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Biomedical subjects

E M Sloand

Publications and source records attributed to E M Sloand.

At least 19 recordsLinked to original sources
PubMed2000

Protease inhibitors stimulate hematopoiesis and decrease apoptosis and ICE expression in CD34(+) cells.

Highly active retroviral therapy has been associated with a decline in the frequency of cytopenia in patients with human immunodeficiency virus (HIV) infection. This may result from lower hematologic toxicity of newer antiviral drugs and their increased efficacy against HIV-1. Protease inhibitors, in addition to their effects on HIV replication, appear to affect various cellular functions. Recently, it was reported that ritonavir inhibited caspase-1 expression in normal CD4(+) cells. It was hypothesized that protease inhibitors may improve hematopoietic function owing to their direct effects on the bone marrow progenitor cells. When ritonavir was added to methylcellulose cultures of bone marrow cells from HIV-infected patients and normal controls, colony formation increased 2.4-fold (n = 5) in control cultures and 4-fold (n = 5) in cultures of cells from HIV-infected patients. In the presence of ritonavir, cultures of CD34(+) cells showed markedly decreased apoptosis in comparison with untreated cultures (45% decrease in apoptotic cell number; n = 6). A synthetic inhibitor of caspase 1 (Ac-Tyr-Val-Ala-Asp-aldehyde [single-letter amino acid codes]), which inhibits activation of several caspases including CPP32 and interleukin 1beta-converting enzyme (ICE or caspase 1), also decreased the rate of apoptosis and enhanced colony formation by progenitor cells derived from HIV-infected patients (3-fold; n = 5). In ritonavir-treated samples derived from HIV-infected individuals, the number of cells expressing ICE also decreased. In conclusion, HIV protease inhibitors may, by blocking the caspase-dependent apoptotic pathway, overcome inhibition of hematopoiesis seen in patients with HIV infection, an effect unrelated to their antiviral activity. (Blood. 2000;96:2735-2739)

Antiretroviral Therapy, Highly Active↗
PubMed2000

Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine production by lymphocytes in vitro and in vivo.

Peripheral blood stem cell (PBSC) transplantation is successful in improving engraftment without increasing acute graft-versus-host disease (GVHD), despite much larger numbers of T cells in unmanipulated PBSCs than in bone marrow grafts. In mouse models and retrospective human studies, granulocyte colony-stimulating factor (G-CSF) therapy has been associated with less acute GVHD. We studied the effect of G-CSF on interferon (IFN)-gamma and IL-4 expression in CD4(+) lymphocytes. CD4(+) cells co-cultivated with G-CSF and stimulated with PHA or CD3 monoclonal antibodies showed significant decreases in IFN-gamma and increases in IL-4 expression (n = 13; P <. 01). G-CSF appeared to have a direct effect on CD4(+) cells independent of monocytes present in the culture because purified CD4(+) cells exposed to G-CSF, washed, and cocultivated with untreated monocytes demonstrated similar changes in IFN-gamma and IL-4 expression, whereas untreated CD4(+) cells cocultured with G-CSF-stimulated monocytes behaved as controls. We then studied peripheral blood mononuclear cells (PBMCs) from G-CSF-mobilized PBSC donors. When their PBMCs were cultured with PHA or CD3 monoclonal antibody, the percent of IFN-gamma-expressing cells decreased by a mean of 55% and 42%, respectively, whereas the percent of IL-4-containing cells increased by a mean of 39% and 58%, respectively, following G-CSF stimulation. Increased apoptosis of IFN-gamma-producing CD4(+) cells was not responsible for the shift in TH1/TH2 subsets. G-CSF-R mRNA was present in both CD4(+) and CD8(+) cells. These results suggest that G-CSF decreases IFN-gamma and increases IL-4 production in vitro and in vivo and likely modulates a balance between TH1 and TH2 cells, an effect that may be important in PBSC transplantation.

Antibodies, Monoclonal↗
PubMed2000

Use of human immunodeficiency virus (HIV) human hyperimmune immunoglobulin in HIV type 1-infected children (Pediatric AIDS clinical trials group protocol 273).

The clinical, immunologic, and virologic effects and the pharmacokinetics of human immunodeficiency virus (HIV) human hyperimmune immunoglobulin (HIVIG) were assessed in 30 HIV-infected children aged 2-11 years. All had moderately advanced disease with an immune complex-dissociated (ICD) p24 antigen >70 pg/mL and were on stable antiviral therapy. Three groups of 10 children received 6 monthly infusions of 200, 400, or 800 mg/kg of HIVIG, and serial immunologic and virologic assays were performed. HIVIG doses as high as 800 mg/kg were safe and well tolerated. The half-life of HIVIG, determined by serial p24 antibody titers, was 13-16 days, the volume of distribution was 102-113 mL/kg, and clearance was 5.6-6.0 mL/kg/day. Plasma ICD p24 decreased during the infusions, but CD4 cell levels, plasma RNA copy number, cellular virus, immunoglobulin levels, and neutralizing antibody titers were minimally affected by the infusions. Clinical status did not change during the 6-month infusion and 3-month follow-up periods.

Cells, Cultured↗
PubMed1999

Human immunodeficiency virus type 1 protease inhibitor modulates activation of peripheral blood CD4(+) T cells and decreases their susceptibility to apoptosis in vitro and in vivo.

CD4(+) T cells from patients with human immunodeficiency virus (HIV) infection undergo apoptosis at an increased rate, which leads to their depletion during disease progression. Both the Fas-Receptor (Fas-R) and interleukin-1beta (IL-1beta)-converting enzyme (ICE; caspase 1) appear to play a role in the mechanism of apoptosis of CD4(+) lymphocytes. Although Fas-R is upregulated on both CD4(+) and CD8(+) cells in HIV-infected patients, results from our laboratory and others indicate that, in patients with advanced disease, CD4(+) cells preferentially express ICE. Protease inhibitors have successfully halted the progression of HIV disease and increased CD4(+) T counts. In this study, we examined the effect of protease inhibitors on Fas-R (CD95), ICE (caspase 1) expression, apoptosis, and cell death in CD4(+) T cells of (1) HIV-infected patients who were receiving protease inhibitors, and (2) normal and patient CD4(+) T cells cultured with a protease inhibitor in vitro. Fifteen patients with advanced HIV disease on treatment showed dramatically decreased CD4(+) T-cell ICE expression, diminished apoptosis, and increased numbers of CD4(+) cells within 6 weeks of institution of protease inhibitor therapy, and before down-modulation of Fas-R (CD95) expression was evident. To determine the role of HIV infection, we studied the effect of ritonavir, a protease inhibitor, on normal and patient cells in vitro. Stimulated and unstimulated normal CD4(+) T cells, cultured with protease inhibitor, demonstrated markedly decreased apoptosis and ICE expression (P =. 01). While Fas-R expression was not significantly altered during short-term culture by such treatment, Fas-Ligand (Fas-L) membrane expression of phytohemagglutinin (PHA)-stimulated blood lymphocytes was decreased by protease inhibitor. In the presence of ritonavir, CD4(+) T cells from HIV-infected patients showed similar changes in ICE intracellular levels without alteration of Fas expression. In conclusion, protease inhibitors appear to decrease CD4(+) T-cell ICE expression and apoptosis before they affect Fas-R expression in HIV-infected patients. This action was independent of HIV infection, as similar effects were seen in CD4(+) T cells from normal controls. Some of the benefit of protease inhibitors may be related to modification of programmed cell death, which increases CD4(+) T-cell number. Whether this is due to directly to the changes effected in the caspase system remains to be determined.

Apoptosis↗
PubMed1998

Correction of the PNH defect by GPI-anchored protein transfer.

Hemolytic anemia is a major feature of paroxysmal nocturnal hemoglobinuria (PNH). Intravascular red blood cell (RBC) destruction is caused by increased sensitivity of the abnormal erythrocyte to complement-mediated lysis, due to the GPI absence of a membrane-bound glycosylphosphatidylinositol (GPI)-linked protein, which functions as an inhibitor of reactive lysis (CD59). Both in vivo and in vitro models have suggested the feasibility of cell-to-cell transfer of GPI proteins, and patients with hemolysis could potentially benefit from transfer of CD59 to their deficient erythrocytes. We studied the ability of RBC components prepared from outdated packed RBC collections, as well as high-density lipoprotein (HDL) preparations, rich in CD55 and CD59, to promote protein transfer, as assessed by flow cytometry, immunoblotting, and susceptibility to complement-mediated lysis. By flow cytometry, CD55 and CD59 were present on RBC-derived microvesicles that stained with an antiglycophorin antibody Ab; in addition, soluble CD59 and CD55 were detected by immunoblot in soluble fractions eluated from RBC units stored for more than 35 days, but not in fresh blood. Both commercial HDL preparations and those prepared in our laboratory contained CD55 and CD59, as assayed by immunoblot. When RBC that were deficient (GPI)-anchored protein, obtained from five patients, with PNH were incubated with HDL preparations for 2 to 4 hours, there was significant transfer of both proteins to the cell surface, as demonstrated by flow cytometry. Washed RBC microvesicles, prepared by ultrasonification, also mediated transfer of GPI-linked proteins to deficient RBC. Pretreatment of microvesicles, RBC eluate preparations, and HDL with phosphatidylinositol-specific, phospholipase C, abrogated protein transfer to deficient cells, indicating that increased cell-associated CD55 and CD59 levels were related to insertion of the intact GPI moiety, rather than to simple adhesion. PNH RBC that were exposed to HDL, RBC eluate preparations, or microvesicles demonstrated decreased in vitro complement-mediated hemolysis in the Ham test. Transfer of GPI-linked proteins from soluble preparations containing CD55 and CD59 to PNH erythrocytes is feasible and may have clinical utility.

CD55 Antigens↗
PubMed1998

Neither human immunodeficiency virus-1 (HIV-1) nor HIV-2 infects most-primitive human hematopoietic stem cells as assessed in long-term bone marrow cultures.

Attempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38-) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.

Adult↗
PubMed1998

The role of interleukin-converting enzyme in Fas-mediated apoptosis in HIV-1 infection.

Apoptosis of CD4+ lymphocytes is partially responsible for the depletion of these cells in HIV-infected individuals. CD4+ lymphocytes from HIV-1-infected patients express higher membrane levels of the Fas receptor, and are particularly susceptible to apoptosis after Fas triggering. IL-1beta- converting enzyme (ICE) is a key enzyme of the apoptotic machinery involved in Fas-mediated apoptosis of normal lymphocytes. The role of ICE in mediating the increased susceptibility of CD4+ lymphocytes from HIV-1-infected patients to apoptosis has not been examined. In this study, we found that ICE mRNA was present in T cells from both HIV-1-infected patients and controls. Active ICE proteins, p10 and p20, were demonstrated by immunoblot in lymphocytes from HIV-1-infected patients and in normal lymphocytes after treatment with Fas agonist, CH11 mAb. Cocultivation of lymphocytes from HIV-1-infected persons with Fas antagonist, antibody ZB4, resulted in decreased expression of ICE protein in lymphocytes from HIV-infected patients, and decreased apoptosis. Similar effects were obtained when cells were treated with synthetic ICE inhibitors, which blocked apoptosis in response to Fas triggering. When CD4+ and CD8+ cells were sorted by flow cytometry and analyzed by reverse transcriptase PCR, ICE mRNA was present in both CD8+ and CD4+ cells. However, flow cytometric analysis of lymphocytes with intracellular staining for ICE demonstrated ICE protein in the CD4+ but not the CD8+ cell fraction derived from blood of HIV-1-infected patients. These data suggest that activation of ICE occurs in vivo in CD4+ lymphocytes from HIV-1-infected individuals, and may account for the increased susceptibility of CD4+ cells to apoptosis.

Apoptosis↗
PubMed1998

Inhibition of interleukin-1beta-converting enzyme in human hematopoietic progenitor cells results in blockade of cytokine-mediated apoptosis and expansion of their proliferative potential.

Inhibitory and stimulatory cytokines regulate the function and survival of hematopoietic progenitor cells. Interactions between cytokines and progenitor cells may result in programmed cell death. Apoptosis of hematopoietic cells ultimately serves to diminish the size of the stem cell compartment in bone marrow (BM) failure, and this has frustrated efforts at ex vivo expansion of hematopoietic stem cells for BM transplantation. We previously demonstrated that triggering of the Fas-receptor, which is expressed on BM CD34+ cells, mediates apoptosis of progenitor cells. Although interleukin-1beta-converting enzyme (ICE) appears to be an important factor in the signaling cascade regulating Fas-mediated apoptosis of lymphoid cells, its role in apoptosis of CD34+ cells has not been explored. In this study, we determined whether ICE protein was present in CD34+ cells and assessed its role in limiting expansion of hematopoietic stem cells by apoptosis. We demonstrated that ICE mRNA was constitutively produced in CD34+ cells, although the active form of ICE protein was not detected in fresh, unstimulated CD34+ cells from healthy donors. ICE protein could be induced in these CD34+ cells when they were cultured for 24 hours in the presence of hematopoietic growth factors. Interferon (IFN)-gamma and Fas agonist (CH11 monoclonal antibody) enhanced ICE expression and triggered CD34+ cell apoptosis and cell death. In both short- and long-term BM cultures, hematopoietic colony-forming cell numbers were increased after ICE blockade with a synthetic ICE inhibitor (Ac-Tyr-Val-Ala-Asp-aldehyde), even in the absence of IFN-gamma, suggesting that ICE regulates the proliferation and cell death of committed and primitive progenitor cells. The suppressive effect of IFN-gamma and Fas agonist on colony formation was also antagonized by ICE inhibitor. The effects of ICE blockade on proliferation of hematopoietic progenitors cells were related to inhibition of apoptosis, as demonstrated by annexin staining and in situ terminal dideoxytransferase apoptosis assays. Our results suggest that ICE protein is present in CD34+ cells after exposure to cytokines, that regulation of the levels of ICE protein in CD34+ cells is posttranscriptional, and that ICE plays a role in the regulation of apoptosis and expansion of primitive hematopoietic cells. ICE inhibition could potentially be used to reverse intrinsic and cytokine-mediated apoptotic signals for the purpose of stem and progenitor cell expansion.

Antigens, CD34↗
PubMed1997

Impaired hematopoiesis in paroxysmal nocturnal hemoglobinuria/aplastic anemia is not associated with a selective proliferative defect in the glycosylphosphatidylinositol-anchored protein-deficient clone.

Paroxysmal nocturnal hemoglobinuria (PNH) results from somatic mutations in the PIG-A gene, leading to poor presentation of glycosylphosphatidylinositol (GPI)-anchored surface proteins. PNH frequently occurs in association with suppressed hematopoiesis, including frank aplastic anemia (AA). The relationship between GPI-anchored protein expression and bone marrow (BM) failure is unknown. To assess the hematopoietic defect in PNH, the numbers of CD34+ cells, committed progenitors (primary colony-forming cells [CFCs]), and long-term culture-initiating cells (LTC-ICs; a stem cell surrogate) were measured in BM and peripheral blood (PB) of patients with PNH/AA syndrome or patients with predominantly hemolytic PNH. LTC-IC numbers were extrapolated from secondary CFC numbers after 5 weeks of culture, and clonogenicity of LTC-ICs was determined by limiting dilution assays. When compared with normal volunteers (n = 13), PNH patients (n = 14) showed a 4.7-fold decrease in CD34+ cells and an 8.2-fold decrease in CFCs. LTC-ICs in BM and in PB were decreased 7.3-fold and 50-fold, respectively. Purified CD34+ cells from PNH patients had markedly lower clonogenicity in both primary colony cultures and in the LTC-IC assays. As expected, GPI-anchored proteins were decreased on PB cells of PNH patients. On average, 23% of monocytes were deficient in CD14, and 47% of granulocytes and 58% of platelets lacked CD16 and CD55, respectively. In PNH BM, 27% of CD34+ cells showed abnormal GPI-anchored protein expression when assessed by CD59 expression. To directly measure the colony-forming ability of GPI-anchored protein-deficient CD34+ cells, we separated CD34+ cells from PNH patients for the GPI+ and GPI-phenotype; CD59 expression was chosen as a marker of the PNH phenotype based on high and homogeneous expression on fluorescent staining. CD34+ CD59+ and CD34+ CD59-cells from PNH/AA patients showed similarly impaired primary and secondary clonogeneic efficiency. The progeny derived from CD34+ CD59- cells were both CD59- and CD55-. A very small population of CD34+ CD59- cells was also detected in some normal volunteers; after sorting, these CD34+ CD59- cells formed normal numbers of colonies, but their progeny showed lower CD59 levels. Our results are consistent with the existence of PIG-A-deficient clones in some normal individuals. In PNH/AA, progenitor and stem cells are decreased in number and function, but the proliferation in vitro is affected similarly in GPI-protein-deficient clones and in phenotypically normal cells. As measured in the in vitro assays, expansion of PIG-A- clones appears not be caused by an intrinsic growth advantage of cells with the PNH phenotype.

Adolescent↗
PubMed1997

Role of Fas ligand and receptor in the mechanism of T-cell depletion in acquired immunodeficiency syndrome: effect on CD4+ lymphocyte depletion and human immunodeficiency virus replication.

Direct killing of CD4+ lymphocytes by human immunodeficiency virus-1 (HIV-1) probably cannot account for the magnitude of the loss of these cells during the course of HIV-1 infection. Experimental evidence supports a pathophysiologic role of the apoptotic process in depletion of CD4 cells in acquired immunodeficiency syndrome (AIDS). The Fas-receptor/Fas-ligand (Fas-R/Fas-L) system mediates signals for apoptosis of susceptible lymphocytes and lympoblastoid cell lines. A number of investigators have recently reported increased expression of the Fas receptor in individuals with HIV infection, along with increased sensitivity of their lymphocytes to anti-Fas antibody mimicking Fas ligand. We attempted to determine the role of Fas-mediated apoptosis in disease progression and viral replication. Increased Fas-receptor (CD95) expression on CD4+ and CD8+ lymphocytes was found in a large group of HIV-1-infected patients compared with normal controls; individuals with a diagnosis of AIDS and a history of opportunistic infection had significantly more Fas receptor expression than did asymptomatic HIV-infected persons and normal blood donor controls (P < .01). Triggering of the Fas-R by agonistic anti-Fas monoclonal antibody, CH11, was preferentially associated with apoptosis in the CD4+ cells; this effect was more pronounced in lymphocytes derived from HIV+ individuals. Soluble and membrane-bound forms of Fas-L were produced in greater amounts in peripheral blood mononuclear cells (PBMC) cultures and in plasma obtained from HIV-1-infected persons than from normal controls. Furthermore, triggering of lymphocytes from HIV-infected persons by CH11 increased levels of interleukin-1beta converting enzyme (ICE), a protein associated with apoptosis. When PBMC were cultured in the presence of CH11, p24 production per number of viable cells was decreased as compared with the same PBMC without CH11 (P < .01). These findings suggest that multiple mechanisms, including increased production of Fas-L by infected PBMC, increased Fas-R expression, and induction of a protease of ICE family, may play roles in the apoptotic depletion of CD4+ cells in HIV infection.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Secondary colony formation after long-term bone marrow culture using peripheral blood and bone marrow of HIV-infected patients.

OBJECTIVE: To determine if defective bone marrow function in HIV infection is associated with decreased numbers of progenitor cells or defective stromal cell function. DESIGN: Defective bone marrow function may in part be responsible for the cytopenias so frequently seen in patients with AIDS. Although a number of investigators have reported impaired growth of committed hematopoietic progenitor cells in HIV-1-infected patients, few studies have examined the most primitive hematopoietic stem cells. Our study was designed to determine the function and quality of the most immature hematopoietic progenitor and stem cells in the peripheral blood and bone marrow of HIV-1-infected patients and to assess stromal cell function. METHODS: For quantification of these cells we used a modified long-term culture-initiating cell (LTCIC) assay in which the number of secondary colony-forming cells after 5 weeks of stromal culture served as a measure of LTCIC. Stromal cells from normal controls and HIV-1-infected patients were used for cross-matching experiments. Normal CD34+ cells or those derived from HIV-infected patients were plated and colony growth assessed. RESULTS: We found that HIV-1-infected patients had a mean of 0.8 secondary colony-forming cells/10(5) peripheral blood mononuclear cells (PBMC), whereas normal controls showed 1.2 secondary colony-forming cells/10(5) PBMC (P = not significant) after long-term culture. Asymptomatic patients showed well preserved numbers of secondary colony-forming cells after long-term culture of PBMC, but a significant reduction was seen in patients with a history of opportunistic infections (P < 0.01), low CD4+ cell count (< 200 x 10(6)/l; P < 0.05), or leukopenia (P < 0.05). Decreased numbers of secondary colony-forming cells have also been found in bone marrow of HIV-1-infected patients with advanced disease. When normal CD34+ cells were cultured on stromal layers from bone marrow of HIV-1-infected patients or normal controls, no differences in the numbers of surviving progenitor cells were found. CONCLUSIONS: Our data indicate that the hematopoietic defect in HIV-1 infection involves the most immature hematopoietic cells and becomes evident in advanced disease. Stromal function of HIV-infected patients appears normal.

Bone Marrow↗
PubMed1997

Transfusion in HIV disease.

Cytopenias often accompany infection with HIV. The pathophysiology responsible for the anemia, neutropenia, and thrombocytopenia in AIDS is complex and incompletely understood. Although HIV-infected patients with advanced disease were once routinely transfused for anemia accompanying zidovudine therapy, this practice has decreased as a result of decreased doses of zidovudine now being used and the more frequent use of HIV protease inhibitors. A more thorough understanding of the adverse effects of transfusion and the potential of immune modulation and facilitation of viral replication has no doubt led physicians to consider more thoroughly other factors before transfusing their patient. The results of trials examining the effect of leukodepletion on transfusion-related immune modulation are eagerly awaited. The availability of hematopoietic growth factors has also led to more effective treatment of the cytopenias without significant complications.

AIDS-Related Opportunistic Infections↗
PubMed1997

Viral risks associated with blood transfusion.

Great strides have been made within the last decade to help ensure the safety of the blood supply. Additional tests to detect infectious agents, as well as development of improved donor screening and deferral techniques have helped make the risk of transfusion-transmitted disease very low. Currently, blood banks perform seven tests to detect infectious agents. Prospective donors are carefully questioned about factors that place them at risk for transfusion-transmitted disease and donors known to test positive for certain viruses are permanently deferred. The risks of receiving a human immunodeficiency virus (HIV)-infected unit is now estimated to be 1 in 493,000, while the risk of hepatitis B is 1 in 63,000. However, changes in prevalence within the blood donor population brought about by changes in the factors that place an individual at risk for a transfusion-transmitted disease could significantly alter these risks. The American public continues to be concerned about the safety of blood transfusion. These concerns coupled with the fears that new viruses or new strains of viruses will be identified that escape detection has created the impetus for development of methods that will remove or inactivate viruses in cellular blood products.

Cytomegalovirus Infections↗
PubMed1997

Studies on platelet membrane glycoproteins and platelet function during hemodialysis.

Hemodialysis only partially corrects the defects in platelet function associated with uremia. Platelet contact with the artificial surfaces of the dialysis filter during hemodialysis can itself cause platelet activation, degranulation, and loss of platelet membrane glycoproteins. Although the transient platelet dysfunction that occurs after platelet contact with foreign surfaces during cardiopulmonary bypass has been well characterized, there has been no such investigation of hemodialysis. In this study of hemodialysis patients, bleeding times (BT) and the response of their platelets to thrombin, ristocetin, and collagen were measured before, immediately after, and in some patients, the day after hemodialysis. In addition, membrane glycoproteins in platelets obtained at these time intervals were studied using fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies (mAb) CD42b (anti-GPIb), CD41a (anti-GPIIb/IIIa), and CD62 (anti-P-selectin), with flow cytometry. BT was significantly prolonged, and response to thrombin and ristocetin was significantly decreased immediately after hemodialysis (P < 0.01). Binding of CD42b mAb to the platelet membrane was decreased in platelets obtained immediately after hemodialysis. Most patients had shortened BT and demonstrated increased response of their platelets to thrombin and increased CD42b binding to their platelets the day after hemodialysis. These findings suggest that in uremic patients, hemodialysis is associated with transient platelet dysfunction and decreased membrane expression of GPIb.

Antibodies, Monoclonal↗
PubMed1996

Analysis of the expression of glycosylphosphatidylinositol anchored proteins on platelets from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH), an acquired clonal disorder is manifested by failure of hematopoietic cells to express phosphatidylinositol glycan-anchored proteins (PIG-AP). Since the PIG-A mutation is present at the stem cell level, all cell lines may be affected. Although the pathogenesis of hemolytic anemia in PNH is related to the absence of CD55 and CD59 molecules on the membrane of red cells, the mechanism responsible for the increased incidence of thrombotic events in PNH is not clear. In this study we measured two glycosylphosphatidylinositol (GPI)-linked molecules on platelets (CD55 and CD59) and two GPI-linked proteins on neutrophils (CD14 and CD16), comparing their expression on normal and PNH patients. Using two-color flow cytometric analysis with antibodies directed against CD42b and CD41a, we found that CD55 and CD59 were constitutively expressed by normal fresh platelets, but that the expression levels decreased during the five day storage of platelets. A substantial population of platelets lacking the GPI-linked proteins were detected in most cases. We demonstrated varying degrees of deficiency in the expression of GPI-anchored molecules with neutrophils, monocytes and platelets with the highest proportion of deficient cells found within monocytic lineage. Similar numbers of platelets with the PNH phenotype and normal platelets expressed activation markers before and after exposure to platelet agonists. Flow cytometry is more sensitive than Ham's test in monitoring expression of PNH in platelets. Differences in the numbers of circulating GPI-deficient platelets and myeloid cells suggest that either the survival of platelets and mature myeloid cells differs or megakaryocytopoeisis is abnormal within the GPI-deficient clone.

Anemia, Aplastic↗
PubMed1994

1-Deamino-8-D-arginine vasopressin (DDAVP) increases platelet membrane expression of glycoprotein Ib in patients with disorders of platelet function and after cardiopulmonary bypass.

1-deamino-8-D-Arginine vasopressin (DDAVP) shortens the bleeding time in some patients with platelet dysfunction and decreases blood loss in some cardiopulmonary bypass patients. We studied platelet membrane glycoproteins in patients with von Willebrand disease (vWD), disorders of platelet function, and in cardiopulmonary bypass patients after infusion of 0.3 microgram/kg of DDAVP. Platelets from 8 cardiopulmonary bypass patients, receiving DDAVP immediately after surgery, were compared to those of 14 patients not receiving DDAVP. We also studied 12 patients with vWD, and 8 patients with platelet dysfunction receiving DDAVP. Fixed platelets, stained with monoclonal fluorescein (FITC)-labeled antibodies directed against GPIb (CD42b antigen), GPIb/IX, GPIIb/IIIa (CD41a antigen), CD63 antigen (a platelet activation protein), and P-selectin (CD62 antigen) were studied by flow cytometry. Binding of CD42b monoclonal antibody (MoAb) and anti-GPIb/IX to platelets from both groups of bypass patients increased during the 18-20 hr after surgery, but the group receiving DDAVP showed the greater increase (P = 0.032). Platelets from patients receiving DDAVP for vWD or for platelet dysfunction, had increases in CD42b MoAb and anti-GPIb/IX binding (P < 0.01) that coincided with shortening of their bleeding time. No changes were seen in binding of other antibodies. When platelets from normal donors were incubated with DDAVP for 20 hr, there were increases in platelet surface CD42b MoAb binding, while immunogold-stained transmission electron micrographs of permeabilized platelets demonstrated decreases in cytoplasmic CD42b MoAb binding. DDAVP increases platelet membrane GPIb expression in a variety of patients and may account for improvement in hemostasis seen in some studies. Redistribution of GPIb from the cytoplasm to the membrane may account for this increased expression.

Adult↗
PubMed1994

Effect of amphotericin B and fluconazole on platelet membrane glycoproteins.

BACKGROUND: Fever, chills, and reduced platelet recovery may result when platelets are transfused simultaneously with amphotericin B. Amphotericin B reportedly increases the pitting of membranes in stored platelets. STUDY DESIGN AND METHODS: The effects of amphotericin B and another antifungal agent, fluconazole, on platelet membrane glycoproteins (GP) were examined by the incubation of split aliquots of fresh and stored platelet concentrates (PCs) with these drugs for 3 days in storage bags. To determine the effect of storage, PCs were stored for 5 days, and aliquots removed on Days 1 through 5 were placed in platelet storage bags with 4 micrograms per mL of amphotericin B for 2 to 6 hours. Membrane glycoprotein expression was assessed by flow cytometry with fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed against the following antigens: GPIb (CD42b), CD63 (an activation protein), P-selectin (CD62), and GPIIb/IIIa (CD41a). RESULTS: Amphotericin B produced a concentration-dependent decrease in the surface binding of CD42b MoAb with no consistent changes in the binding of CD41a, CD63, or CD62 MoAbs after a 3-day exposure. Stored but not fresh PCs showed decreased binding of MoAb CD42b after a 6-hour exposure to amphotericin B (4 micrograms/mL). Fluconazole produced no changes. When the binding of MoAb CD42b to permeabilized platelets was used to measure total platelet content, amphotericin B (4 micrograms/mL) decreased MoAb CD42b binding to a similar degree in fresh and stored platelets. Inhibition of aggregation to ADP and collagen and ADP and epinephrine was seen in stored but not fresh PCs. CONCLUSION: Therapeutic levels of amphotericin B resulted in partial loss of total platelet GPIb in fresh and stored PCs, but decreased surface expression of platelet membrane GPIb only in stored platelets. This difference between fresh and stored platelets may be related to the limited reservoir of GPIb available for redistribution to the membrane in the previously stored PCs and may account for the decreased recovery of transfused platelets observed in some patients receiving amphotericin B.

Amphotericin B↗