ALBERT

Description: Cases of acute leukemia showing both terminal deoxynucleotidyl transferase (TdT) and myeloperoxidase (MPO) activities are usually classified as acute myelogenous leukemia (AML). Although some reports have implied overlap of TdT and MPO based on population percentages, direct evidence for simultaneous expression of TdT and MPO by a leukemic blast is lacking. By use of a simple new technique developed in our laboratory for identifying TdT and MPO in individual cells by light microscopy, we examined three cases of acute leukemia with both TdT and MPO positivity and found that the incidence of cells positive for both TdT and MPO was 0%, 1%, and 23%. Cytogenetic analysis showed a single leukemic clone in all patients, providing additional evidence that these leukemias arose from a single cell capable of expressing both MPO and TdT. These findings have implications for understanding the relation between MPO and TdT expression in leukemia.

Description: Biosynthesis and molecular structure of major histocompatibility complex (MHC) class II antigens of DR2/DR7 hairy cells were analyzed by two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE). Two anti- human Ia monoclonal antibodies (mAb) were used to immunoprecipitate DR and DR-linked DC/DS molecules. Monoclonal antibody VI 15 C recognizes DR (I-E-like) molecules and CA 2.06 precipitates DR and DR-linked DC/DS (I-A-like) molecules in DR7 allotypes. Studies were performed on a pure population of hairy cells before and after culture with phorbol ester: 12-O-tetradecanoyl phorbol-13-acetate (TPA), 5 azacytidine (5 Aza), sodium butyrate (NA-BU), and phytohemagglutinin (PHA-P). Before any treatment, hairy cells expressed and synthesized DR antigens: DR alpha and beta subunits appeared both qualitatively and quantitatively normal by 2D-PAGE profile. In contrast, the hairy cells failed to express and synthesized any DC/DS molecule. The lack of DC/DS molecular expression was restored after culture in presence of TPA, sodium butyrate, and 5 azacytidine, but not after PHA-P treatment. Differential molecular expression of MHC class II antigens in leukemic cells provides a model to define further discrete stages of hemopoietic differentiation and study the role of these molecules in the cellular interactions occurring during differentiation.

Description: The activity of delta-aminolevulinic acid (ALA) dehydratase, an enzyme involved in heme biosynthesis, has been shown to increase in Friend virus-transformed murine erythroleukemia (MEL) cells during erythroid differentiation. In this study, the nature of the increase in ALA dehydratase activity in MEL cells was examined using a monospecific antibody directed to the enzyme. A sevenfold increase in ALA dehydratase activity was observed after cells had been treated with 1.5% Me2SO for 5 days. Ouchterlony double immunodiffusion analysis showed that lysates from untreated and Me2SO-treated MEL cells formed a single precipitin line with rabbit IgG directed to the normal mouse liver ALA dehydratase. A single arc of identity was also observed with the lysates from normal mouse erythrocytes, spleen, liver, and lysates from both uninduced and induced MEL cells. Rocket immunoelectrophoresis demonstrated that lysates from both uninduced and induced cells formed rockets with the IgG and that the peak height of the rocket was proportional to the ALA dehydratase activity applied. The slope of linear plots of rocket peak heights v ALA dehydratase activity was identical for lysates from uninduced and Me2SO-induced cells. Succinylacetone, a potent inhibitor of ALA dehydratase, was shown to markedly inhibit the activity of the enzyme, but did not interfere with the synthesis of ALA dehydratase induced by Me2SO treatment. Me2SO- induced increases in ALA dehydratase activity and the enzyme protein were both blocked by the simultaneous treatment of cells with 5-bromo- 2′-deoxyuridine (BrdU). BrdU-mediated repression of ALA dehydratase was partially overcome by treating the cells with thymidine. These data demonstrate that increased ALA dehydratase activity in MEL cells undergoing erythroid differentiation after Me2SO treatment is due to de novo synthesis of the same enzyme protein present in uninduced MEL cells as well as in normal erythrocytes. This represents the first direct demonstration of an increase in a heme biosynthetic pathway enzyme protein in erythroid cells undergoing differentiation.

Description: Hebbel and colleagues have proposed that increased adherence of sickle red cells to vascular endothelium may initiate vasoocclusive events in sickle cell disease. We have developed a micropipette technique to obtain direct, quantitative measure of the adherence of individual red cells to vascular endothelial cells. Using this technique, we found that the vast majority of sickle cells suspended in autologous plasma were strongly adherent to endothelial cells, whereas only a small fraction of normal cells were weakly adherent. Influence of plasma factors on adherence was determined by measuring adherence of sickle cells suspended in normal plasma and normal cells suspended in sickle plasma. Although over 90% of sickle cells adhered to endothelial cells in autologous plasma, the percentage of adherent cells decreased dramatically to less than 20% when the same sickle cells were suspended in normal plasma. In contrast, adhesion of normal red cells suspended in sickle plasma was only modestly increased compared to adhesion in autologous normal plasma. Our results provide direct evidence for markedly enhanced adherence of sickle cells to endothelial cells. In addition, they suggest that both cell membrane changes and plasma factors contribute to this interaction. The requirement for sickle plasma further implies that temporal changes in plasma factors may play an important role in determining the onset of vasoocclusive crisis.

Description: The effect of splenectomy on the response to random donor platelet transfusion in 15 multitransfused thrombocytopenic patients is presented. Eight patients responded poorly, with low corrected platelet count increments at 1 and 24 hours posttransfusion. These eight patients were clinically alloimmunized and had lymphocytotoxic antibody ( LCTAb ) in their sera. They responded well to closely HLA-matched transfusions. In contrast, seven splenectomized patients responded well to random donor platelets. Five of these patients had no LCTAb and no other evidence of immunization. Two patients who responded well to random donor platelets had “weak” LCTAb , and one responded to platelets presplenectomy in the presence of this antibody. Splenectomy does not improve the response to random donor platelets in alloimmunized recipients.

Description: Prenatal diagnosis of hematologic diseases can now be performed with fetal blood, fetal amniotic fluid cell DNA, and fetal chorionic villi DNA. Some hemoglobinopathies can be detected by all three methods, and the choice will depend on the available obstetric and laboratory techniques, as well as the time of presentation of the pregnancy. Hopefully, further development of molecular probes and techniques will soon expand these options to all of the globin disorders. Detection of coagulation disorders in utero currently requires samples of pure fetal blood. Gene cloning is accomplished for some (factor IX and antithrombin III) and is underway for others (factor VIII), and further investigation is necessary to determine whether deficiencies in these gene products are due to gene deletion or to mutant genes linked to polymorphic restriction enzyme sites of diagnostic use. Thus, molecular biology may be applied to prenatal diagnosis of the clotting problems, but this has not yet been accomplished. Disorders affecting the number and/or function of erythrocytes, leukocytes, and platelets can be diagnosed by analysis of fetal blood. Blood samples will continue to be required until more is known about the molecular biology of hematopoiesis. Syndromes that can be diagnosed by chromosome studies should be revealed in cultures of amniotic fluid cells, fetal blood lymphocytes, and chorionic villi cells. Cultured cells can be examined for karyotypes, Y-chromatin, spontaneous or induced chromosome breakage, DNA repair, SCEs, and translocations. The techniques for culturing amniotic cells and fetal blood white cells are established, and those for growing cells from chorionic villi are improving rapidly. Direct preparations of cells from villi only may suffice for some of the above analyses. The study of hematologic disease in utero has thus come full circle, from the use of amniotic cells to determine the sex in X-linked disorders, to fetal blood sampling for the analysis of gene products, then back to amniocentesis for DNA, and now earlier in gestation to chorionic villi. All of this has occurred in less than ten years, and it is anticipated that developments in the next ten years will be equally dramatic. The future should bring all prenatal testing into the first trimester, use molecular probes, and provide for both early diagnosis and early treatment of genetic hematologic disease.

Description: Methotrexate has been used as the mainstay therapy to prevent or ameliorate graft-versus-host disease (GVHD) in allogeneic bone marrow transplantation. We began a nonrandomized study in which methotrexate was not given routinely. Fifty-five patients underwent transplant for acute leukemia (44 patients), aplastic anemia (6 patients), and other malignancies (5 patients). Methotrexate was given to 34 patients (MTX +) and was withheld in 21 patients (MTX -). Median (range) age of patients was 12 (0.8–43) years in the MTX + group, and 16 (3–45) years in the MTX- group. Mean days (+/- SEM) to engraftment (neutrophils greater than 500/microL, and platelets greater than 20,000/microL untransfused) occurred earlier in the MTX- patients (19.6 +/- 1.4 v 24.9 +/- 1.8 days for granulocytes, and 19.3 +/- 1.5 v 27.4 +/- 2.8 days for platelets, P less than .05). There were no statistically significant differences between the patient groups for the incidence or severity of GVHD (10/34 in the MTX + group had grade O-l GVHD compared to 9/21 in the MTX- group). The interstitial pneumonitis occurred at a significantly increased rate in patients who received methotrexate (15/34) compared to those patients who did not (3/21) (P = .02). However, there was also a significant relationship between the interstitial pneumonitis and the preparative regimen: if the preparative regimen contained 1,000 rad single fraction total body irradiation, 8/14 patients were affected compared to 5/22 patients affected when 1,200 rad fractionated total body irradiation was used (P = .03). Because methotrexate significantly retards hematopoietic reconstitution, randomized trials for GVHD prevention are recommended.

Description: Acute graft-versus-host disease (GVHD), a major complication of allogeneic bone marrow transplantation (BMT), is probably mediated by T lymphocytes present in the marrow graft. In this study, the repopulation of the peripheral blood with T4+ and T8+ T cells was investigated during the period preceding the occurrence of acute GVHD. Twenty-four allogeneic and 11 autologous BMT recipients were monitored from day 4 post-BMT onward by the use of monoclonal antibodies, indirect immunofluorescence, and flow cytometry. The recipients of allogeneic transplants received methotrexate as GVHD prophylaxis. Similar recovery patterns for T4+ and T8+ T cells were found following autologous and allogeneic BMT. However, lymphoid repopulation occurred at a clearly faster rate after autologous BMT. T4+ T cells were the first to reappear in the peripheral blood, followed by T8+ T cells 4–7 days later. The T8+ T cell reconstitution occurred at an even faster rate in patients who were to develop grade II-IV GVHD, as compared with those with grade O-I GVHD, thus leading to an earlier decrease in the T4/T8 ratio. Of 10 patients with a T4/T8 ratio less than 2.5 at day 19, 9 developed grade II-IV GVHD and 1 showed no GVHD. Of 14 patients with a ratio greater than 2.5 at that time, only 2 developed grade II-IV and 12 grade O-I GVHD (p less than 0.001). In the 11 patients developing grade II-IV GVHD, the T4/T8 ratio decreased to values less than 2.5 before the first clinical symptoms of GVHD in 9; it coincided in one and occurred later in another patient. Thus, early monitoring of the T4/T8 ratio can distinguish patients at risk of developing grade II-IV GVHD.

Description: We have studied the role of factor VIII-von Willebrand factor (FVIII- vWF) in both platelet adherence to subendothelium and ristocetin- induced platelet aggregation using monoclonal antibodies to human FVIII- vWF. Twenty-five monoclonal antibodies were obtained, two of which were directed to the factor VIII moiety of FVIII-vWF; one of these two completely inhibited the procoagulant activity (FVIII:C). The remaining 23 monoclonal antibodies were directed to the von Willebrand factor moiety of FVIII-vWF. The ability of the latter monoclonal antibodies to inhibit platelet adherence to arterial subendothelium was investigated with a perfusion model. According to the number of platelets adhering to the subendothelium, three groups of monoclonal antibodies could be discerned: (A) antibodies not affecting platelet adherence; (B) antibodies that inhibited platelet adherence to the level as observed when von Willebrand's disease plasma was tested; and (C) antibodies that completely inhibited both platelet adherence to subendothelium and ristocetin-induced platelet aggregation. The two antibodies present in group C competed for the same or closely related epitope(s) present on FVIII-vWF. These results demonstrate that a domain is present on the FVIII-vWF molecule that is associated both with ristocetin-induced aggregation and with the ability of FVIII-vWF to support platelet adherence to the subendothelium. Based on these observations, it is concluded that ristocetin-induced binding of FVIII-vWF to platelets reflects, at least in part, a physiologic mechanism regulating the function of FVIII-vWF in primary hemostasis.

Description: We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O- thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O- thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5′ flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta- mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.