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Biomedical subjects

V Blanchette

Publications and source records attributed to V Blanchette.

At least 19 recordsLinked to original sources
PubMed2002

Anti-D (WinRho SD) treatment of children with chronic autoimmune thrombocytopenic purpura stimulates transient cytokine/chemokine production.

Intravenous anti-D is often used in the treatment of autoimmune thrombocytopenic purpura (AITP), but little is known about its mechanisms of action. To investigate anti-D's potential in vivo mechanism(s) of action, a small group (N = 7) of children with chronic AITP was studied. The children initially received either 25 or 50 microg/kg of WinRho-SD in a four-cycle cross-over trial, and peripheral blood samples from the first and third cycles were assessed for cytokine levels at pre-treatment, 3 hr, 1 day, and 8 days post-treatment. Results showed that platelet counts significantly increased in all the children by day 8 post-treatment. Analysis of serum by ELISA showed that there was a significant but transient rise in both pro- and anti-inflammatory cytokine/chemokine levels (e.g., IL1RA, IL6, GM-CSF, MCP-1 alpha, TNF-alpha and MCP-1) by 3 hr post-treatment in both cycles which returned to baseline levels by 8 days post-treatment. These results suggest that anti-D administration may initially activate the RES in the form of cytokine/chemokine secretion, which is subsequently followed by an increase in platelet counts. It is possible that the induced cytokine/chemokine storm may have an effect on several physiological processes such as those mediating either adverse effects or potentially RES phagocytic activity.

Chemokines↗
PubMed2002

Childhood chronic immune thrombocytopenic purpura (ITP).

Chronic immune thrombocytopenic purpura (ITP) develops in approximately 20% of children with acute ITP. Treatment requires differentiating the truly idiopathic state from thrombocytopenias associated with other disorders. Treatment is recommended in cases where platelet counts drop below 20 x 10(9)/L. Therapeutic options include corticosteroids, intravenous immunoglobulin TG(i.v.Ig), and anti-D, as well as splenectomy. The benefits and disadvantages of each modality are reviewed, as well as the management of postsplenectomy failures.

Child↗
PubMed2000

Approach to the investigation and management of immune thrombocytopenic purpura in children.

Childhood immune thrombocytopenic purpura (ITP) is typically a benign, self-limiting disorder occurring in young (<10 years of age) previously healthy children. More than 80% of such children enter a complete sustained remission within a few weeks to a few months of initial presentation, irrespective of any therapy given. The major concern is the small but finite (0.1 to 0.9%) risk of intracranial hemorrhage, which occurs in children with very low platelet counts (<20 x 10(9)/L), and is the justification for treatment to increase the circulating platelet count. Effective treatment strategies are single-dose intravenous immunoglobulin G (IVIgG; approximately 1 g/kg) and medium to high-dose corticosteroids, administered orally or parenterally. The necessity for initial bone marrow aspiration and hospitalization continues to be debated. In children with chronic ITP, defined by persistence of thrombocytopenia for > or =6 months, splenectomy should be considered for the relatively small subgroup with symptomatic, severe thrombocytopenia who have either failed an adequate trial (> or = 12 months) of primary therapy (IVIgG, intravenous anti-D, corticosteroids) or are intolerant of such therapy. Laparoscopic splenectomy is preferred over open splenectomy. Children who fail to respond to splenectomy ( < or = 20% of cases) should be evaluated for the presence of accessory spleens; their management is often difficult and must be individualized. In severe refractory cases, second-line therapies (such as azathioprine or vinca alkaloids) need to be considered. Secondary ITP in children is relatively rare and is sometimes associated with other autoimmune cytopenias (Evan's syndrome, ITP with autoimmune neutropenia). These cases often respond poorly to conventional medical therapies and response rates to splenectomy are considerably lower than in children with primary chronic ITP.

Acute Disease↗
PubMed2000

Improved survival in severe acquired aplastic anemia of childhood.

Multi-agent immunosuppressive therapy has produced improved survival for severe acquired aplastic anemia in children. Recently, some investigators have suggested that immunosuppressive therapy may replace bone marrow transplantation as first-line therapy for this disorder. To assess its validity, we compared the outcomes of bone marrow transplantation vs immunosuppressive therapy in one institution from 1987 to 1997. We studied 46 consecutive patients less than 18 years of age who presented between January 1987 and April 1997. Inherited marrow failure syndromes and myelodysplastic syndromes were excluded. Patients received immunosuppressive therapy vs bone marrow transplantation based on availability of HLA-matched donors. The main outcome measures were survival, complete marrow and hematological remission, or partial remission but achieving independence from transfusional support. Twenty patients received multi-agent immunosuppressive therapy (cyclosporine, antithymocyte globulin and methylprednisolone); 11 attained complete remission and three partial remission for a transfusion-independent survival of 70%. Six patients died of infectious and hemorrhagic complications. Twenty-six patients were transplanted and 24 (93%) achieved complete remission; one achieved a PR, 25 remain transfusion independent with a median follow-up of 5.9 years or 70 months. One patient developed AML 34 months after successful transplant and one patient died due to graft failure and complications of transplant. There has been a striking improvement in survival for pediatric patients treated with multi-agent immunosuppression in the last decade. However, transplantation results have also improved and this remains the definitive first-line therapy for severe acquired aplastic anemia in this age group.

Adolescent↗
PubMed2000

Effect of premedication guidelines and leukoreduction on the rate of febrile nonhaemolytic platelet transfusion reactions.

Platelet transfusion reactions were prospectively studied in haematology/oncology patients at five university teaching hospitals over three consecutive summers. The initial summer study provided baseline information on the use of premedications and the rate of platelet transfusion reactions (fever, chills, rigors and hives). Most (73%) platelet recipients were premedicated and 30% (95% CI 28-33%) of transfusions were complicated by reactions. The second study followed implementation of guidelines for premedicating platelet transfusions. Despite a marked reduction in premedication (50%), there was little change in the platelet transfusion reaction rate, 26% (95% CI 24-29%), or the type of reactions. The third study followed implementation of prestorage platelet leukoreduction while maintaining the premedication guidelines. The reaction rate decreased to 19% (95% CI 17-22%). For nonleukoreduced platelets, there was a statistically significant association between the platelet age and reaction rate (P = 0.04). For leukoreduced platelets, there was no statistically significant association between platelet age and reaction rate (P = 0.5). Plasma reduction of nonleukoreduced platelet products also reduced the reaction rate. These prospective studies document a high rate of platelet transfusion reactions in haematology/oncology patients and indicate premedication use can be reduced without increasing the reaction rate. Prestorage leukoreduction and/or plasma reduction of platelet products reduces but does not eliminate febrile nonhemolytic platelet transfusion reactions.

Blood Preservation↗
PubMed1999

Antibody-mediated inhibition of the human alloimmune response to platelet transfusion in Hu-PBL-SCID mice.

Severe combined immune deficient (SCID) mice were engrafted with human (Hu) peripheral blood lymphocytes (PBL) from a previously alloimmunized donor and transfused with HLA-mismatched platelets. We have previously shown this to be a useful model for platelet transfusion. These engrafted mice (Hu-PBL-SCID mice) produced high levels of alloantibody in response to standard platelet preparations. However, when the first platelet challenge was presensitized with anti-HLA antibody and then transfused there was a marked reduction in the amount of alloantibody produced to five subsequent untreated platelet transfusions. Platelets pretreated with platelet-specific anti-HPA-1a (PL(A1)) sera did not induce a decrease in the anti-HLA alloantibody response. This demonstrated that platelet-induced HLA alloimmunization can be blocked by anti-HLA antibody-sensitized cells.

Animals↗
PubMed1999

Characterization of platelet glycoproteins and platelet/endothelial cell antibodies in patients with thrombotic thrombocytopenic purpura.

Platelets and sera from 12 patients with thrombotic thrombocytopenic purpura (TTP) and 12 healthy normal control subjects were examined. As determined by quantitative flow cytometry, prior to plasma exchange therapy platelet surface glycoprotein (GP) Ib levels were similar in TTP patients and normal controls (mean 20 188 and 20 226 molecules/platelet, respectively). Platelets from patients with TTP did, however, have significantly reduced levels of GPIIb/IIIa prior to plasmapheresis (mean 36 348 v 52 505 molecules/platelet in controls; P = 0.0004) and of GPIV (mean 13 321 v 26 212 molecules/platelet in controls; P = 0.0002). An increase in activated platelets, as determined by CD62 expression, was observed in 82% of patients. Increased platelet-associated immunoglobulins and/or complement was also seen in approximately 60% of the patients. In general, with return of platelet counts to normal levels following seven plasmaphereses, the above abnormalities were reversed, although often not to normal levels. Western blot analysis indicated the presence of antibodies reactive to platelet GPIV (88 kD) in 70% of pretreatment sera from patients with TTP; a similar band was observed in 80% of patient sera against microvascular endothelial cells. Immunofluorescence microscopic examination indicated the presence of antibody in pretreatment sera from patients with TTP to microvascular (73%) and large vessel (36%) endothelial cells. As measured by an indirect flow cytometric assay, pretreatment sera from 55% of patients with TTP were reactive with large vessel endothelial cells and 100% reacted with microvascular endothelial cells; reactivity was significantly greater against the microvascular endothelial cells (P = 0.0048) and was reduced following plasma exchange therapy. These results indicate abnormalities in platelet glycoprotein expression in TTP and suggest that anti-platelet and anti-endothelial cell antibodies play a role in the thrombocytopenia and vasculitis characteristic of this disorder.

Adult↗
PubMed1999

Inhibition of a secondary human alloimmune response via the soluble active component of CD154 (CD40L) in severe combined immune-deficient mice engrafted with human lymphocytes.

BACKGROUND: Alloimmunization requires a process known as co-stimulation. An important co-stimulatory pathway for most immune responses is mediated by the interaction of CD40 on antigen-presenting cells with CD154 (CD40L) on host T cells. Blockade of this co-stimulatory pathway simultaneous with exposure to challenge with HLA-incompatible cells is hypothesized to inhibit alloimmunization. STUDY DESIGN AND METHODS: Severe combined immune-deficient (SCID) mice were reconstituted with human peripheral blood lymphocytes (Hu-PBL-SCID mice) from a subject primed to HLA antigens and challenged with HLA-incompatible lymphocytes. Mice were challenged in the presence or absence of an 18-kDa soluble recombinant active form of human CD154 (18-kDa CD154). Human IgG production, alloimmunization, and in vitro T-cell responsiveness were assessed. RESULTS: There was no significant effect of 18-kDa CD154 on human IgG levels in these mice, but it inhibited the development of HLA-specific alloantibody in this model to five subsequent untreated white cell challenges. In vitro T-cell proliferation in a mixed lymphocyte culture was also prevented by 18-kDa CD154. CONCLUSION: The recombinant protein 18-kDa CD154 inhibited the ability of the Hu-PBL-SCID mice to mount a secondary immune response to allostimulation. This implies that transfusion-induced alloimmunization utilizes CD40-CD154 co-stimulation and that blockade of this pathway can inhibit T-cell function and interfere with the development of alloimmunization.

Animals↗
PubMed1998

Permeabilization and fixation conditions for intracellular flow cytometric detection of the T-cell receptor zeta chain and other intracellular proteins in lymphocyte subpopulations.

Expression of the T-cell receptor (TCR) zeta chain in normal individuals was studied by two-color flow cytometric analysis using digitonin-permeabilized human peripheral blood lymphocytes. Optimal detection of the TCR zeta chain involved fixation of cells in 0.25% paraformaldehyde for 2 min and permeabilization with 500 microg/ml of digitonin at 4 degrees C. Permeabilized lymphocytes and monocytes displayed decreased forward light scatter properties. Neutrophils did not survive this permeabilization/fixation/washing procedure. Permeabilization did not affect the ability of antibodies to CD3, CD4, CD8, CD16, and CD19 to detect CD antigens on lymphocytes, and the percentage of lymphocytes reacting with these antibodies was comparable between untreated and permeabilized cells. Staining of the TCR zeta chain was accomplished by incubating permeabilized cells with unconjugated antibody to the zeta chain, followed by an FITC-conjugated F(ab')2 goat anti-IgG. Following TCR zeta chain staining, cells were blocked with 25 microg/ml of mouse IgG for 20 min to saturate the goat anti-mouse antibody and then incubated with a phycoerythrin-conjugated mouse antibody to a variety of lymphocyte surface antigens. The TCR zeta chain was observed in lymphocytes displaying CD3, CD4, CD8, CD16, or TCRgammadelta markers. This permeabilization/fixation/washing procedure was also validated for detection of another intracellular T-cell protein known as the cytolytic granule-associated protein, recognized by the TIA-1 antibody. Thus the technique can be applied to detection of at least two different intracellular T-cell markers.

Antibodies, Monoclonal↗
PubMed1998

Platelet activation induced by porcine factor VIII (Hyate:C).

We studied the effects of porcine factor VIII (P-FVIII; Hyate:C) and other coagulation products employed in the management of patients with hemophilia A, on platelet activation in vitro. Exposure of normal resting platelets to P-FVIII resulted in platelet activation, as manifested by increased expression of the platelet surface activation markers CD62, CD63, and activated-GPIIbIIIa, and by activation-induced modulation of expression of normal platelet membrane glycoproteins CD41, CD42, and CD36. In contrast, platelet activation was not observed after exposure of the platelets to human FVIII, FEIBA, recombinant FVIIa, or cryosupernatant plasma. As with thrombin, exposure of platelets to P-FVIII resulted in the generation of platelet microparticles, an effect not seen not with the other products. In contrast to the characteristic reduction in expression in the number of CD42 molecules detected on thrombin-activated platelets, P-FVIII-stimulated platelets showed a small increase in CD42 expression. In contrast to thrombin, P-FVIII did not cause platelet dense granule release. The results indicate that therapeutic P-FVIII activates platelets, likely in ways that are different from the platelet activation seen with thrombin. The observed platelet activation and microparticle generation may provide a "hypercoagulable" mechanism for hemostasis with P-FVIII therapy separate from, and additional to, that due to increased circulating FVIII levels.

Animals↗
PubMed1998

Intravenous immunoglobulin G and anti-D as therapeutic interventions in immune thrombocytopenic purpura.

Immune thrombocytopenic purpura (ITP) is a disorder caused by accelerated destruction of antibody-coated platelets in the reticuloendothelial system (RES), especially the spleen. Inhibition of RES function following intravenous administration of high-dose immunoglobulin G (IVIG) or intravenous anti-D leads to rapid, albeit usually temporary, reversal of thrombocytopenia in the majority of children and adults with ITP. In emergency situations high-dose IVIG is preferred over anti-D because of the more rapid rate of platelet response; for maintenance therapy in Rh positive ITP patients (e.g. children with chronic ITP pre-splenectomy) anti-D is preferred because of its comparable efficacy to IVIG plus ease of administration and lower cost. In children with typical acute ITP and platelet counts < 20 x 10(9)/L IVIG is preferred over anti-D; however other approaches in this patient cohort should be considered before high-dose IVIG, specifically careful observation alone with therapy given only to children with clinically significant haemorrhage or short course oral prednisone at a starting dose of approximately 4 mg/kg/day. Studies are required to define the short and longer term effects of both IVIG and anti-D on the immune system in order to plan more rational use of these immunomodulatory therapies in this model autoimmune disorder.

Acute Disease↗
PubMed1998

Characterization of HIV-1-specific antibodies and HIV-1-crossreactive antibodies to platelets in HIV-1-infected haemophiliac patients.

Sera from HIV-1-infected haemophiliacs were examined for human immunodeficiency virus (HIV) specific antibodies and for platelet crossreactive antibodies. Using HIV sepharose 4B affinity columns for serum absorption, antibodies against various HIV antigens, including HIV lysate. HIV-p24 and HIV-gp120, were eluted either by low or by high pH buffer. The eluates were examined by ELISA for HIV specificity and by flow cytometry for platelet crossreactivity. Two types of HIV antibodies could be eluted, i.e. acid-sensitive and alkaline-sensitive antibodies. HIV antibodies were obtained in 26/29 acid eluates and in 25/29 of the alkaline eluates from HIV-lysate columns; 96% (25/26) of the acid-eluted antibodies were HIV-specific but 48% (12/ 25) of the alkaline-eluted antibodies also showed crossreactivity to platelets. Of the 20 alkaline-eluted HIV-p24 antibodies, 40% (8/20) reacted specifically with HIV-p24 and 60% (12/20) were platelet crossreactive. In contrast, of the alkaline-eluted HIV-gp120 antibodies (n=17), 88% (15/17) were HIV gp120-specific and only 12% (2/17) were platelet crossreactive. Western blot analysis of platelets demonstrated that the anti-p24 antibodies recognized three bands with approximate molecular weights of 72000 to 95000. 69% of the serum antiplatelet antibodies showed platelet glycoprotein IIbIIIa specificity. Anti-HIV antibodies could be eluted from platelets. Hence, platelet crossreactive antibodies in HIV infection are primarily alkaline-sensitive and are associated predominantly with HIV p24 antibody; these antibodies may play a role in the immune thrombocytopenia of HIV-infected haemophiliacs.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Management of chronic immune thrombocytopenic purpura in children and adults.

Chronic immune thrombocytopenic purpura (ITP) is characterized by macrophage destruction of platelets, primarily in the spleen, and occurs in both adults and children. Historically, splenectomy is often required, especially in adults with chronic ITP; however, several medical options have been used successfully prior to splenectomy or when this procedure has either failed or been refused by the patient. These include corticosteroids, intravenous immunoglobulin (IVIg), intravenous anti-D, danazol, vinca alkaloids, and other immunosuppressive agents such as azathioprine and cyclophosphamide. Platelet transfusions have been used in emergency situations. Several therapies that have been reported, but that are rarely used in chronic ITP, include cyclosporine A, interferon-alpha (IFN-alpha), plasma exchange, staphylococcal protein A immunoadsorption, combination chemotherapy, dapsone, ascorbic acid, and colchicine. Each of these approaches to treatment of chronic ITP are discussed.

Adrenal Cortex Hormones↗
PubMed1997

A survey of Canadian neonatal blood transfusion practices.

In 1990, the Pediatric Hemotherapy Committee of the American Association of Blood Banks developed and distributed a questionnaire addressing neonatal blood transfusion practices. The same questionnaire was subsequently sent to Canadian university-affiliated hospitals (n = 92). This report describes the results of the Canadian survey. Seventy-two percent (n = 66) of institutions contacted responded. Of these 42% (n = 28) had sufficient experience with neonatal transfusions and provided sufficient data for analysis. Although the majority of stated practices did follow published guidelines, several areas of variability and/or suboptimal practices were identified. With respect to component selection and preparation, suboptimal practices included excessive pretransfusion testing, unnecessary routine washing of RBC concentrates for small-volume transfusions, routine volume reduction of platelet concentrates and the use of suboptimal granulocyte preparations. With respect to transfusion practices, a disturbingly high percentage of respondents indicated that frozen plasma would be given in situations generally considered inappropriate. There was a great deal of variability in the provision of blood components at low risk for CMV, in the use of gamma irradiation and in the platelet count used for prophylactic platelet transfusions. The data collected in this survey provide information concerning practices that require improvement, identify areas where further research is desirable and provide a basis for comparison with current and future neonatal blood transfusion practices.

Blood Transfusion↗
PubMed1997

Maternal DRB1*1501, DQA1*0102, DQB1*0602 haplotype in fetomaternal alloimmunization against human platelet alloantigen HPA-6b (GPIIIa-Gln489).

Fetomaternal incompatibility of platelet alloantigens may lead to alloimmunization and neonatal alloimmune thrombocytopenia (NAIT). Human platelet alloantigen (HPA) 6b, which associates with residue Gln 489 of platelet membrane glycoprotein IIIa, has been described as a cause of NAIT. We have studied the MHC genes of all available family members in the six thus far reported families with a thrombocytopenic newborn and fetomaternal HPA-6b incompatibility. Maternal HPA-6b antibodies could be detected in five mothers to the altogether seven thrombocytopenic male infants. The MHC genes HLA-DRB, -DQA1, -DQB1, -DPB1, TAP1,2 and HSP70-Hom were studied by using polymerase chain reaction (PCR)-based DNA analysis methods. All five mothers with detectable circulating HPA-6b antibodies at the time of delivery shared an identical DRB1*1501, DQA1*0102, DQB1*0602 haplotype. The sixth, HPA antibody negative mother and a HPA-6b-negative mother to a healthy HPA-6b+ child were negative for this haplotype. The frequency of DRB1*15-positive haplotype was increased in immunized mothers (100%) as compared with the general Finnish population (27%), but the association was not statistically significant after correction. We conclude that there is a potential association between the MHC haplotype DRB1*1501, DQA1*0102, DQB1*0602 and alloimmunization to the HPA-6b antigen and that this alloimmunization probably involves different HLA class II molecules from immunization to HPA-1a.

Antigens, Human Platelet↗
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↗