Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice [see comments]

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that "niches" need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.     

Binding of heparin‐dependent antibodies to PF4 modified by enoxaparin oligosaccharides: evaluation by surface plasmon resonance and serotonin release assay

Summary. Background: The minimal structural requirements of low‐molecular‐weight heparins that determine the risk of developing heparin‐induced thrombocytopenia (HIT) are not fully defined.Objectives: The ability of enoxaparin‐derived oligosaccharides (OS) to induce platelet activation and exposure of platelet‐factor 4 (PF4) epitopes recognized by antibodies developed in HIT was studied by surface plasmon resonance (SPR) and serotonin release assay.Results: Decasaccharides with ≥ 11 sulfate groups induced platelet activation in the presence of plasma from patients with confirmed HIT. Serotonin release of > 80% without full inhibition at 100 μg mL^?1 was achieved with decasaccharides containing 14 or 15 sulfate groups, 2 dodecasaccharides and 2 tetradecasaccharides. An SPR method was developed using purified PF4 immobilized on carboxymethylated dextran. Antibodies from all HIT samples bound to PF4/heparin in SPR assays with resonance units (RU) ratio of 109–173 with HIT plasma vs. 88–93 with control plasma. RU ratios > 100 were measured when PF4 was pre‐incubated with OS with ≥ 10 saccharide units and one octasaccharide containing 10 sulfate groups. RU ratios > 140, similar to those measured when PF4 was pre‐incubated with unfractionated heparin or enoxaparin, were obtained with purified dodeca‐ and tetradecasaccharides. RU values strongly correlated with the number of sulfate groups in the decasaccharides tested (r = 0.93, P = 0.02).Conclusions: LMWHs with fragments > 10 saccharides and a large number of sulfate groups are more likely to be associated with a higher risk of HIT. These structure‐activity relationships were independent of the ability of the OS to bind antithrombin.     

Membrane abnormality in red blood cells with weak type B expression

The mechanisms of unusually weak blood group (A and B) expressions are not yet well understood. We examined properties of blood group galactosyltransferase (B-enzyme) and characteristics of red cell membrane components obtained from family members with A2Bm character. B- enzyme activity of the A1Bm plasma is in normal range, and kinetic properties (i.e., Km for UDP-Gal, Km for 2'-fucosyllactose, and pH optima) of B-enzyme from the A1Bm subjects are identical to that of normal B-enzyme. When A1Bm red cell were incubated with UDP-Gal and B- enzyme, the cells became strongly agglutinable with anti-B. When A1Bm membranes were incubated with B-enzyme or A-enzyme (i.e., blood group N- acetylgalactosaminyltransferase) and the appropriately labeled nucleotide sugar (UDP-Gal3H for B-enzyme and UDP-GalNAc3H for A- enzyme), significant incorporation of the sugar was observed. The amounts of the sugar incorporated into A1Bm membranes were about 40%- 50% of that incorporated into O membranes at saturation, indicating that about one-half of H-sites remained unglycosylated in A1Bm red cells. Examination of radioactive components by isoelectric focussing revealed that the labeled components of A1Bm membranes were distinctively different from that of O membranes. Therefore, one can conclude that the weak B expression is not due to direct mutation of ABO locus, but due to a secondary consequence of genetic abnormality of a membrane component (or components) associated with blood group substances.     

Early changes in phosphatidylcholine metabolism in human acute promyelocytic leukemia cells stimulated to differentiate by phorbol ester

The HL-60 leukemia cell line derived from a human acute promyelocytic leukemia is stimulated to differentiate into macrophages within 24-28 hr after exposure to the phorbol ester, 12-O-tetradecanoylphorbol-13- acetate (TPA). We studied early alterations (within 90 min of exposure to TPA) in phosphatidylcholine metabolism in HL-60 cells and found that phosphatidylcholine synthesis by methylation is phosphatidylethanolamine was inhibited in a dose-dependent fashion. In contrast, synthesis of phosphatidylcholine from endogenous choline was enhanced and correlated inversely with the degree of inhibition of the methylation pathway. Phorbol ester congeners of TPA caused similar alterations in phosphatidylcholine metabolism in direct relationship to their capacity to induce differentiation in HL-60 cells. Perturbation of phosphatidylcholine metabolism is an early membrane even in TPA- induced HL-60 cell differentiation.     

Monoclonal antibodies recognizing epitopes on the extracellular face and intracellular N-terminus of the human erythrocyte anion transporter (band 3) and their application to the analysis of South East Asian ovalocytes

This report describes the production and characterization of 13 rodent monoclonal antibodies to the human erythrocyte anion transport protein AE1 (syn. band 3). Eleven antibodies (4 murine and 7 rat) recognize epitopes dependent on the integrity of the third extracellular loop of the protein. Two antibodies (1 murine and 1 rat) recognize epitopes on the N-terminal cytoplasmic domain. Quantitative binding studies using radioiodinated IgG and Fab fragments of antibodies to extracellular epitopes on AE1 ranged from 77,000 to 313,000 (IgG) and from 241,000 to 772,000 (Fab) molecules bound at saturation. The results indicate that the epitopes recognized by different antibodies vary in their accessibility and suggest that there is heterogeneity in the organization of individual AE1 molecules in the red blood cell membrane. Quantitative binding studies on South East Asian ovalocytes using several antibodies to AE1 and an anti-Wrb show a marked reduction in the number of antibody molecules bound at saturation. These results are consistent with the existence of highly cooperative interactions between transmembrane domains of AE1 in normal erythrocytes and the disruption of these interactions in the variant AE1 found in South East Asian ovalocytes.     

Long-acting propranolol in the prophylaxis of migraine: a comparative study of two doses

A randomized double-blind, cross-over study using treatment periods of 12 weeks with a 2-week washout, comparing two long-acting formulations of propranolol ('Inderal' LA 160 mg daily and Half-'Inderal' LA 80 mg daily) was performed after a placebo run-in of 4 weeks on 51 patients. The study indicated that both long-acting formulations were significantly better than placebo in reducing the frequency of migraine attacks (p less than 0.01). After 12 weeks there was a significantly lower (p = 0.03) frequency of migraine attacks in patients on the higher dose formulation than in those on the lower dose formulation. There was no significant difference in the frequency of side effects produced by the two formulations.