood components. These transfusion data will assist in blood program planning based on known disease trends, demographics, and population changes.

Cross-Sectional Studies↗
PubMed1995

Indirect allorecognition of platelets by T helper cells during platelet transfusions correlates with anti-major histocompatibility complex antibody and cytotoxic T lymphocyte formation.

To study the cellular immunology of platelet-induced alloimmunization, a murine transfusion model was developed. BALB/c (H-2d) recipient mice were transfused weekly with 2 x 10(8) platelets or 10(3) leukocytes from C57BL/6 (H-2b) donor mice. Recipient antidonor major histocompatibility complex (MHC) class I alloantibodies could be detected in flow cytometric assays by the fifth platelet transfusion. In contrast, when leukocytes only were transfused, alloantibodies were not detected. In vitro assays demonstrated that murine H-2b platelets were positive for MHC class I expression but lacked MHC class II molecules on their membranes and were unable to stimulate proliferation or cytokine production when incubated with naive H-2d spleen cells. In vivo, however, platelet transfusions induced two distinct patterns of cell-mediated reactivity. First, during the initial transfusions and before alloantibody formation, there was induction of T-cell anergy, characterized by the inability of recipient T cells to respond to Concanavalin A (ConA) or to proliferate in an antidonor mixed lymphocyte reaction (MLR), together with suppressed natural killer (NK) cell activity. This unresponsiveness was associated with a transient increase in nitric oxide (NO)-dependent cytotoxicity and interleukin-1 (IL-1) production. Second, once alloantibodies developed, significantly increased antidonor CD8+ cytotoxic T lymphocyte (CTL) and NK cell responses were observed. At this time, when recipient spleen cells were depleted of CD8+ T cells and incubated with only donor platelets in 7-day antigen-presenting cell (APC) assays, enhanced proliferation and IL-2 production occurred. These cellular responses were not seen when 10(3) allogeneic leukocytes were transfused. Thus, the results suggest that leukoreduced platelet transfusions induce antidonor MHC antibodies and CD8+ CTL responses in recipient mice. At the same time, the transfusions induced recipient CD4+ T-cell activation when incubated with donor platelets in the presence of syngeneic APCs, an indirect recognition pathway that correlates with the time of alloantibody production.

Animals↗
PubMed1995

Flow cytometric evaluation of platelet activation in blood collected into EDTA vs. Diatube-H, a sodium citrate solution supplemented with theophylline, adenosine, and dipyridamole.

With platelet activation, there is modulation of platelet surface molecule expression. In flow cytometric analyses of in vivo platelet activation, results are often confounded by activation induced in vitro by the preparative procedures. It is particularly important therefore to prevent or retard platelet activation as soon as possible after withdrawal of the blood sample. Taking blood into paraformaldehyde, or fixing the cells with paraformaldehyde as soon as possible after withdrawal, has been employed to prevent platelet activation in vitro, but paraformaldehyde-fixed platelets cannot be further used in functional studies. We investigated the efficacy of Diatube-H, a commercially available combination of platelet antagonists (theophylline, adenosine, and dipyridamole), in preventing or retarding platelet activation in vitro, along with its effects on modulation of platelet membrane glycoproteins (GP) and adhesion molecules. In contrast to blood taken into EDTA, blood taken into Diatube-H vacutainer tubes could be stored at room temperature for up to 4 hr prior to paraformaldehyde fixation without significant in vitro platelet activation, as measured by CD62P, CD63 and modulation of GPIb and GPIIbIIIa surface expression. Hence, paraformaldehyde fixation could be deferred for several hours, permitting transport of samples from distant sites. Studies of thrombin-induced platelet activation indicated that platelets taken into Diatube-H remained functional i.e. were able to be activated. Expression of the CD29, CD49b and CD31 adhesion molecules on the platelet surface was unaffected by storage in Diatube-H. The results suggest that Diatube-H may be a useful reagent for flow cytometric studies of platelets when the samples cannot be processed immediately.

Adenosine↗
PubMed1995

Platelet activation in warm and cold heart surgery.

Recent studies suggest that patients undergoing warm heart surgical procedures have reduced postoperative bleeding. To determine if this is due to differences in platelet activation, we measured platelet membrane glycoproteins (GPIb, GPIIb/IIIa, GMP 140), platelet fragments, and platelet counts before, during, and after normothermic (37 degrees C) or hypothermic (28 degrees to 30 degrees C) cardiopulmonary bypass. Cardiopulmonary bypass was associated with a significant decrease in platelet count, platelet membrane GPIb, and platelet fragments, and an increase in GMP 140 (p < 0.05). Normothermic cardiopulmonary bypass induced an early significant increase in granulocytes, whereas this was delayed until after rewarming in the hypothermic group. Mean 24-hour postoperative blood loss was 786 +/- 226 mL in the cold group versus 547 +/- 56 mL in the warm group (p = not significant). We conclude that cardiopulmonary bypass affects platelet activation and integrity and that these changes are similar in direction and magnitude for hypothermic and normothermic techniques.

Aged↗
PubMed1995

Programmed cell death induced by HIV type 1 antigen stimulation is associated with a decrease in cytotoxic T lymphocyte activity in advanced HIV type 1 infection.

The immune competence of peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus-seropositive (HIV+) patients was studied by assessing cytotoxic T lymphocyte (CTL) activity following recall HIV antigen stimulation. Target cells were HLA-A-matched EBV-transformed B cells expressing HIV-1 antigen. In the presence of recombinant IL-2 (rIL-2, 2 or 10 U/ml), about 50% of PBMCs from HIV+ asymptomatic patients responded to HIV-1 antigen stimulation in vitro with increased cytotoxic activity. In contrast, PBMCs from patients with overt AIDS, cultured in medium containing rIL-2 (2 U/ml) and HIV-1 antigen, showed no increase in cytotoxic activity; in the presence of rIL-2 (10 U/ml) and HIV-1 antigen, an inhibitory effect on CTL activity was observed. This inhibitory effect was associated with programmed cell death (apoptosis) of CD8+ lymphocytes and cells of both gamma/delta TcR-positive and -negative phenotypes. However, prior to the apoptosis, different TcR phenotypes of T lymphocyte reacted differently to HIV-1 antigen stimulation. The HIV-1 antigen initially appeared to cause gamma/delta TcR-positive T lymphocytes to proliferate and/or differentiate and later induced cell death. Whereas, prior to the apoptosis, no proliferation of gamma/delta TcR-negative T lymphocytes induced by HIV-1 antigen was observed.

Acquired Immunodeficiency Syndrome↗
PubMed1995

A randomized, double-blind comparison of methoxamine and epinephrine in human cardiopulmonary arrest.

The beneficial effect of epinephrine has been attributed to its alpha-adrenergic properties. The present study was designed to compare the effects of epinephrine and methoxamine in witnessed cardiac arrests. Consecutive, witnessed cardiac-arrest victims presenting to the emergency room or from the inpatient population of our institution were enrolled in this study. Patients were randomized to receive either epinephrine (2 mg bolus followed by 2 mg every 4 min) or methoxamine (40 mg bolus followed after 4 min by 40 mg) in a blind design. Patients were followed prospectively for survival and neurologic outcome. A total of 199 patients were randomized into the study, but 54 had to be retrospectively dropped from analysis for failure to comply with the study protocol. Of the 145 patients remaining, 77 received methoxamine (M) and 68 epinephrine (E). There was no difference in rate of successful resuscitation (42% versus 53%, M versus E, respectively), or in neurologic outcome as measured by the Glasgow-Pittsburgh Coma Score (GPCS). This study failed to demonstrate any difference in the rate of initial resuscitation, survival to discharge from the hospital, or neurologic status with methoxamine as opposed to epinephrine in the setting of cardiac arrest.

Adult↗
PubMed1994

Thrombin binding to platelets and their activation in plasma.

The interactions of alpha-thrombin with platelets are critical in haemostasis and arterial thrombosis. This study established methods for characterizing the binding of alpha-thrombin to platelets and some of its consequences in platelet-rich plasma. The binding of alpha-thrombin to platelets and the subsequent platelet activation were quantified by flow cytometry, using affinity purified polyclonal antibodies to human alpha-thrombin and a monoclonal antibody to GMP-140, respectively. Dose-dependent binding of alpha-thrombin to platelets and their activation occurred in parallel, both reaching the maxima for each enzyme concentration within 10s after > or = 1.0 nM alpha-thrombin was added to recalcified PRP containing 1 microM recombinant tick anticoagulant peptide. The tick anticoagulant peptide abrogated prothrombin activation in the platelet-rich plasma. alpha-Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antibody to the hirudin tail-like domain of the seven transmembrane thrombin receptor on platelets. Therefore this receptor represents an important site for alpha-thrombin binding to platelets suspended in plasma. D-Phe-Pro-ArgCH2-alpha-thrombin only bound to platelets when its concentration was > or = 100 nM, and it did so without inhibiting platelet activation by alpha-thrombin. Whereas concentrations of hirudin equimolar to those of alpha-thrombin failed to abrogate alpha-thrombin-mediated activation of platelets, a 10-fold molar excesses of hirudin over alpha-thrombin abrogated alpha-thrombin binding to platelets. The demonstration that > or = 1.0 nM alpha-thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and arterial thrombosis contribute to platelet activation in vivo.

Blood Platelets↗
PubMed1994

The effect of rabbit antithymocyte serum (RATS) and OKT3 on peripheral blood mononuclear cell subsets following renal transplantation.

Fifty-five renal transplant recipients were studied prospectively for changes in monoclonal antibody-defined mononuclear cell subsets (MCS) over the first 45 days posttransplant. Patients received induction immunotherapy with either monoclonal OKT2(n = 29) or polyclonal RATS (n = 26) preparation. Sequential examinations showed characteristic patterns of MCS depletion, which differed according to the type of therapy received and which subset was examined. In general, induction therapy with RATS resulted in greater and more sustained reduction of most MCS than was seen with OKT3 therapy. In recipients who received OKT3 induction there was a correlation between allograft rejection and an increase in lymphocytes expressing pan-T cell markers and in natural-killer cells. In contrast, rejection episodes in patients receiving RATS were associated with increases in subpopulations of T cells including helper, inducer and suppressor/cytotoxic T-cell subsets. There was no correlation of rejection with B cells or T-cell activation markers. The different patterns of MCS depletion with different antilymphocyte preparations and the association between changes in different MCS and rejection warrant further investigation.

Animals↗
PubMed1994

Idiopathic thrombocytopenia and neutropenia in childhood.

PURPOSE: Between 1975 and 1990, 13 patients seen at The Hospital for Sick Children, Toronto, Ontario, were noted to have concurrent idiopathic thrombocytopenia and neutropenia (ITN; platelet count < 150 x 10(9)/L; absolute neutrophil count < 1.5 x 10(9)/L). These patients all had normal marrow function and no evidence of systemic disease. PATIENTS AND METHODS: A detailed chart review was performed to define the clinical and laboratory features of these patients. RESULTS: Although this was a heterogeneous group of patients, they shared several common characteristics. The disease followed a chronic course with thrombocytopenia or neutropenia or both that persisted or recurred over the entire period of follow-up in all patients (18 months to 15 years). The disease was associated with splenomegaly in eight patients and lymphadenopathy in nine patients. Boys were affected more frequently than were girls (ratio 11:2). Thrombocytopenia improved temporarily during treatment with corticosteroids, i.v. immunoglobulin G, RhoGAM, and a variety of immunosuppressive agents; however, neutropenia tended to be much more resistant to these therapies. Splenectomy was ineffective in two children in whom the procedure was performed. Platelet of and neutrophil antibodies or both were detected in five of six patients who were tested, suggesting an autoimmune cause for the cytopenias. CONCLUSIONS: These findings suggest that ITN in childhood is distinct from immune thrombocytopenic purpura, particularly in terms of its chronicity and poor response to therapy.

Adolescent↗
PubMed1993

Downregulation of the anti-HLA alloimmune response by variable region-reactive (anti-idiotypic) antibodies in leukemic patients transfused with platelet concentrates.

Approximately 30% to 40% of patients with acute leukemia receiving repeated pooled random-donor platelet transfusions develop anti-HLA alloantibodies. Over time, however, serum anti-HLA concentrations decrease in approximately 50% of these patients, despite continued exposure to platelet and/or red blood cell transfusions. Using an enzyme-linked immunosorbent assay to measure serum Igs, the present study demonstrates that the sera of 67% of 82 transfused patients exhibiting a decrease in anti-HLA contain antibodies (anti-idiotypes) that react with the variable (V) region of anti-HLA antibodies. Anti-HLA binding to platelet membranes could be inhibited by these serum antibodies in 36% of the patients, indicating they had paratope-related reactivity. Protein G sepharose absorption showed that the anti-HLA V region-reactive antibodies were IgG. Of the 43 patients who had a decrease in anti-HLA levels, that were 16 whose anti-HLA decreased to undetectable levels; 7 (44%) developed anti-idiotypic antibodies that could specifically inhibit their own previously anti-HLA-positive serum. In contrast, antibodies with reactivity to the V region of anti-HLA antibodies (anti-idiotypes) were not demonstrable in patients who developed anti-HLA that did not decrease or disappear. The findings suggest that the development of anti-HLA V region-reactive antibodies (anti-idiotypic antibodies) correlates with a decrease in anti-HLA antibody formation in patients multiply transfused with platelet concentrates. The observations indicate that anti-idiotypic antibodies may downregulate alloimmune responses in patients undergoing repeated allostimulation during platelet transfusion therapy.

Acute Disease↗