Leukocyte depletion of random single-donor platelet transfusions does not prevent secondary human leukocyte antigen-alloimmunization and refractoriness: a randomized prospective study

We studied the value of leukocyte depletion of platelet transfusions for the prevention of secondary human leukocyte antigen (HLA)- alloimmunization in patients with a high-risk of prior immunization induced by pregnancies. Seventy-five female patients with hematologic malignancies (mostly acute leukemia) and a history of pregnancy were randomized to receive either standard random single-donor platelet transfusions (mean leukocytes, 430 x 10(6) per transfusion) or leukocyte-depleted random single-donor platelet transfusions. Leukocyte depletion to less than 5 x 10(6) leukocytes per platelet transfusion (mean leukocytes, 2 x 10(6) per transfusion) was achieved by filtration. Of the 62 evaluable patients, refractoriness to random donor platelets occurred in 41% (14 of 34) of the patients in the standard group and in 29% (8 of 28) of the patients in the filtered group (P = .52); anti-HLA antibodies developed in 43% (9 of 21) of individuals in the standard group and 44% (11 of 25) of cases in the filtered group. The time toward refractoriness and development of anti- HLA antibodies was similar for both groups. We conclude that leukocyte depletion of random single-donor platelet products to less than 5 x 10(6) per transfusion does not reduce the incidence of refractoriness to random donor platelet transfusion because of boostering of anti-HLA antibodies.     

Monoclonal antibodies against porcine platelet membrane glycoproteins Ib and IIb/IIIa

We prepared murine monoclonal antibodies to porcine platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3-4C, blocked Ristocetin-induced platelet agglutination and caused 80% inhibition of Ristocetin-induced 125I-von Willebrand factor (vWF) binding to porcine platelets at a concentration of greater than or equal to 12 micrograms IgG/mL. PP3-4C did not affect adenosine diphosphate (ADP)- or collagen-induced platelet aggregation. Binding of 125I-Fab fragments of PP3-4C to platelets was saturable at 3.7 x 10(4) +/- 0.8 x 10(4) molecules per platelet. Another monoclonal antibody, designated PP3-3A, blocked ADP- or collagen-induced platelet aggregation at 6 micrograms IgG/mL. At a concentration of 10 micrograms IgG/mL, PP3-3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated platelets. PP3-3A did not affect Ristocetin-induced platelet agglutination nor 125I-vWF binding to platelets in the presence of Ristocetin. Binding of 125I-Fab' fragments of PP3-3A to platelets was saturable at 9.8 x 10(4) +/- 1.2 x 10(4) molecules per platelet. PP3-4C antibody (anti-GP Ib) did not bind to human platelets; however, PP3-3A antibody (anti-GP IIb-IIIa) had partial cross-reactivity with human platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that PP3-4C recognized a GP with an apparent molecular weight of 160,000 (nonreduced), and 140,000 (reduced). PP3-3A recognized GPs with apparent molecular weights of 130,000 and 80,000 (nonreduced), and 115,000 and 95,000 (reduced). These monoclonal antibodies to porcine platelet membrane GPs, which are structural and functional analogues of human GP Ib and GP IIb/IIIa, will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.     

Multilineage hematopoietic growth factor interleukin 3 and direct activators of protein kinase C stimulate phosphorylation of common substrates

In order to investigate early signal transduction events in myeloid cells, the phosphosubstrates of an interleukin 3 (IL 3)-dependent cell line, FDC-P1, have been analyzed. Using synthetic diacylglycerol as a direct activator of the unique calcium-phospholipid-dependent phosphotransferase protein kinase C (PK-C) and genetically engineered homogeneous IL 3, we have demonstrated a common element to signal transduction events associated with these stimulants. One novel substrate, p68 (68,000 kd), was rapidly phosphorylated in either IL 3- or diacylglycerol-stimulated cells. The phosphorylation of p68 was dose- dependent, with both the physiological ligand and diacylglycerol inducing the same maximal level of phosphorylation. Phosphorylation of p68 occurred in a time-dependent manner analogous to previously described kinetics of PK-C subcellular redistribution in the FDC-P1 cell line. The p68 substrate was also phosphorylated in a cell-free system under conditions designed to activate PK-C. Phosphoamino acid analysis demonstrated that the p68 molecule phosphorylated in intact cells as well as in a calcium-phospho-lipid-dependent cell-free system was phosphorylated on threonine residues, not tyrosine. These data support the hypothesis that the activation of PK-C that occurs after IL 3-receptor interaction which leads to the rapid phosphorylation of cellular proteins is an important element of the signal transduction mechanism in FDC-P1 cells. We propose that phosphorylation of the p68 molecule is a physiochemical marker for the activation of PK-C in myeloid cells, in response to the growth-promoting physiological ligand.     

In situ hybridization histochemistry localization of interleukin-3 mRNA in mouse brain

The hematopoietic growth factor interleukin-3 (IL-3) promotes the proliferation and maturation of pluripotent myeloid progenitor cells. In the immune system, IL-3 is synthesized by mitogen or antigen- stimulated T lymphocytes. We demonstrate the expression of IL-3 mRNA in mouse brain by in situ hybridization histochemistry and Northern blot analysis. The IL-3 mRNA is localized in discrete areas of the brain and can be found in neuronal cell body and astrocytes. Northern analysis of cerebellar RNA, compared with mRNA extracted from WEHI-3 cells, showed a single hybridization band, approximately 1.2 kb, suggesting similar processing between brain and myeloid cells. The molecular evidence and previous observations of IL-3-like biologic activity found in the brain suggest a potential role for IL-3 in the neurobiology of the CNS.     

CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa

Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen. We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa. Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies. On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media. Populations of approximately 100,000 cells with greater than or equal to 95% viability and containing 70% to 90% megakaryocytes were obtained reliably for study. After the radioactive pulse, the cell suspension was solubilized with nonionic detergent. To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose. The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1-1D-Sepharose) or on a control monoclonal antibody resin. Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. Autoradiograms of diethylamine eluates from HP1-1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions. Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1-1D-Sepharose. Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material. This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.     

Idiotype as a tumor-specific marker in childhood B cell acute lymphoblastic leukemia

Immunoglobulin (Ig) or idiotype (Id) is a tumor-specific target in those B cell malignancies that express this molecule on their surface. We explored the biology of B cell acute lymphoblastic leukemia (B cell ALL) using Id as a tumor marker. In this report we describe the development of anti-Id monoclonal antibodies (MAB) for two children with B cell ALL. These reagents were used retrospectively to study tumor kinetics and to detect residual disease after chemotherapy. In both cases serum Id values were strikingly high at diagnosis (1.2 mg/mL and 10.8 mg/mL), suggesting that the tumor cells were relatively mature B cells capable of significant antibody production. In both patients the serum Id levels fell with the institution of therapy and confirmed that the patients were in remission. Increasing serum Id predicted relapse four months before conventional methods in patient 1, and Id proved to be a more sensitive measure of tumor burden than Southern blot analysis of rearranged Ig genes in bone marrow samples. Surprisingly, low levels of Id were redetected in the second patient just before completing therapy and have persisted for over a year despite the absence of clinical evidence of recurrent disease. Thus, serum Id levels reflect tumor burden during initial therapy but may not necessarily predict tumor progression after a complete clinical remission.     

Involvement of PKCα and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of μ-opioid receptors in mature brain neurons

Background and purpose:

The ability of an agonist to induce desensitization of the µ-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist).

Experimental approach:

MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)α knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs).

Key results:

Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCα, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCα knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM.

Conclusions and implications:

In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCα-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.     

Bisphosphonates and atrial fibrillation: Bayesian meta-analyses of randomized controlled trials and observational studies

Background  

Occurrence of atrial fibrillation (AF) amongst bisphosphonate users has been increasingly reported but results are conflicting. We performed a Bayesian meta-analysis to address the possible association between the occurrence of AF and bisphosphonate use and estimated the posterior probability of development of AF with bisphosphonate use.     

Viscoelastic properties of bovine articular cartilage attached to subchondral bone at high frequencies

Background  

Articular cartilage is a viscoelastic material, but its exact behaviour under the full range of physiological loading frequencies is unknown. The objective of this study was to measure the viscoelastic properties of bovine articular cartilage at loading frequencies of up to 92 Hz.