Heterogeneity of human red cell autoantibodies assessed by isoelectric focusing

Human red cell (RBC) autoantibodies may be the products of a single lymphocyte clone or of a restricted number of clones. For insight into the clonal distribution of human RBC autoantibodies, serum fractions from 28 individuals with various forms of autoimmune hemolytic anemia (AHA) and two nonanemic individuals with positive direct antiglobulin tests were separated by isoelectric focusing (IEF), and RBC binding in each fraction was quantitated with a solid-phase radioimmunoassay. IEF fractions of serum from normal volunteers and patients with nonimmune hemolytic anemia served as controls. These studies indicate that RBC antibodies are found in a restricted number of IEF fractions in sera from some patients with immune hemolytic anemia. IEF fractions containing RBC-binding activity vary among patients with idiopathic AHA, and distinct patterns of binding activity are found in serum from some patients with AHA associated with alphamethyldopa and procainamide or with B-cell immunoproliferative diseases. These findings suggest that the mechanism leading to autoantibody production may differ among patients with the various forms of immune hemolytic anemia.     

Linkage of a familial platelet disorder with a propensity to develop myeloid malignancies to human chromosome 21q22.1-22.2

Linkage analysis was performed on a large pedigree with an autosomal dominant platelet disorder and a striking propensity in affected family members to develop hematologic malignancy, predominantly acute myelogenous leukemia. We report the linkage of the autosomal dominant platelet disorder to markers on chromosome 21q22. Four genetic markers completely cosegregate with the trait and yield maximum logarithm of difference scores ranging from 4.9 to 10.5 (theta = .001). Two flanking markers, D21S1265 and D21S167, define a critical region for the disease locus of 15.2 centimorgan. Further analysis of this locus may identify a gene product that affects platelet production and function and contributes to the molecular evolution of hematologic malignancy.     

High-molecular-weight kininogen is exclusively membrane bound on endothelial cells to influence activation of vascular endothelium

An important biologic function of high-molecular-weight kininogen (HK) is to deliver bradykinin (BK) to its cellular receptors. Internalization and degradation of HK may provide a mechanism by which endothelial cells modulate the production of BK and control its activities. Therefore, we investigated the binding and subsequent distribution of biotinylated-HK (biotin-HK) associated with human umbilical vein endothelial cells (HUVEC). HUVEC bound 3 to 4 times more HK and with greater avidity at 1 to 3 hours at 37 degrees C than at 4 degrees C (Bmax = 1.0 +/- 0.02 x 10(7) molecules/cell, kd = 7 +/- 3 nmol/L v Bmax = 2.6 +/- 0.2 x 10(6) molecules/cell, kd = 46 +/- 8 nmol/L). However, there was no evidence that the difference was caused by internalization of HK at the higher temperature. First, the same amount of biotin-HK was associated with nonpermeabilized and permeabilized HUVEC using buffers containing 20 to 50 mumol/L zinc ion in the absence or presence of 2 mmol/L calcium ion. Second, binding of biotin-HK to HUVEC was approximately 92% reversible at 1 hour when the cells were maintained at both 37 degrees C and 4 degrees C. Third, neither chloroquine nor primaquine altered the amount of biotin-HK bound to HUVEC. Fourth, biotin-HK bound to HUVEC was almost completely removed by pronase. Fifth, the nonpermeable dye, crystal violet, almost completely quenched the fluorescence signal emitted by HUVEC-associated fluorescein isothiocyanate (FITC) HK. Finally, the localization of HUVEC-bound FITC-HK was restricted to the membrane as shown by laser scanning confocal microscopy. The expression of HK binding sites had an absolute requirement for metabolic energy, but was not dependent on new protein synthesis. Membrane-bound HK contributed to the anticoagulant nature of endothelial cells by blocking human alpha-thrombin binding and its resultant induction of prostacyclin formation. These studies indicate that HK is not internalized by HUVEC, but remains primarily on cell surfaces to be accessible for BK liberation and to modulate the binding and actions of alpha-thrombin.     

Analysis of optimal conditions for retroviral-mediated transduction of primitive human hematopoietic cells

We sought to define optimal conditions for retroviral-mediated transduction of long-lived human hematopoietic progenitors from bone marrow and peripheral blood. CD34+ cells were transduced by the LN and G2 retroviral vectors in the presence or absence of stromal support and with or without cytokine addition. After transduction, a portion of the cells was plated in methylcellulose colony-forming assay, with or without G418, to assess the extent of gene transfer into committed progenitors. The remaining cells from each experiment were transplanted into immunodeficient mice to allow analysis of transduction of long- lived progenitors. Human colony-forming cells contained within the murine bone marrow were analyzed after engraftment periods of 2 to 11 months. Cells were plated in a human-specific colony-forming assay with and without G418 to assess the extent of transduction of primitive progenitors. Individual human colonies were also analyzed by polymerase chain reaction for the presence of provirus. Bone marrow progenitors were efficiently transduced only when stroma was present, whereas mobilized peripheral blood progenitors were effectively transduced in the presence of either stroma or cytokines. Inclusion of the cytokines interleukin-3, interleukin-6, and stem cell factor did not further augment the extent of gene transfer in the presence of a stromal support layer. Additionally, human CD34+ progenitors from bone marrow or mobilized peripheral blood that had been transduced for 3 days in the absence of stroma failed to produce sustained, long-term engraftment of bnx mice. Mice transplanted with the same pools of human progenitors that had been transduced in the presence of stroma for 3 days had significant levels of human cell engraftment at the same timepoints, 7 to 11 months after transplantation. Our data show loss of long-lived human progenitors during 3-day in vitro transduction periods in the absence of stromal support. Therefore, the presence of bone marrow stroma has dual benefits in that it increases gene transfer efficiency and is essential for survival of long-lived human hematopoietic progenitors.     

Chondromalacia patellae: assessment with MR imaging

Magnetic resonance (MR) images of the posterior patellar hyaline articular cartilage were obtained in 23 subjects to determine if MR imaging could accurately demonstrate the patellar cartilage. Arthroscopy was used as the standard of reference. Three subjects were asymptomatic volunteers. In the remaining 20 who had patellofemoral pain, arthroscopy was performed before MR imaging in seven and afterward in 12; one did not undergo arthroscopy. MR imaging showed focal areas of swelling of the patellar cartilage, focal hypointensity, surface irregularity, areas of thinning, and areas of cartilage loss with exposure of subchondral bone. The surgical findings agreed with those from MR images in all seven patients who underwent arthroscopy before MR imaging and in ten of the 12 who underwent surgery afterward. MR imaging is an accurate means of examining the posterior patellar cartilage and should be considered as an alternative to diagnostic arthroscopy when chondromalacia patellae is suspected.     

Assessment of testicular metabolic integrity with P-31 MR spectroscopy

To evaluate the reliability of phosphorus-31 magnetic resonance (MR) spectroscopy in the assessment of acute testicular ischemia, vascular integrity, and spermatogenesis, the authors studied in vivo canine and primate testicles grouped as follows: group 1 testes (n = 8), in situ canine controls; group 2 (n = 11), canine testes subjected to warm ischemia; group 3, canine (n = 4) and primate (n = 4) testicles from hormone-treated animals. Group 1 control testicles showed high monophosphoester (MP) levels; low levels of inorganic phosphate (Pi), phosphodiester (PD), and phosphocreatine; and high levels of adenosine triphosphate (ATP). Group 2 testes revealed a time-dependent decay of MP/Pi ratios (from 2.1 to 0.70). Regeneration of ATP was noted in the acute reperfusion period. After 6 weeks of pituitary gonadotropin suppression, group 3 testes showed a significant decrease (P less than .05) in MP/PD ratios from a control level of 2.6 +/- 0.3 and a decrease in the MP/beta-ATP ratio from 2.4 +/- 0.1 to 1.8 +/- 0.3. P-31 MR spectroscopy appears to be a potential method for noninvasively assessing testicular ischemic injury and the metabolic integrity of spermatogenesis.