ALBERT

Description: Of eight patients with progressive hairy cell leukemia who were treated with daily doses of an alkylating agent (chlorambucil, 4 mg) for at least 6 mo, seven have had an objective response, as measured by improved blood counts. Two patients were pancytopenic and had almost total bone marrow replacement with hairy cells at the initiation of chemotherapy; approximatley 6 mo later, their blood counts and bone marrow had improved dramatically. The five other patients had the leukemic form of the disease, and all responded to therapy. It is important to identify postsplenectomy patients with progressive disease in order to initiate low-dose single-agent chemotherapy.

Description: Since studies of the giant platelets in the Bernard-Soulier syndrome have shown decreased electrophoretic mobility, decreased sialic acid, and an abnormality in a membrane glycoprotein, we performed similar studies on the giant platelets from two patients with the May-Hegglin anomaly. The patients' platelet electrophoretic mobilities did not differ from control. Although the total sialic acid contents of the patients' platelets were greater than control when calculated per platelet, they were very similar to control when normalized for differences in platelet volume and surface area. When platelet proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis there were no differences between the glycoproteins of control and patient platelets as judged by the patterns of periodic acid Schiff staining and fluorescein-labeled concanavalin A binding. Similarly, patterns of surface glycoprotein labeling by neuraminidase/galactose oxidase/KB3H4 were identical. We conclude that unlike the giant platelets in the Bernard-Soulier syndrome, those of the May-Hegglin anomaly are not associated with a membrane abnormality detectable by these techniques.

Description: Biochemical and morphological evidence is presented to support the characterization of a rat bone-marrow-derived cell line (RPM) as an analog of the promegakaryoblast. The conditions for in vitro growth and maturation of the RPM line are described. Rapid proliferation of the RPM line is readily achieved when cultures are supplemented with moderate levels of fetal bovine serum (FBS). The proliferative compartment is a small blast-like cell. Immunofluorescent staining demonstrates that the RPM cells contain factor VIII:antigen and fibrinogen in their cytoplasm. The cells secrete, into their conditioned medium, a potent mitogenic activity for rat aortic smooth muscle cells. When incubated under conditions of relative serum deprivation, the cells stop proliferating and undergo a process of maturation. The sequence of maturation is described as stage I (promegakaryoblast--the proliferative compartment); stage II (immature megakaryocyte or promegakaryocyte); stage III (mature megakaryocyte). The stage III cells release, from their cytoplasm, small membrane-bound vesicular bodies containing lavender granules and cytoplasmic organelles. These have been designated stage IV. The RPM line may provide a useful model for the in vitro study of megakaryocyte maturation and the synthesis of megakaryocyte-specific proteins.

Description: Adenylate cyclase (AC) and guanylate cyclase (GC) activities were studied in normal B-enriched and T-enriched lymphocytes, in lymphocytes of children with acute lymphocytic leukemia (ALL), and in lymphocytes of adults with chronic lymphocytic leukemia (CLL). AC activity was greater in normal B than T lymphocytes (215 pmole/min/mg protein versus 80 pmole in the membrane-enriched fraction) and i both increased greatly after stimulation with isoproterenol and more so with prostaglandins E and F2 alpha. In leukemic lymphocytes, AC showed depressed activity (20 pmole in ALL cells and 55 pmole in CLL cells) and was less sensitive to hormonal stimulation: this loss of sensitivity occurred to a greater extent in ALL than in CLL lymphocytes. GC activity was greater in normal T than B cells (in membrane-enriched fraction: 10.2 pmole versus 5.3 pmole). It increased little with isoproterenol and prostaglandins stimulation, and much more with sodium azide and dehydroascorbic acid stimulation. GC activity was increased in both types of leukemic lymphocytes (23 pmole for ALL cells and 18 pmole for CLL cells) and was insensitive to stimulation. Possible derangement of cyclase and cyclic nucleotide regulation in leukemic cells is suggested.

Description: A glycoprotein antigen has been isolated from cured tobacco leaves (TGP- L) Nicotiana tabacum) and from cigarette smoke condensate (TGP-CSC) to which approximately one-third of human volunteers, smokers and non- smokers, exhibit immediate cutaneous hypersensitivity. TGP-L and TGP- CSC contain polyphenol haptens that activate the factor XII (Hageman factor) dependent pathways of coagulation, fibrinolysis, and kinin generation in normal human plasma. The purpose of this communication is to describe the isolation antigens from cocoa powder (Theobroma cacao), ground coffee (Coffea arabica), and ragweed (Ambrosia eliator) pollen that are immunologically cross-reactive with TGP-L and TGP-CSC, contain similar polyphenol haptens, and are capable of activating factor-XII- dependent pathways in normal human plasma.

Description: To study whether the culture time at which the burst populations mature influences the expression of fetal hemoglobin in bursts, we measured hemoglobin synthesis in cohorts of fully hemoglobinized erythroid bursts maturing sequentially in cultures of adult peripheral blood BFU- Es. In 13 of 15 experiments, a decline in gamma/gamma + beta ratio was noted as the culture time advanced. On the average, erythroid bursts that mature during the third culture week showed lower levels of fetal Hb synthesis compared to bursts that are already mature in the second culture week. The decline of gamma/gamma + beta ratio with culture time was also noted in erythroid bursts composed of immature erythroblasts. The enhanced HbF formation in peripheral blood BFU-E cultures is thus most pronounced among the bursts that become hemoglobinized early, and there is a tendency for normalization of HbF synthesis in bursts that mature in late culture days. These results can be interpreted by several alternatives, including the possibility that the expression of high HbF levels in the early days of adult BFU-E cultures is a reflection of premature commitment to terminal differentiation of progenitors that possess an active HbF program. The present data indicate that the variation of HbF synthesis with culture time should be taken into consideration when the influence of various culture conditions of HbF synthesis is studied in BFU-E cultures.

Description: Acetylcholinesterase (AchE), an essentially specific and early marker of rodent thrombocytic series, in several tissues acts both as a constituent of the cellular membrane and as a secretory enzyme. This study presents the ultrastructural transport and fate of this substance in the megakaryocytes of mouse bone marrow, using Tranum-Jensen and Behnke's adaptation of the indirect thiocholine method. It is shown that megakaryoblasts and megakaryocytes undergo a typical secretory cycle consisting of (1) enzyme synthesis and segregation on the endoplasmic reticulum and nuclear envelope, (2) enzyme concentration in AchE-vesicles and AchE-granules formed from the cisternae of the Golgi apparatus, and (3) discharge in the demarcation membrane system and extracellular space. The AchE-vesicles and granules appear to be hitherto unrecognized megakaryocytic organelles as they do not resemble alpha nor the dense granules, and their mode of formation and fate differ from those of primary lysosomes and peroxidase granules. Released platelets reveal AchE activity in the open canalicular system. The data are compatible with the hypothesis that by controlling acetylcholine concentration in hematopoietic tissues, the secretion of AchE by megakaryocytes can modulate the proliferative activity of megakaryocytes progenitors.

Description: Using either hexazotized pararosaniline or new fuchsin as coupling agents, we investigated the ultrastructural localization of alpha- naphthyl acetate esterase (ANAE) activity in guinea pig bone marrow and peritoneal exudates, and in human peripheral blood cells. DFP- inhibitable ANAE activity was present on the cell surface of lymphocytes, monocytes, macrophages, neutrophils, eosinophils, basophils, megakaryocytes, platelets, and blasts. Demarcation lines in megakaryocytes and the perinuclear cisternae in normoblasts were also positive. In addition, lymphocytes, monocytes, and macrophages displayed ANAE activity associated with cytoplasmic-vesicle clusters (CVC). Reaction product was always present on the cytoplasmic surfaces of these vesicles and in the adjacent cytoplasm; vesicle interiors were invariably ANAE-negative. Small lymphocytes generally had a single large paranuclear ANAE-positive CVC, whereas mononuclear phagocytes had multiple discrete foci of similar appearing ANAE-positive CVC that sometime became confluent. ANAE activity was also found in the Gall bodies of human lymphocytes and in coated vesicles of macrophages. Cytoplasmic ANAE activity was increased in oil-induced guinea pig peritoneal macrophages. Both surface and cytoplasmic esterase activities had a neutral pH optimum. An identical distribution of reaction product was observed when alpha-naphthyl butyrate was employed as substrate. The function of these esterases, and their relation to known surface and cytoplasmic neutral proteases, awaits further investigation.

Description: Severe pulmonary reactions have been reported in patients receiving leukocyte transfusion and amphotericin-B. To study the interaction of amphotericin-B with polymorphonuclear leukocytes (PMN), purified human PMN were incubated with 200 mg of nylon wool fiber for 60 min either in the absence or presence of 2 mM EDTA. PMN were recovered in acid citrate dextrose solution and were suspended in balanced salt solution for determination of their aggregation properties. The cells exposed to nylon wool fibers without EDTA aggregated in response to concentration as low as 1.25 micrograms/ml of amphotericin-B. Cells initially treated with EDTA, however, failed to aggregate. Serum from a patient treated with amphotericin-B aggregated PMN exposed to nylon wool fiber but not control cells, whereas serum taken before amphotericin was given without effect on the PMN treated with nylon wool fiber. Amphotericin-B at 5 micrograms/ml failed to potentiate the release of beta- glucocuronidase or lactic dehydrogenase by PMN treated by nylon wool beyond that seen with exposure to the fibers alone. Rabbit peripheral blood was similarly incubated with nylon wool fibers and the recovered PMN were infused into recipient rabbits that had received 1 mg/kg of amphotericin-B intravenously 1 hr prior to the infusion of the leukocytes. Rabbits were sacrificed 30 min after transfusion of PMN, and their lungs were excised for histologic sectioning. Those rabbits receiving a combination of amphotericin-B and 4 x 10(7) nylon-wool- fiber-treated PMN had evidence of pulmonary hemorrhage and accumulation of leukocytes in the pulmonary vasculature whereas those animals who received such cells alone had normal appearing lung tissue. In summary, amphotericin-B at concentrations achievable in vivo enhanced the aggregation of PMN damaged by incubation with nylon fiber with subsequent accumulation of the phagocytes in pulmonary tissue.

Description: Two fractions of human prothrombin can be isolated from single donor plasma by the technique of heparin-agarose chromatography in (sodium) citrate buffer, pH 7.5, as previously reported for pooled plasma. The two fractions, designated H-II1 and H-II2, are found in a ratio of approximately 4:1. Both forms comigrate in sodium dodecyl sulfate gel electrophoresis; however, under nondenaturing electrophoretic conditions, each fraction migrates as a discrete entity with a different mobility. The larger fraction (H-II1) has a faster mobility towards the anode. Isoelectric focusing in urea of H-II1 reveals that it has two components, a minor component with a pl of 5.25 (H-II1a) and a major component with a pl of 5.40 (H-II1b). H-II2 has a pl of 5.6 H- II1 and H-II2 possess the same amino terminal residue (alanine, 0.87- 0.92 mole/mole) and the same number of gamma -carboxyglutamic acid residues (9.8–10.5). Their amino acid composition is indistinguishable. However, the two fractions of prothrombin differ in their content of neutral sugar and of sialic acid residues. Removal of sialic acid with neuraminidase abolishes the electrophoretic heterogeneity. Thus, the charge heterogneity of the three variants of prothrombin found in normal human plasma appears to result exclusively from differences in the number of sialic acid residues attached to the protein moiety of the molecule.

Description: Bleeding times were performed on 71 hemophiliacs using the Simplate II device. Eight patients receiving Indocin or Motrin for hemophilic arthropathy were evaluated separately from the remaining 63 who had a mean bleeding time of 7.65 +/- 3.20 min (1 SD) compared to the control group of 5.35 +/- 1.49 min (p less than 0.005). No difference was found when 26 mild hemophiliacs who had received less than 10,000 U of clotting factor concentrate the previous year and no infusions in at least 3 mo were compared with 28 severe hemophiliacs who had received greater than 20,000 U of clotting factor concentrate the previous year and had been infused within 1 mo of testing. Ten patients (16%) had bleeding times greater than 10 min. Bleeding times remained prolonged on repeat evaluations in 7 of these patients, 3 of whom had mild disease and all of whom had normal platelet aggregations in response to arachidonic acid. We conclude that the bleeding time is longer than normal in hemophilia. This abnormality is not related to disease severity, recent transfusions, or the use of nonsteroidal antiinflammatory drugs.

Description: Lymph node cells obtained from a case of non-Hodgkin's lymphoma, of follicular center cell histology, were seen by immunofluorescence to express immunoglobulin mu heavy chains but no detectable light chain on their surfaces. Antibody specific for determinants expressed only on uncombined mu-chain reacted with these cell surfaces, but not with normal or neoplastic cells expressing surface IgM. Analysis of radioiodinated surface material revealed mu-chains which, without reduction, migrated on SDS-gel electrophoresis as normal mu-chain monomers, and again failed to detect light chains. Lysates of cells incubated with 3H-leucine revealed the synthesis of mu-chains but not of light chains. The labeled mu-chains were not secreted into the culture supernatant, a finding confirmed in parallel cultures by radioimmunoassay. The findings are discussed with particular reference to current concepts of the “mu-chain only” phenotype.

Description: Microdensitometric measurement of the DNA content of individual megakaryocytes was performed using megakaryocyte colonies obtained following culture, in soft agar, of hematopoietic cells from C57BL/6J mice. Two types of colonies were detected. After 7 days of culture, the big cell type contained 16 /+- 2.3 acetylcholinesterase (AChE) positive cells/colony, with a mean ploidy level of 16.8 /+- 0.8/cell and the ploidy distribution characteristic of recognizable megakaryocytes in bone marrow. The heterogeneous type contained 44 /+- 9.6 cells/colony (some of which were AChE negative), with a mean ploidy level of 6.8 /+- 0.7/cell. The ploidy distribution of heterogeneous colonies differed markedly from big cell colonies, with preponderance of 2N and 4N cells. Colony-forming cells, obtained 4–5 days after induction of acute thrombocytopenia, gave big cell colonies with a marked increase in DNA content. Mean ploidy level increased to 21.5 /%- 1.8/cell; the frequency of 32N cells increased from 17% to 30% and 64N cells from 0% to 6%. This is the pattern of change observed in bone marrow, in vivo, 24 to 48 hr after induction of acute thrombocytopenia. The number of cells/colony did not increase. In contrast, acute thrombocytopenia did not alter the ploidy of heterogeneous colonies. The different responses to the stimulus of acute thrombocytopenia suggest that there are at least two types of Meg-CFC. The delayed appearance of altered Meg-CFC that produced big cell colonies indicates that the pool of stem cells, from which committed megakaryocyte precursors are derived, may respond indirectly to the stimulus of platelet depletion.

Description: The recent finding that retinoic acid induces terminal granulocytic differentiation of the human promyelocytic leukemia cell line, HL-60, prompted an investigation of the sensitivity to this inducer of human myelocytic leukemia cells in primary suspension culture. Of the 21 leukemic specimens, only cells from the two patients with acute promyelocytic leukemia differentiated in response to retinoic acid. After an incubation period of 5--7 days in 1 microM retinoic acid, the cells from these two patients showed extensive morphological and functional maturation. Thus, because it appears that retinoic acid specifically induces granulocytic differentiation of leukemic promyelocytes, this compound may have therapeutic utility in the treatment of acute promyelocytic leukemia.

Description: The human leukemia K562 cell line can be induced by 20 micro M hemin to reversibly accumulate embryonic and fetal hemoglobins without any change in the rate of cell division. When we reduced the rate of cell division by glutamine starvation or addition of hydroxyurea, the cells increased by tenfold the basal hemoglobin level of 0.3–0.5 pg Hb/cell. The combined effects of hemin and inhibitors of cell division permitted K562 cells to attain levels of hemoglobin (26–34 pg Hb/cell) close to that found in normal red cells. This superinduction was reversible and cells could be recycled indefinitely. Furthermore, electrofocusing experiments show that the three primary hemoglobin species produced by these cells (Hb Gower 1, Hb Portland, and fetal Hb), were induced, or reinduced, synchronously by inhibitors of cell division but asynchronously by hemin. Differing effects of hemin and inhibitors of cell division were observed in the absence of irreversible differentiation and suggest different molecular mechanisms controlling globin gene expression.

Description: Alloimmunization is the major complication of platelet transfusion therapy in patients with acute leukemia. To evaluate whether alloimmunization continues to be a long-term problem in patients surviving induction therapy, 114 patients with acute nonlymphocytic leukemia (ANLL) who survived more than 6 mo and who received multiple courses of chemotherapy and abundant platelet transfusions were studied. Clinical response to random donor platelets and lymphocytotoxic antibody (LCTAb) were measured pretreatment and serially throughout the study period. Fourteen patients (12%) were alloimmunized upon admission, 34 (30%) patients became alloimmunized during remission induction therapy, and 66 (58%) patients did not become alloimmunized during that period. Sixty-one of these 66 patients (92%) never became alloimmunized and responded to random donor platelets during their subsequent course despite the fact they received multiple further platelet transfusions, whereas the alloimmunized patients tended to remain alloimmunized for their entire clinical course. There was no difference in age or sex between groups, and prognostic factors predicting alloimmunization could not be detected. In greater than 90% of patients not alloimmunized at admission, the presence or absence of LCTAb after induction predicts later alloantibody production. This information can be used to plan the type of platelet transfusions (HLA-matched or random donor) needed for subsequent maintenance and induction therapy. It may also help to identify a group of patients to whom more aggressive maintenance chemotherapy may be more safely administered.

Description: Antibody-secreting hybridomas were produced by fusion of P3-NS1/1Ag--4- 1 mouse plasmacytoma cells with splenocytes from a mouse immunized with HL-60 human promyelocytic leukemia cells. A cloned hybridoma cell line was identified that secreted antibody against a cell surface antigen expressed on all normal human peripheral blood granulocytes, on bone marrow granulocytic precursor cells, and on blast cells from 3 of 15 patients with nonlymphoid leukemia. However, the antibody did not react with normal peripheral blood lymphocytes, monocytes, platelets, or red cells, or with blast cells from 20 patients with acute lymphoblastic leukemia or from 5 patients with lymphoma. This monoclonal antibody identified a granulopoietic differentiation antigen (designated My-1) and may prove useful in the subclassification on nonlymphoid leukemia and in the investigation of hematopoiesis.

Description: A system of 3H-thymidine incorporation by lymphocytes in culture for 3 wk has been utilized for quantitative assessment of the ability of T lymphocytes to inhibit outgrowth of autologous Epstein-Barr virus (EBV) transformed B lymphocytes. Lymphocytes from EBV-seronegative individuals lack the ability to suppress outgrowth of autologous EBV- transformed B lymphocytes. This capability appears during the course of primary EBV-induced infectious mononucleases (IM) as the atypical lymphocytosis is subsiding and persists for years after recovery from primary EBV infection. The ability of T lymphocytes from EBV- seropositive subjects or convalescent IM patients to inhibit B- lymphocyte outgrowth is not HLA restricted. Thus, T lymphocytes capable of inhibition of in vitro EBV-induced B-cell outgrowth emerge during the acute stage of IM and may represent an important control mechanism of EBV-induced B-lymphocyte proliferation in vivo. The system provides a highly sensitive quantitative means for in vitro assessment of cell- mediated immunity to EBV.

Description: Four patients with acute nonlymphocytic leukemia who were heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) were studied to determine the numbers and types of progenitor cells in which the disease arose. Both forms of enzyme were found in normal tissues, but the malignant blast cells showed only one G6PD, indicating that the disease was clonal at the time of testing. The observations that normal erythroid cells were present in two young patients at diagnosis and relapse indicate that the clone suppressed expression of normal granulopoiesis but did not prevent normal erythroid differentiation. In contrast to this situation, in two elderly patients, the disease involved stem cells multipotent for granulocytes, red cells, and platelets. These results indicate that acute nonlymphocytic leukemia is heterogeneous. In some patients, the disease is expressed in cells with differentiation restricted to the granulocyte-monocyte pathway; in others, it involves stem cells capable of differentiating to granulocytes-monocytes, platelets, and erythrocytes. This heterogeneity may reflect differences in causation and could have prognostic and therapeutic importance.

Description: Subjects with neutrophil myeloperoxidase (MPO) deficiency have been rarely reported. In part this may be due to the lack of a simple screening technique that would detect them. With the routine use of a cytochemical leukocyte differential counter that employs MPO stains, over a 40-mo a period 8 unrelated probands with partial MPO-deficiency and one with complete deficiency were identified. Family studies have identified 23 additional, partially deficient subjects. The largest pedigrees demonstrate that the carrier state or partial MPO deficiency is inherited in an autosomal dominant pattern. Leukocytes from a subject with complete MPO deficiency and from some subjects with partial deficiency have impaired bactericidal activity against S. aureus. Superoxide generation was increased and chemiluminescence decreased in MPO-deficient leukocytes. Eleven subjects were prospectively followed for 18–30 mo. Only two partially deficient subjects have had serious infections consisting of recurrent streptococcal cellulitis and aseptic meningitis. These data suggest that leukocyte MPO deficiency is a common inherited defect that results in minimal clinical problems, supporting the concept of multiple leukocyte bacterial killing systems.

Description: The behavior of rat transferrin has been investigated employing acrylamide gel electrophoresis and isoelectric focusing. In vitro trace labeling with iron chelates at 30 min was 93%-98% effective, whereas binding by simple ferric salts was reduced to 71%-76%. Complete and specific binding of 59FeSO4 by the iron binding sites of transferrin was demonstrated after in vitro or in vivo addition of ferrous ammonium sulfate in pH 2 saline up to the point of iron saturation. In vitro the radioriron transferrin complex in plasma was stable and its iron had a negligible exchange with other transferrin binding sites over several hours. The distribution of radioiron added in vitro or through absorption was shown to be random between the binding sites of slow and fast transferrin molecule. Iron distribution among body tissues was similar for mono- and diferric transferrin iron and was not affected by the site distribution of iron on the transferrin molecule. The only important aspect of transferrin iron binding was the more rapid tissue uptake of iron in the diferric form was compared to monoferric transferrin. Additional in vivo effects on internal iron exchange were produced by changes in the iron balance of the animal. In the iron loaded animal, monoferric transferrin injected into the plasma was rapidly loaded by iron from tissue and thereby converted to diferric transferrin. Injection of diferric transferrin in the iron deficient animal was associated with a rapid disappearance from circulation of the original complex and a subsequent appearance of monoferric transferrin as a result of iron returning from tissues. These observations support the concept that plasma iron behaves as a single pool except that diferric iron exchange occurs at a more rapid rate than dose monoferric iron exchange.

Description: An indirect immunofluorescence assay was used to quantitate TdT- containing (TdT+) cells in the mononuclear leukocyte fraction of peripheral blood from normal subjects and patients with acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LL). In normal children (10) and adults (10), 0.036% +/- 0.014% (mean +/- SD) and 0.030% +/- 0.015% TdT+ cells were found. In peripheral bloods from 10 children receiving chemotherapy for tumors other than ALL or LL, 0.040% +/- 0.039% TdT+ cells were found. Serial determinations were performed on 15 patients with ALL or LL who were in clinical remission. Eight of these patients remained in continuous remission and always had fewer than 0.11% TdT+ cells in their peripheral blood. Three patients who developed systemic relapse were found to have progressively rising numbers of TdT+ cells in their peripheral blood prior to clinical evidence of relapse. All 3 of these patients had greater than 0.1% TdT+ cells in their peripheral blood from 3 to 8 wk prior to clinical relapse. In 3 other patients, localized extramedullary relapse developed, but no trend was found on serial TdT determinations. Thus, the indirect immunofluorescence assay for TdT detects a small population of cells in normal peripheral blood. In patients with ALL, progressive increases above this normal level were associated with subsequent bone marrow relapse.

Description: Washed human platelets were labeled with 125I by the lactoperoxidase- catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.

Description: A 65,000 dalton T-cell specific antigen previously demonstrated to be present on the surface of normal and malignant T cells, but not normal B cells, has been detected on the surface of leukemic cells from patients wih nonsecretory, surface immunoglobulin-positive chronic lymphocytic leukemia (CLL). By means of immunofluorescence and flow cytometry, the relative surface density of the T65 antigen on CLL cells was compared to that on normal peripheral blood T cells and human thymocytes, as well as cell lines of T-cell lineage. In all cases, the CLL cells had a more homogeneous and a lower median fluorescence intensity than that of normal circulating T cells. Thymocytes were composed of three populations, two with low surface density of T65 resembling the CLL cells and the other with higher density similar to normal T cells. The staining of cell lines varied from bright, heterogeneous staining (8402) to uniform, low-density staining (Molt- 4). The implications of these findings with regard to lymphocyte differentiation are discussed.

Description: Three types of immature megakaryocytes, detected by their morphological properties, have been characterized in bone marrow of normal C57BL/6 mice. Morphological classification of these cells was carried out by determining (1) presence and relative amount of acetylcholinesterase, (2) cell size, (3) nuclear/cytoplasm ratio, and (4) nuclear shape. The immature megakaryocytes were classified as: (A) cells distinguished by a round nucleus (10.6 +/- 1.1 mu diameter; mean +/- SEM), which had the highest nucleus / cytoplasm ratio and lowest content of acetylcholinesterase; (B) cells with an indented nucleus (13.0 +/- 1.9 mu diameter), which had increased acetylcholinesterase content and reduced nucleus/cytoplasm ratio compared to the round-nucleus cell type; and (C) lobed-nucleus cells (14.5 +/- 2.9 mu diameter), which showed further increase in acetylcholinesterase content and reduction in nucleus/cytoplasm ratio. Increased numbers of immature megakaryocytes were detected, indicating that a proportion of these cells are undetected using conventional staining techniques. Based on the observed alterations in size, acetylcholinesterase content, and nuclear complexity, it was concluded that these cells constitute part of a progressive maturation sequence intermediate between the progenitor cell (CFU-Mk) and mature easily recognizable megakaryocytes.

Description: A systematic approach to the optimization of centrifugal elutriation is described for the purification of rat femoral marrow megakaryocytes, which has permitted the first direct demonstration of thromboxane synthesis in these cells. The rapidity, simplicity, and capacity of this technique has made it possible to process 2 x 10(9) marrow cells in the absence of platelet aggregation inhibitors in 20 min to obtain 10(6) megakaryocytes at a recovery of about 80% with a concentration of up to 282-fold. Incubation of these isolated megakaryocytes with 14C- arachidonic acid and subsequent thin-layer radiochromatography has revealed thromboxane B2 as the major cyclooxygenase pathway metabolite of arachidonic acid in megakaryocytes.

Description: We have systematically investigated a variety of fixation and plastic embedding procedures and arrived at a method that allows processing of approximately 2-micron sections of bone marrow biopsies for examination by light microscopy. More importantly, this method permits the use of enzyme histochemical and immunohistochemical procedures that are rapidly becoming mandatory in the diagnosis of hematologic malignancies. Over 200 full-length bone marrow biopsy specimens were fixed in a mixture of paraformaldehyde, glutaraldehyde, and acrolein, dehydrated in acetone, and embedded in a mixture of methyl and glycolmethacrylate. All procedures were carried out at 4 degrees C. Decalcification was unnecessary. Sections 2-micron thick were cut and incubated for peroxidase, naphthol AS-D chloroacetate esterase, alpha- naphthyl butyrate esterase, acid phosphatase (with and without tartrate), or alkaline phosphatase and then examined by light microscopy. Specimens could be prepared for examination within 48 hr. This approach, which provides definitive markers for various hematopoietic cell lines in intact tissues, is invaluable when aspirated material is unavailable. It is also useful in the analysis of focal lesions of bone marrow due to inflammation or neoplasia and shows potential as an investigative tool. For example, we have discovered that early myelofibrosis is accompanied by a marked increase in the number of alkaline-phosphatase-positive reticulum cells.

Description: Acute myelofibrosis (AM) or malignant myelosclerosis is a myeloprofilerative syndrome in which bone marrow fibrosis is associated with a proliferation of immature myeloid cells. In four patients with typical AM, investigated by the platelet-peroxidase reaction at ultrastructural level, the blast cells were found to be megakaryoblasts. One patient, treated with the drug combination DAT, achieved a complete remission of 5 mo duration. This study supports the view that megakaryoblastic leukemia is the most frequent underlying cause of AM and proposes that it should be classified as a form of acute myeloid leukemia.

Description: An autoantibody that reacted with nuclei of polymorphonuclear neutrophils (PMN) was detected at titers of greater than 10 in sera of 25 of 50 patients with rheumatoid arthritis and 36 of 50 with autoimmune chronic active hepatitis but in none of 160 controls comprising 24 patients with alcoholic cirrhosis, 36 with multiple myeloma, and 100 healthy subjects. Through the use of enriched populations of hemopoietic cells, this antibody was shown to be cell- specific, reacting only with the nucleus of the mature neutrophil. It was unreactive with nuclei of progenitor cells in the myeloid series and with nuclei of eosinophils, monocytes, lymphocytes, and thymocytes. It reacted with a determinant that appeared to be a differentiation antigen. This cell-specific autoantibody may prove to be of value in analytical studies of granulocyte maturation.

Description: Hemopoietic progenitor cells (colony- and cluster-forming cells in semisolid agar) were purified from light density CBA murine fetal liver cells using fluorescein-conjugated pokeweed mitogen (PWM) and a rhodamine-conjugated antineutrophil serum sandwich (alpha N) and three- parameter fluorescence-activated cell sorting. All clonable progenitor cells were highly enriched (36–50-fold) in PWM-positive (greater than channel 15), alpha N-negative (less than channel 30) fractions with relatively high intensity (greater than 100) low angle light scatter. No separation was achieved between different types of progenitor cells (granulocyte-macrophage and erythroid colony-forming cells). The enriched fraction was a pure population of large, basophilic, undifferentiated blast cells, and in agar cultures stimulated with colony-stimulating factors, up to 90% of the enriched cells were hemopoietic progenitor cells capable of varying levels of clonal proliferation. Further fractionation based on increasing fluorescence with PWM separated into discrete populations, nonproliferative morphologically recognizable erythroid cells, late erythroid progenitor cells (day 2 CFU-E), and cells forming pure or mixed erythroid burst colonies. In addition, the majority of pluripotential hemopoietic stem cells (CFU-SS) were clearly separated from progenitor cells forming colonies in vitro. The present techniques provide suitable numbers of enriched progenitor cells for a variety of biological and biochemical studies.

Description: Auxiliary bone marrow cells are required for optimal murine megakaryocyte colony formation in addition to progenitor cells and a colony stimulating activity (CSA) present in WEHI-3 cell conditioned medium. These auxiliary cells are adherent, with a sedimentation rate of 5.8 mm hr-1 and buoyant density of 1.065–1.078 gcm-3. The activity from bone marrow cells is loss at irradiation doses above 900 rad. Bone marrow cells with these characteristics, and supernatants from lung, bone shafts, and peritoneal exudate cells were all active in enhancing megakaryocyte colony incidences in mouse bone marrow cultures above those stimulated by an obligatory activity in WEHI-3 cell conditioned medium. Certain macrophage cell lines (J774, P388D1) could elaborate the activity. This study confirms that a potentiation activity enhances CSA stimulation of megakaryocyte colony formation. The potentiator is elaborated by bone marrow cells in limiting amounts requiring either high cell concentrations or an exogenous source of the activity for optimal colony growth.

Description: Eosinophils, which may be associated with allergic, parasitic, or neoplastic disease, have a potent oxidative burst that may be activated by particulate or soluble stimuli. Eosinophils from normal persons and patients with hypereosinophilia were compared with respect to their ability to produce the active oxygen product, superoxide anion. Normal eosinophils produced large amounts of superoxide anion under resting conditions (0.53 +/- 0.15 nmoles cyto-c/10(5) eos/hr) and when stimulated by preopsonized zymosan (0.85 +/0 1.10 nmoles cyto-c/10(5) eos/hr) or phorbol myristate acetate (PMA) (2.38 +/- 0.46 nmoles cyto- c/10(5) eos/hr). Considerable variation was observed in superoxide production by eosinophils from patients with hypereosinophilia. Eosinophils from a group of four patients with hypereosinophilia associated with neoplastic disease produced less superoxide anion than normal eosinophils when stimulated by preopsonized zymosan or PMA (p less than or equal to 0.05). Eosinophils from a group of 5 patients with other causes of hypereosinophilia produced more superoxide anion than normal eosinophils when stimulated by PMA (p less than or equal to 0.01). These studies demonstrate metabolic heterogeneity of eosinophils from patients with hypereosinophilia, and further emphasize that normal eosinophils and eosinophils from hypereosinophilic patients are not functionally equivalent.

Description: We describe a method for preparing purified erythroblastic nests in large numbers (approximately 10(6)/run) in three steps: (1) induction of splenic erythropoiesis in mice, (2) preparative differential centrifugation for the removal of erythrocytes and single cells from spleen cell suspensions, and (3) sedimentation in an isokinetic gradient of Ficoll 400 in Joklik's modification of minimum essential medium. Viability of isolated EN is very high, as demonstrated by the trypan blue exclusion and in vitro erythrocyte formation methods.

Description: It is increasingly clear that histamine mediates a variety of lymphocyte functions. Further understanding of these mechanisms requires a method for the analysis of histamine membrane receptors on the lymphocyte surface. We report now a biochemical technique for the identification and quantitation of specific histamine H1 and H2 receptors of lymphocytes. The method can be performed on small numbers of formaldehyde-fixed cells. The data this assay yields, together with that resulting from the flow cytometric analysis of histamine receptor distribution (a technique we have previously described), will be a powerful tool in the study of histamine mediation of lymphocyte function.

Description: A woman with lymphoblastic lymphoma was treated with combination chemotherapy. She subsequently became febrile while granulocytopenic and was given unirradiated granulocyte transfusions from normal, unrelated donors. She recovered, but 12 days later noted the onset of progressive skin rash, hepatic dysfunction, diarrhea and pancytopenia and, 22 days after her last granulocyte transfusion, died of gram negative septicemia. Histologic examination of multiple tissues including the skin, liver, and intestinal tract showed changes characteristic of acute graft-versus-hose disease (GVHD). Y-chromatin analysis of the patient's peripheral blood just before death indicated the presence of male cells. HLA typing of lymphocytes and skin fibroblasts from the patient and lymphocytes from the family and granulocyte donors was also consistent with engraftment of cells from one of the male granulocyte donors. This donor most likely was homozygous for one of the patient's halotypes, perhaps facilitating engraftment of his cells and subsequent development of transfusion- induced acute GVHD. Until more precise guidelines can be established, we recommend that all cellular blood products given to patients receiving intensive chemotherapy be irradiated with 1500 rad.

Description: Twenty-four patients with aplastic anemia or acute leukemia were treated by marrow grafts from HLA-identical donors after conditioning with high doses of cyclophosphamide and/or today body irradiation. They were studied between 4 and 63 mo (median 14.2) after transplantation. Seventeen patients had chronic graft-versus-host disease (C-GVHD) and 7 were healthy. They were studied for defects in their T- and B-cell function using and indirect hemolytic plaque assay for Ig production after 6 days of culture in the presence of pokeweek mitogen. T or B cells from the patients with or without C-GVHD were cocultured with T or B cells from their HLA-identical marrow donors or unrelated normal controls. Intrinsic B-cell defects, lack of helper T-cell activity, and suppressor T-cell activity were more frequently found in patients with C-GVHD than in healthy patients. Fifteen of the 17 patients with C-GVHD showed on or more defects in their T-and B-cell function compared to only 3 of the 7 patients without C-GVHD. None of the healthy controls, including the marrow donors, showed defects in their T- and B-cell functions. These in vitro findings may be helpful in assessing the process of immune reconstitution and the immunologic aberration found after human marrow transplantation.

Description: White blood cells from 22 patients with leukemia and lymphoma were studied for the presence of terminal deoxynucleotidyl transferase with a peroxidase-antiperoxidase method. The enzyme was detected in leukemic cells of 5 patients with acute lymphoblastic leukemia and 1 patient with chronic myelogenous leukemia, whereas 16 patients with different forms of leukemia or lymphoma were negative for this enzyme. Comparative studies using a biochemical and an indirect immunofluorescence assay revealed complete concordance between these three methods.

Description: To determine whether kinase (TK) isozyme status adds clinically useful information in adult non-Hodgkin's lymphoma (NHL), we have analyzed peripheral blood plasma and lymphocytes of 44 patients with NHL for either TK1 or TK2 isozyme activity. On the basis of isozyme status, patients could be divided into two groups that did not differ significantly with respect to known determinants for survival. The median survival of patients exhibiting peripheral blood TK1 thymidine kinase activity was 40 wk and that of individuals with TK2 activity was in excess of 200 wk. These data suggest that peripheral blood TK1 isozyme is a useful independent biochemical marker for a subgroup of NHL who respond poorly to current therapy and thus require new therapeutic approaches.

Description: Spleen cell production of granulocyte-macrophage colony stimulating activity (CSA) and colony forming capacity (CFU-GM) from 59 patients with Hodgkin's and non-Hodgkin's lymphoma, acute (AML) and chronic myeloid leukemia (CML), and control subjects was quantified to evaluate local cellular potential for modulating splenic granulocytopoiesis. Mononuclear spleen cell conditioned media stimulated myeloid CFU-GM by human nonadherent marrow target cells. In contrast to conditioned media produced by marrow and peripheral blood cells, the vast majority of spleen CSA was generated by nonadherent lymphoid cells rather than adherent monocytic cells. The nonadherent cells producing CSA were non- T cells (assessed by sheep erythrocyte rosetting), with 98% +/- 2% CSA produced by the nonrosetted fraction (B lymphocytes and null cells), and had a peak density heavier than that of the adherent spleen CSA- producing cells. Dose response curves demonstrated significantly increased cellular CSA production from patients with lymphomas and AML in remission. In a high proportion of patients, foci of immature granulocytic cells were found by specific cytochemical staining of histologic sections of spleens. A limited degree of splenic granulocytopoiesis was demonstrated morphologically and by CFU-GM incidence. CSA was not detectable in conditioned medium prepared from nonadherent spleen cells from 5 patients with CML, due to a nondialyzable substances(s) produced by the nonadherent cells which inhibited normal CFU-GM response to CSA. The high CFU-GM incidence and extensive leukemic granulocytopoiesis present in the CML spleens suggests diminished effect of this inhibitor on leukemic as opposed to normal granulocytic precursor cell proliferation.

Description: We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

Description: The presence of complement and Fc receptors on neutrophils from patients with acute leukemia was investigated at different stages of the leukemic process. Both Fc receptors (Fc-H and Fc-R) and one complement receptor (C3d) were normal when patients were studied at diagnosis, in relapse, and in remission. In contrast, the C3b receptor was significantly reduced on te neutrophils at the time of diagnosis, but returned to normal levels when patients entered remission. Variable amounts of C3b activity were observed in patients relapse, with approximately one-half of the patients showing decreased C3b activity. To determine whether the reduction inC3b receptors was specific for patients at diagnosis, three patients were studied at different times during their diseases. Normal receptor levels were detected in these patients during remission, but the C3b receptor was markedly reduced at diagnosis and at relapse.

Description: Platelets from patients with several bleeding disorders (congenital afibrinogenemia, Glanzmann's thrombasthenia, gray platelet syndrome, and Hermansky-Pudlak syndrome) were evaluated for both platelet-bound and platelet-free hemagglutination activities. Thrombin and A23187 activated afibrinogenemic, Hermansky-Pudlak, and thrombasthenic platelets had normal platelet-bound hemagglutination activity. Gray platelets activated by the same agents had deficient platelet-bound hemagglutination activity. In contrast, thrombin-activated afibrinogenemic, gray, and thrombasthenic platelets lacked platelet- free hemagglutination activity. Only thrombin-activated Hermansky- Pudlak platelets had a normal level of platelet-free hemagglutination activity. On the basis of these results and the distinguishing characteristics of the defective platelets, it is concluded that the alpha-granules are the origin of the enhanced hemagglutination activity. Furthermore, it is suggested that the insufficiency of the platelet-bound agglutinin may be the cause of the inability of gray platelets to aggregate normally in response to thrombin.

Description: Treatment of human PMNs with cytochalasins (CE, CD, CB, and H2CB) results in alteration of cell morphology and inhibition of cell motility. Morphological changes are similar to those reported for nonamoeboid fibroblasts--rounding, zeiosis, and arborization. Mean cell velocity of PMNs, as measured by quantitative analysis of time-lapse videotape recordings, was reduced to 0.1 micron/min (control, 7.3 +/- 4.2 micron/min). Phagocytosis by PMNs, as measured by phagocytosis of latex beads, was inhibited by 75%. The relative potency of the cytochalasins for inducing morphological change or for inhibiting locomotion and phagocytosis is similar to their relative potencies for affecting non-amoeboid cells: CE greater than CD greater than CB greater than or equal to H2CB. Quantitative binding of 3H-CB to purified PMNs under equilibrium conditions reveal two types of specific CB binding sites: high-affinity sites (KD approximately 3 x 10(-7) M, 3 x 10(6) sites/cell) and low affinity sites (KD approximately 2 x 10(-6) M). The relative affinities of the cytochalasins for the high-affinity and low-affinity CB binding sites parallel their relative potencies for inducing biologic effects (i.e. CE greater than CD greater than CB greater than or equal to H2CB).

Description: The migration in vitro of neutrophils from six patients with monosomy-7 or partial deletion of the long arm of chromosome 7 was studied by two methods: the Millipore filter assay and the migration under agarose assay. Four of the patients had preleukemia, one had subacute myelomonocytic leukemia, and one polycythemia vera. In four patients, chemotaxis (migration towards a higher concentration of chemoattractant) and chemokinesis (stimulated migration without a gradient) were shown to be defective by both methods. In the remaining two patients, this defect could be demonstrated only by the Millipore filter assay or by the agarose assay. Under agarose, random locomotion (no chemoattractant present) of the patients' neutrophils was less than that of the control subjects in four patients, whereas no clear difference could be shown by the Millipore filter method. This study demonstrates that the previously described defect of neutrophil migration in monosomy-7 involves not only chemotaxis but all stimulated migration and, at least in some patients, random locomotion as well. Defective migration in two patients with an apparently terminal deletion of the long arm of one chromosome 7 indicates that the distal half of 7q carries genetic material important for neutrophil locomotion.

Description: A pathway for the synthesis of membrane phosphatidylcholine involving the N-methylation of phosphatidylethanolamine has been detected in several types of mammalian cells. Furthermore, it has been implicated in the coupling of agonist binding to cell response. We examined whether human platelets exhibit this synthetic pathway and whether platelet agonists influence its activity. When washed platelets were incubated with 0.15 microM L-[methyl-3H]methionine at 37 degrees C, they incorporated methyl-3H into their phospholipids linearly at the rate of 1 pmole/10(9) platelets/hr. When incubated with 20 microM radiolabeled methionine, they incorporated about 15 pmole/10(9) platelets/hr. The radioactivity was found predominantly in phosphatidyl- N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine. Thrombin caused an immediate (within 15 sec) and sustained (up to 30 min) decrease in the rate and extent of N- methylation of platelet phospholipids. This was accounted for by a decrease in synthesis of methylated phospholipids rather than an increase in their degradation. This thrombin effect correlated with serotonin release and could be dissociated from platelet aggregation and prostaglandin synthesis. Thrombin also decreased the synthesis of phosphatidylcholine when choline was used as the radiolabeled substrate. Other agonists such as epinephrine, adenosine diphosphate (ADP), or A23187 also decreased phospholipid methylation under conditions in which they stimulated serotonin release. These data demonstrate that platelets are capable of synthesizing phosphatidylcholine from phosphatidylethanolamine by N-methylation and that agonists perturb this pathway as they induce platelet secretion. The precise role of phospholipid methylation in either resting or stimulated platelets remains to be established.

Description: In multiple myeloma, tumor cell mass and labeling index correlate with subsequent survival duration, but do not predict for response to treatment. In the present study was have autoradiographically measured the incorporation of 3H-thymidine as determined by the number of grains over the myeloma nuclei in bone marrow aspiration samples. In 33/37 patients with less than 50% tumor regression or progressive disease, the pretreatment grain count was greater than or equal to 20/myeloma nucleus. Conversely, values of less than 20 were found in 27/29 patients who had greater than or equal to 50% cell mass reduction. Survival duration was significantly better than (p less than 0.001) in patients with grain counts less than 20. Sixty percent of the patients with both a low labeling index (less than or equal to 3%) and grain count (less than 20) were alive at 48 mo, whereas 15/17 patients with a high labeling index and grain count had a median survival of less than 6 mo. In a subset of 22 patients, there as a significant correlation between in vitro resistance to melphalan, adriamycin, and vincristine as tested in the myeloma stem cell colony assay system and a grain count of greater than 20. We can only speculate as to the reasons for the increased 3H-thymidine uptake by myeloma cells resistant to treatment, however, it could be associated with accumulation of excess DNA and /or increased unscheduled DNA synthesis following injury from alkylating agents.

Description: Adenosine deaminase (ADA) and ecto-5′-nucleotidase (5′-N) activities were examined in peripheral leukocytes from patients with leukemias, including nine patients with chronic myeloid leukemia (CML) in blast crisis. Four of none cases of CML in blast crisis were myeloid and the remaining lymphoid morphologically. The diagnosis of CML in lymphoid blast crisis was further contributed by the measurement of terminal deoxynucleotidyl transferase (TdT) activity. In all four cases of lymphoid blast crisis and one of myeloid blast crisis, leukemia cells had high 5′-N activity, while there was a little or no detectable activity in those from four cases of myeloid blast crisis and all of CML in chronic phase. ADA activity was high in seven of nine patients with blast crisis. Taken together, leukemia cells from two cases of lymphoid blast crisis had high ADA and 5′-N activities comparable to those in acute lymphocytic leukemia (ALL) cells. In contrast, the enzyme activities of leukemia cells from all but one patient in myeloid blast crisis were in a range similar to acute myeloid leukemia cells. The implications of these findings are as follows: (1) 5′-N may be used as a new biochemical marker of CML in lymphoid blast crisis. (2) Some lymphoid cells of CML in blast crisis have high ADA, 5′-N, and TdT activities and thus are very similar to ALL cells.

Description: Platelet-associated IgG (PAIgG) was measured by a simple radial immunodiffusion technique using washed solubilized platelets and commercially available immunoplates. Subjects with normal platelet counts had PAIgG levels of 1.5--7.0 fg/platelet. Subjects with idiopathic immune thrombocytopenic purpura (ITP) had levels ranging from 5.7 to 70.5 fg/platelet. All patients with recurrent ITP and 85% of patients with acute ITP had elevated PAIgg. Elevated PAIgG was also found in 17% of patients with recovered ITP, 40% of patients with SLE and thrombocytopenia, 57% of patients with thrombocytopenia occurring during the course of septicemia, and 100% of patients with IgG myeloma in whom the serum IgG level was clearly elevated, regardless of the platelet count. The results are similar to reports that used more complex techniques.

Description: The electrophoretic mobility distributions of hairy cells, normal monocytes. CLL, and normal lymphocytes isolated from blood were determined by electrophoretic light scattering. Values obtained for hairy cells, 1.52 X 10(-4) cm2/V . sec, were indistinguishable from that of normal monocytes. The mobility of CLL lymphocytes was similar to that of normal B cells. After exposure to neuraminidase, hairy cells revealed a homogeneous distribution with a reduced mobility of 0.55 X 10(-4) cm2/V . sec, while normal monocytes showed a heterogeneous distribution of electrophoretic mobilities suggestive of subpopulations. The electrokinetic behavior of hairy cells thus differs from that or normal and CLL lymphocytes before, and from that of monocytes after, treatment with neuraminidase. The hairy cell therefore possesses a distinct pattern of surface charge properties that clearly distinguish it from the circulating B cells, T cells, or monocytes.

Description: The in vitro granulopoietic effects of adherent bone marrow fibroblastic cells (FC) were studied in normal humans and in patients with acute myelogenous leukemia (AML) and myeloproliferative disorders (MPD). To determine their influence on granulopoiesis, we established FC in liquid-phase cultures, overlaid the adherent FC with normal bone marrow cells in agar, and subsequently measured the growth of CFU-C. When using target marrows containing few spontaneous colonies, increased numbers of CFU-C were found above the FC obtained from normals. No growth greater than controls was observed in those areas lacking FC. If target marrows contained large numbers of spontaneous CFU-C, actual inhibition of colony formation was produced by FC co- incubation. In contrast to normals, FC obtained from untreated AML and MPD patients typically failed to enhance granulopoiesis. Regardless of source, FC were not synergistic with the effects of placenta- conditioned media (typically being inhibitory) for colony number, but were synergistic for colony size. Conditioned media obtained from FC cultures did not enhance colony formation and actually inhibited spontaneous colony formation. Thus, microenvironmental abnormalities in interactions between “stromal cells” and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans.

Description: One-hundred-one adult patients with ALL were analyzed to determine the prognostic implications of splenomegaly occurring at any time during the course of their illness. The clinical status of the spleen at presentation was not found to be of major prognostic significance. Complete response rates, remission durations, and survivals did not differ between patients with and without splenomegaly at presentation. An enlarged spleen accompanied relapse in four patients. In six additional patients, splenomegaly was present during complete remission, and splenectomies performed in five of these patients revealed no evidence of leukemia to account for the splenomegaly. Splenectomy does not appear to be detrimental, as all five patients are currently in complete remission from 20 to 63 mo after splenectomy. Evidence implicating the spleen as a source of an antibody directed against autologous leukemia cells in one patient is reviewed.

Description: A subject with HbG Philadelphia-HbH disease exhibited an unusually high alpha/beta synthesis ratio; when peripheral blood was tested in vitro on several occasions, ratios of 0.63 - 0.89 were obtained after incubations of 30–120 min. HbH amounted to 5%-8% of the circulating hemoglobin. Rapid destruction of excess newly synthesized beta-globin was demonstrated in kinetic and pulse-chase experiments. After 2 min of incubation, the alpha/beta synthesis ratio was 0.48; this figure rose to 0.89 by 30 min. The zero time alpha/beta ratio was estimated to be 0.35. The degradation of beta-chains was calculated to proceed at approximately one-half the rate of beta-globin synthesis; this result was confirmed by the loss of 50% of the specific activity in beta- chains during 9 min of a chase experiment following a 10-min radioactive pulse. The results suggest that efficient proteolysis may be responsible, in some blacks, for the low levels of excess beta- globin chains in HbH disease as well as for the mildness of the clinical disorder.

Description: Phagocytic cells generate superoxide in response to stimulation by opsonized particles. A continuous assay for opsonized zymosan- stimulated granulocyte superoxide production shows that there is a lag time between the addition of particles and the onset of detectable superoxide production. Superoxide production is preceded by membrane potential depolarization. Neither superoxide production nor membrane depolarization occurs in granulocytes from patients with chronic granulomatous disease. The extent of activation by opsonized zymosan is affected by the dose of zymosan from 0.5 to 4.5 mg/ml, but the time necessary for activation (lag time) is not. Similarly, the extent of depolarization but not the time necessary for attaining maximum depolarization is concentration-dependent. Effects of temperature, divalent cations, 2-deoxyglucose, cyanide, and N-ethyl maleimide on superoxide production are similar for granulocytes treated with soluble stimuli and with opsonized zymosan. Thus, zymosan stimulates granulocytes to generate superoxide and undergo membrane depolarization in a manner similar to that elected by soluble stimuli.

Description: Normal and plethoric bone marrow cells were grown in plasma clot diffusion chambers (PCDC) implanted into the peritoneum of normal mice or mice submitted to 7 her of hypoxia (23,000 ft) daily, on a single day or on 2 consecutive days at different times after implantation of the PCDC's. Daily discontinuous hypoxia (DDH) produced more 6-day bursts than other treatments. Hypoxia on days 1 and 2 after implantation was nearly as effective as DDH on day-6 bursts. Later bouts of hypoxia or a singly hypoxic exposure on day 1 or 2 was less effective. Erythropoietin (Ep) levels were measured by bioassay on both diffusion chamber (DC) contents and serum. Serum Ep levels peaked at 160 mU/ml after a 7-hr hypoxic exposure while the DC content Ep levels were in the nondetectable range (less than 50 mU/ml). The data implies that either higher than normal Ep levels or a companion molecules (s) produced by hypoxia are required for 1–2 days early in the culture period of force an increasing number of BFU-d-e down the erythrocytic pathway and thus increase red cell production at times of need in vivo.

Description: Normal human plasma contains a complex of two proteins that are important in hemostasis and coagulation. The factor VIII procoagulant protein (antihemophilic factor) and the factor VIII-related protein (von Willebrand factor) are under separate genetic control, have distinct biochemical and immunologic properties, and have unique and essential physiologic functions. While the nature of their interaction and the details of the biochemical structures remain to be determined, the information now available permits a preliminary understanding of the molecular defects in hemophilia and von Willebrand's diseases.

Description: A simple in vitro method has been developed for the assessment of ineffective erythropoiesis by measuring the release of heme from a labeled cohort of erythroblasts in short-term suspension culture. The release of labeled heme was shown to correlate with the death of erythroblasts in culture determined by cell counting. Heme release was markedly increased in conditions where there is known to be excessive ineffective erythropoiesis, while in hematologic disorders where ineffective erythropoiesis is thought to be normal, heme release was within the normal range.

Description: The aggressive clinical course and the distinctive histologic, cytochemical, and cytogenetic features of an adult non-Sezary T-cell lymphoma with suppressor activity have been investigated. Morphological and ultrastructural analysis of neoplastic cells from peripheral blood and involved lymph nodes revealed cells with convoluted nuclei, prominent cytoplasmic azurophilic granules, well developed Golgi apparatus, short strands of endoplasmic reticulum, and moderate numbers of ribosomes and mitochondria. Cytochemical reactions showed acid phosphatase (ACP) positivity in virtually all of the neoplastic cells; and a substantial percentage of the cells, the tartrate-resistant acid phosphatase (T-ACP) isoenzyme was observed. Granular naphthyl acetate esterase (A-EST) reactivity was not present. The histological and cytochemical features of these neoplastic suppressor cells were compared with those recently described for the suppressor T-cell fraction isolated from normal peripheral blood T-cell by Fc gamma- rosette formation. The aneuploid clone had 47 chromosomes with multiple complex abnormalities, including a 14q + chromosome formed by the tandem translocation of two no. 14 chromosomes and translocations involving the long arms of no. 2 and no. 9 at band 9q34. These latter changes are particularly common in T-cell disorders. The extensive analysis of this histologic, cytochemical, and cytogenetic features of this adult T-cell suppressor lymphoma should help to clarify the criteria for distinguishing among the subsets of T-cell neoplasms with definable immunologic function.

Description: A retrospective blind study of 32 patients with the hypereosinophilic syndrome was undertaken utilizing a hematologic scoring system that was based on peripheral blood and bone marrow findings, cytogenetics B12 levels, and leukocyte alkaline phosphatase determinations. In addition to the grading system, which allowed formulation of a hematologic score, the date could also be normalized for individuals who did not have all tests performed by use of the hematologic quotient. This study clearly defined two groups of patients within the idiopathic hypereosinophilic syndrome. One group were those individuals with low hematologic scores and quotients who did not require therapy or who responded to prednisone therapy, while the second group of patients required cytotoxic therapy. These patients had significantly higher hematologic scores and quotients and a significant number of abnormalities similar to those seen in myeloproliferative syndromes, such as myelofibrosis and cytogenetic abnormalities. This type of hematologic scoring seems useful in predicting therapy and/or evaluating individuals or groups of patients with the hypereosinophilic syndrome.

Description: Negative charges on the external surface of red cells were visualized by colloidal iron hydroxide labelling of 50% of the membrane area after osmotic hemolysis and glutaraldehyde fixation. Counts were made over randomly selected areas on electron micrographs at 350,000 x magnification. Statistical analyses showed that at the 95% level of confidence there was no significant difference between oxygenated normal (AA) and sickle (SS) cells in either the distribution or the density of negative charges.

Description: Essential thrombocythemia is characterized by proliferation of hematopoietic tissue predominantly involving megakaryocytes and resulting in marked thrombocytosis. The disorder has some clinical and laboratory features that resemble those seen in the clonal multipotent stem cell disorders chronic myelogenous leukemia, polycythemia vera, and agnogenic myeloid metaplasia. It has been argued that essential thrombocythemia should be classified together with those disorders as a myeloproliferative syndrome. However, without knowledge of the numbers and types of cells that are involved in essential thrombocythemia, this suggestion remains speculative. Three patients with thrombocytosis were studied. The diagnosis of essential thrombocythemia was considered to be firm in two patients and probable in the third one. The X-linked glucose-6-phosphate dehydrogenase locus was used as a cell marker. Whereas both A and B types of glucose-6-phosphate dehydrogenase were found in nonhematopoietic tissues, only a single-enzyme type was found in the granulocytes, red cells, and platelets from each patient. These data indicate that the disorders in these three patients are clonal and involve multipotent stem cells.

Description: Lymph node cells obtained from a case of non-Hodgkin's lymphoma, of follicular center cell histology, were seen by immunofluorescence to express immunoglobulin mu heavy chains but no detectable light chain on their surfaces. Antibody specific for determinants expressed only on uncombined mu-chain reacted with these cell surfaces, but not with normal or neoplastic cells expressing surface IgM. Analysis of radioiodinated surface material revealed mu-chains which, without reduction, migrated on SDS-gel electrophoresis as normal mu-chain monomers, and again failed to detect light chains. Lysates of cells incubated with 3H-leucine revealed the synthesis of mu-chains but not of light chains. The labeled mu-chains were not secreted into the culture supernatant, a finding confirmed in parallel cultures by radioimmunoassay. The findings are discussed with particular reference to current concepts of the “mu-chain only” phenotype.

Description: Platelet concentrates were prepared at twice the normal concentration and stored at room temperature for 7 days in either standard bags (controls) or bags to which 1 or 2 g of Amberlite resin beads charged with dibasic phosphate had been added. The resin beads served as a buffer system by providing a “slow release” form of phosphate ions as well as by binding CO2 produced during platelet metabolism. Control platelets demonstrated rapid falls in pH, ATP content, morphology score, and thrombin-induced nucleotide release after 24 hr of storage with a fall in pH to less than 6.0 by day 3. Profound ultrastructural changes and a rise in pO2, suggesting loss of platelet viability, accompanied these changes. In contrast, the resin-stored platelets remained near normal after 24 hr of storage, with preservation of discoid morphology, 95% of ATP levels, excellent ultrastructural appearance, and evidence of continued oxygen consumption after 3 days of storage. Even after 7 days of storage, ATP levels remained greater than 50% of baseline and ultrastructurally intact platelets were seen. In the 1-g resin bags the pH remained at baseline levels (6.9–7.0), while there was a rise in pH in the 2-g resin bags. These results demonstrate the beneficial effects of maintaining a higher pH during platelet storage and provide a new approach to studying the metabolic changes that occur during longer term storage.

Description: Cell surface antigens of the human bone marrow CFU-C have been studied. Human marrow cells were incubated with a variety of monoclonal antisera and complement prior to culture in semisolid media. By using indirect immunofluorescent studies, the percentage of bone marrow cells binding the antibodies was determined. The CFU-C phenotype is HLA+, la+, 4F2+, 3A1-, and DUALL-1-. This study provides information that is useful in the study of myeloid cell ontogeny and necessary for the use of some of these reagents in the treatment of bone marrow cells prior to human bone marrow transplantation in various clinical settings.

Description: The regeneration of hemopoietic precursor cells (colony-forming cells, CFC) was monitored in spleen organ cultures from lethally irradiated mice injected with 10(7) normal syngeneic or allogeneic bone marrow cells. The important role of the microenvironment in supporting hemopoiesis was confirmed by the failure of mutant S1/S1d spleens to support CFC regeneration in organ cultures. However, the extent and quality of the CFC regeneration was clearly dependent on the genetic properties of the injected cells. Evidence for this was obtained from the regeneration patterns of various CFC types in organ cultured spleens derived from different mouse donor-recipient strain combinations (CBA/CBA, CBA/C57BL, CBA/BALB/c, C57BL/C57BL, C57BL/CBA, C57BL/BALB/c) that maintained the differences in the bone marrow frequency of various CFC types characteristic of the donor strain.

Description: Adenylate cyclase (AC) and guanylate cyclase (GC) activities were studied in normal B-enriched and T-enriched lymphocytes, in lymphocytes of children with acute lymphocytic leukemia (ALL), and in lymphocytes of adults with chronic lymphocytic leukemia (CLL). AC activity was greater in normal B than T lymphocytes (215 pmole/min/mg protein versus 80 pmole in the membrane-enriched fraction) and i both increased greatly after stimulation with isoproterenol and more so with prostaglandins E and F2 alpha. In leukemic lymphocytes, AC showed depressed activity (20 pmole in ALL cells and 55 pmole in CLL cells) and was less sensitive to hormonal stimulation: this loss of sensitivity occurred to a greater extent in ALL than in CLL lymphocytes. GC activity was greater in normal T than B cells (in membrane-enriched fraction: 10.2 pmole versus 5.3 pmole). It increased little with isoproterenol and prostaglandins stimulation, and much more with sodium azide and dehydroascorbic acid stimulation. GC activity was increased in both types of leukemic lymphocytes (23 pmole for ALL cells and 18 pmole for CLL cells) and was insensitive to stimulation. Possible derangement of cyclase and cyclic nucleotide regulation in leukemic cells is suggested.

Description: We observed several patients with chronic idiopathic thrombocytopenic purpura (ITP) whose bleeding times were more prolonged than would have been expected from their platelet counts. To investigate this further, we performed in vivo and in vitro platelet function studies, assessed arachidonate metabolism, and measured platelet-associated IgG (PAIGG) in seven patients with chronic ITP. The bleeding times of three of the patients were prolonged for greater than 7 min, and all of these patients had impaired platelet aggregation and abnormal platelet arachidonic acid metabolism as reflected by increased production of the lipoxygenase product HETE and a concomitant decrease in cyclooxygenase products, TXB2 and HHT (p less than 0.001). The abnormalities noted were not due to concomitant drug ingestion, since they were present on repeated evaluation. There was no relationship between the platelet count and the bleeding time; however, there was a significant inverse correlation between the bleeding time and TXB2 production in all patients evaluated (r = 0.81; p less than 0.05). There was no relationship between the level of platelet-associated IgG and any parameter of platelet aggregation or arachidonate metabolism. The abnormalities noted should be looked for in the individual patient with chronic ITP, since the bleeding tendency is exacerbated by the superimposed impairment of platelet function even at platelet counts of greater than 50,000/cu mm, levels generally regarded as “safe”.

Description: A specially designed rotational viscometer was used to investigate the effects of the antiplatelet agent PGI2 in combination with theophylline on the response of human platelets subjected to shear stress. Samples of citrated platelet-rich plasma (PRP) were exposed to shear stress in the viscometer for a period of 5 min at 23 degrees C. The levels of stress studied ranged from 50 to 300 dynes/sq cm. Pretreatment of the platelets with 0.01 microM PGI2 and 500 microM theophylline before exposure to shear stress caused a large reduction in shear-induced platelet aggregation. However, it was also observed that the PGI2-- theophylline pretreatment concomitantly caused a large increase in shear-induced platelet lysis and serotonin release at stress levels equal to or greater than 150 dynes/sq cm. This observed increase in platelet fragility may have important implications for clinical applications of PGI2. The results are discussed and compared to those obtained in prior work in which platelets were pretreated with acetylsalicylic acid or with PGE1.

Description: A newborn without any signs of Down's syndrome was found to have an acute proliferation that remitted without drug therapy. Chromosomal analysis of blood, bone marrow, and skin cells revealed that the child was a constitutional mosaic with normal cells and a low number of cells in which one no. 21 chromosome was replaced by a probably isochromosome for the no. 21 long arm: 46,XY/46,XY,i(21q). The abnormal cell line of the mosaic appeared to be selectively involved in this proliferation.

Description: Peripheral blood mononuclear cells (PBMC) from 18 untreated patients with non-Hodgkin's lymphoma (NHL) were studied to characterize the cellular mechanisms contributing to impaired in vitro lymphocyte responses after stimulation by the mitogen conconavalin A (Con-A). In vitro reactivity was quantitated by the 3H-thymidine incorporation in response to an optimal dose of Con-A. All patients demonstrated impaired in vitro reactivities compared to normal controls. These in vitro impairments were partially reversible since patient's cells precultured in media alone for 3 days demonstrated enhanced Con-A responses. In greater than half of the patients, the hyporeactive PBMC suppressed the enhanced reactivities of autologous precultured PBMC when assayed in cocultures. Suppressor activity was detected mainly in those untreated patients presenting with either constitutional symptoms or diffuse histology and in general was not marked compared to the severity of impairments. Adherent monocytes were shown to participate in the suppression of autologous lymphocyte reactivity but only appeared partially responsible for the in vitro impairments. In those patients lacking detectable suppressive activity, preculturing also enhanced Con-A reactivities and was compatible with the presence of a reversible, inhibitory mechanism differing from active suppression. Many patients' hyporeactive PBMC, however, failed to demonstrate normal responses after preculturing. This failure could not be directly attributed to aberrant regulatory populations, but rather appeared to possibly represent an additional intrinsic impairment of potentially reactive populations.

Description: Platelets are activated by many different agonists that act synergistically. Since there is a characteristic pattern of responses to each agonist, and since there is a clear distinction between weak and strong agonists, understanding the nature of the synergism and its physiologic significance requires characterization of the pattern of responses to the synergistic action of the various agonists. Shape change, aggregation, and secretion of ATP by human platelets in citrated plasma were analyzed after activation by ADP, epinephrine, arachidonic acid, gamma-thrombin, or collagen, either singly or in pairs. The patterns of responses were characteristic of the agonist in higher concentration relative to its threshold concentration; if neither was clearly higher, the pattern of responses was intermediate between the responses characteristic of each agonist. No combination of weak agonists had the characteristics of a strong agonist. These results help define the extent to which platelet responses can be attributed to the synergistic actions of weak agonists.

Description: We assessed the integrity of the prostaglandin synthetic pathway by measuring malondialdehyde (MDA) production and studied platelet aggregation responses to arachidonic acid and PGG2 in 12 patients with storage pool deficiency (SPD). Eight patients were deficient only in dense granules (delta-SPD) and four were deficient in both dense and alpha-granules (alpha delta-SPD). Production of MDA in response to arachidonic acid (AA), epinephrine, and collagen suggested that the transformation of AA to prostaglandin metabolites was normal in delta- SPD but abnormal in alpha delta-SPD and that the liberation of AA from phospholipids were abnormal in the majority of patients with SPD. Since the content of secretable adenosine diphosphate (ADP) is diminished in SPD platelets, the aggregation responses of these platelets to AA and PGG2 were studied to help answer the question whether these agents aggregate platelets directly or through release of endogenous ADP. Among patients with delta-SPD, aggregation by both AA and PGG2 was decreased in four albinos whose platelets were markedly deficient in ADP. In contrast, normal, or less strikingly abnormal, responses were observed in patients whose platelets either contained higher levels of platelet ADP or showed increased sensitivity to ADP. The more marked impaired responses to AA and PGG2 in patients with alpha delta-SPD suggest that substances derived from alpha-granules may also play a role in platelet aggregation by these agents. The aggregation responses in these patients with various types of SPD is consistent with a theory that granule-derived ADP mediates platelet aggregation by AA and PGG2.

Description: Plasma fibrinopeptide A (FPA) levels were determined in 20 unselected adult patients with acute nonlymphocytic and lymphocytic leukemia. The mean FPA level in patients with active disease (15.0 ng/ml) was significantly higher than during clinical remission (2.4 ng/ml, p less than 0.01). Elevated FPA levels were observed in patients with all morphological forms of acute leukemia. In the group of patients in clinical remission, 20/47 FPA values remained elevated beyond the normal range, suggesting that low-grade intravascular coagulation was present even when no leukemic cells were observed. Sequential studies revealed reduction of FPA levels to the normal range in five patients who entered clinical remission after chemotherapy and rapid elevation of the levels in eight patients who entered relapse after clinical remission. FPA levels rose significantly in five patients studied during induction chemotherapy. Thus, subclinical activation of blood coagulation, as defined by elevation of plasma FPA level, may occur commonly in acute leukemia. Plasma FPA generation may relate to leukemic disease activity.

Description: The lymphoblasts of three patients with childhood acute lymphoblastic leukemia (ALL) were analyzed for their immunologic surface markers. Blasts from two of these patients did not form rosettes with sheep erythrocytes and the third did so marginally, suggesting these patients had non-T-cell leukemia. These blasts were also tested with monoclonal antibodies that detect thymocyte differentiation markers, and all three patients were highly reactive with at least two of these reagents. We anticipate the availability of multiple standardized monoclonal reagents will necessitate a recategorization of ALL phenotypes. Some of these leukemic phenotypes may not correspond to normal stages of lymphoid differentiation. Therefore, we suggest that it may be inappropriate to attempt to identify and categorize leukemic cells by the pathways of normal differentiation.

Description: Reed-Sternberg cells in the lymph nodes from five patients with Hodgkin's disease were studied. Indirect immunofluorescence on fixed sections with a monospecific anti-serum to fibronectin revealed abundant cytoplasmic fibronectin in approximately 90% of the Reed- Sternberg cells. In addition, the cells were shown by immunofluorescence to contain polyclonal IgG; however, factor VIII antigen, albumin, fibrinogen, alpha-2-macroglobulin, anti-thrombin III, and ceruloplasmin were not present. The abundant cytoplasmic fibronectin suggests that Reed-Sternberg cells are derived from tissue macrophages.

Description: Treatment of human PMNs with cytochalasins (CE, CD, CB, and H2CB) results in alteration of cell morphology and inhibition of cell motility. Morphological changes are similar to those reported for nonamoeboid fibroblasts--rounding, zeiosis, and arborization. Mean cell velocity of PMNs, as measured by quantitative analysis of time-lapse videotape recordings, was reduced to 0.1 micron/min (control, 7.3 +/- 4.2 micron/min). Phagocytosis by PMNs, as measured by phagocytosis of latex beads, was inhibited by 75%. The relative potency of the cytochalasins for inducing morphological change or for inhibiting locomotion and phagocytosis is similar to their relative potencies for affecting non-amoeboid cells: CE greater than CD greater than CB greater than or equal to H2CB. Quantitative binding of 3H-CB to purified PMNs under equilibrium conditions reveal two types of specific CB binding sites: high-affinity sites (KD approximately 3 x 10(-7) M, 3 x 10(6) sites/cell) and low affinity sites (KD approximately 2 x 10(-6) M). The relative affinities of the cytochalasins for the high-affinity and low-affinity CB binding sites parallel their relative potencies for inducing biologic effects (i.e. CE greater than CD greater than CB greater than or equal to H2CB).

Description: Lactic dehydrogenase (LDH) was quantitated and the isozyme pattern studied in lymphocyte subpopulations from normal people and patients with chronic lymphocytic leukemia (CLL). Normal T lymphocytes differed from normal B lymphocytes in having greater total LDH activity (597.2 versus 252.1). Total LDH activity in CLL T cells (347.1) was lower than normal T cells., but not significantly different than normal B cells. Total LDH activity in CLL B cells (124.6) was lower then normal B cells and normal T cells. The isozyme pattern of normal T lymphocytes showed a higher activity in the LDH-1 band (26.7% versus 5.4%) but showed a lower activity in LDH-5 band (4.3% versus 16.3%) compared to normal B cells. Chronic lymphocytic leukemia T cells could be distinguished from CLL B cells by a high LDH-5 band (22.3% versus 7.6%) and from normal T cells by a high LDH-5 band (22.3% versus 4.3%) and a low LDH-1 band (7.3% versus 26.7%). CLL B cells could be distinguished from normal B cells by a low LDH-5 band (7.6% versus 16.3%). Thus, the LDH isozyme pattern distinguishes normal T lymphocytes from normal B lymphocytes, and normal T and B lymphocytes from CLL T and B lymphocytes.

Description: Removal of iron from biologically important ligands by desferrioxamine (DF), in vitro, is exceedingly slow because of a kinetic barrier. This kinetic barrier can be overcome by a variety of a small molecular weight anions, of which nitrilotriacetate (NTA) is a prototype. We explored the effect of NTA on DF iron mobilization in vivo. Iron mobilization was increased and the effect was synergistic.

Description: We describe three patients who had typical features of hairy cell leukemia (HCL) and multiple myeloma (MM) at the same time. In two, both diagnoses were made within a short period of time, and in the third, HCL had been present for 2 yr before the appearance of a paraprotein, bone lesions, and plasma-cell infiltrates established the diagnosis of MM. Although this association has not been previously reported, cases of HCL with osteolytic lesions or a paraprotein band have been described. The cases described may represent clinical manifestations of closely related disorders arising from divergent differentiation from a common B-cell precursor rather than a chance association.

Description: Forty-eight patients with fever greater than 38.3 degrees C for at least 24 hr despite broad spectrum antibiotics and an absolute granulocyte count less than 1000/microliter were randomly allocated to 4 days of granulocyte transfusions obtained by leukapheresis using filtration (n = 27) or gravity (n = 21) techniques, the latter permitting simultaneous nonmechanical collection of granulocytes and platelets utilizing hydroxyethyl starch as a sedimenting agent. Patient characteristics and dose of granulocytes obtained from both techniques were similar. Complete response to granulocyte transfusions was established by a reduction in temperature to less than 37.2 degrees C sustained for at least 48 hr after the fourth transfusion with sterilization of cultures where previously positive and diminution of measurable infection when present. This occurred in 6/21 (29%) for gravity leukapheresis and 9/27 (33%) for filtration leukapheresis. An additional group had diminution in temperature and clinical improvement during transfusions (6/21 gravity leukapheresis versus 10/27 filtration leukapheresis). Eighty-six percent of patients transfused with gravity leukapheresis cells were alive at day 20 compared with 81% for filtration leukapheresis cells. Transfusion reactions were comparable. Thus, gravity leukapheresis appears to be as efficacious as filtration leukapheresis for treating granulocytopenic febrile patients, with the added advantages of availability to any blood bank without new equipment, of having platelets as by-products, and of not requiring donor heparinization.

Description: Peripheral blood samples from 57 children with newly diagnosed E- rosette-negative, surface-immunoglobulin negative acute lymphocytic leukemia (ALL) were studied for the presence of a leukemia-associated antigen (ALLA). Ficoll-Hypaque separated cells were tested using a rabbit antiserum to human null lymphoblasts and an indirect immunofluorescent assay. The percentage of ALLA-positive cells were compared to the percentage of lymphoblasts determined by differential counts of a Wright-Giemsa-stained smear of a concurrently obtained peripheral blood sample. The mean ratio of percentage of lymphoblasts to percentage of ALLA-positive cells was 0.90. However, in 13 patients, the ratio of percent of ALLA-positive cells to percent of lymphoblasts was equal to or greater than 2:1. In the blood of 6 additional children (5 newly diagnosed, 1 relapsed patient) in whom no morphologically identifiable lymphoblasts were detected. ALLA-positive cells were present (7%-49%). These results indicate that testing for ALLA-positive cells in a sensitive technique for detection of leukemic cells in children with ALLA-positive ALL.

Description: A clonal method in cell culture is described that permits the quantitation of blast precursors in common (non-T, non-B) ALL; the method also yields information about progenitor properties, based on analysis of cells in colonies. The technique is identical to that used successfully for normal and malignant B-cell progenitors except that it requires culture at below O2 tension (5%-7%); mononuclear cells from blood or marrow are depleted of T cells and cultured with media conditioned by T cells in the presence of phytohemagglutinin and irradiated T cells. Cultures are incubated for 5–7 days in a moist atmosphere at 5% CO2 and 5%-7% O2. Colonies were obtained from marrow or blood of 16 of 18 ALL patients. Cells in colonies had the same characteristics (E-, slg-, cALL+ and clgM+ or clgM-) as the cells in the patient. By replating pooled colonies, self-renewal of progenitors was shown. The findings are considered in light of a model of leukemic blasts that depicts such populations as lineages maintained by progenitors that either renew themselves or give rise to blast cells with little or no proliferative capacity.

Description: 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.

Description: The action of an alkyl-lysophospholipid (ALP), ET180CH3, on clonogenicity, 3H-TdR uptake, and cell numbers was tested in two human leukemic cell lines, HL60 and K562, and short-term human leukemic bone marrow cultures. ALP eliminated clonogenicity in HL60 but not in K562 cultures; 3H-TdR uptake and cell numbers were depressed at low concentrations of ET180CH3 in HL60, but not K562 cultures. The action of the lysophospholipid analog on human leukemic bone marrow short-term cultures at low concentrations was similar to its action on HL60 cultures; clonogenicity and 3H-TdR uptake were depressed, but cell numbers were not significantly affected. The demonstration of differential action of ALP on two cell lines should significantly simplify the investigation of the mechanism of the reported differential action of ET180CH3 on normal and leukemic cell membranes.

Description: We have analyzed the multimeric structure of factor VIII/von Willebrand factor in plasma by sodium dodecyl sulfate electrophoresis using gels of varying porosity and a discontinuous buffer system. Factor VIII/von Willebrand factor bands were identified by reaction with 125I-labeled affinity-purified antibody and subsequent autoradiography. In 1% agarose gels, normal plasma displayed a series of sharply defined oligomers. However, increasing the agarose concentration to 2.0% or utilizing mixtures of 0.8% agarose--1.75% acrylamide revealed two bands of lesser intensity interposed between the major bands. When the acrylamide concentration in the gels was increased to 2.5%, bands with a faster mobility than IgM and fibronectin were now evident. Type IIA von Willebrand's disease showed not only an absence of the larger multimers but also a relative increase in several of the newly identified bands as compared to type IIB, type I, and normal. These studies suggest that factor VII/von Willebrand factor in IIA von Willebrand's disease is structurally different from that in other forms of the disorder. They also indicate that the multimeric composition of factor VII/von Willebrand factor is more complex than can be explained by simple linear polymerization of a single protomer.

Description: Washed human platelets were labeled with 125I by the lactoperoxidase- catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.

Description: We have examined the biologic and physical properties of a human T- lymphocyte granulocyte-macrophage colony-stimulating factor (CSF). The source of the factor is a T-lymphoblast cell line (Mo) that was derived from a patient with a T-cell variant of hairy-cell leukemia. The Mo line constitutively produces a number of lymphokines that are normally produced by mitogen-stimulated T lymphocytes. Medium conditioned by Mo cells grown in the absence of serum is especially rich in CSF activity, and using this source we have purified the CSF to a specific activity of about 3.5 x 10(6) colonies per 10(5) Ficoll-Hypaque-separated human bone marrow cells plated per mg protein. The Mo CSF stimulates the formation of both granulocyte and macrophage colonies in vitro (in about equal numbers) and it has a relatively steep dose-response curve. Both the crude and purified preparations stimulated the formation of eosinophil as well as neutrophil colonies; it is unclear whether this is due to the presence of multiple factors with similar physical properties or a single factor with multiple activities. The CSF has little stimulating activity for mouse bone marrow progenitors. Physically, the Mo CSF is an acidic glycoprotein of molecular weight about 34,000. It binds to concanavalin A-Sepharose, is unusually resistant to denaturing agents and heat treatment, and is not inactivated in the presence of sulfhydryl reagents. The Mo CSF is distinct from factors stimulating erythroid colony formation and inhibiting neutrophil migration that are also produced by Mo cells. It differs in several physical and biologic properties from other human CSFs that have been characterized.

Description: We have studied the effects of medium conditioned by the human progranulocytic leukemia cell line, HL-60, on the subsequent growth of new inocula of HL-60 cells. When HL-60 cells were cultured at high cell density, optimal growth rate occurred in liquid suspension and confluent colony growth was observed in viscous medium without the addition of conditioned medium. However, when cells were cultured at lower cell density, growth rate was reduced and colony growth was nil unless conditioned medium from HL-60 culture was added. All HL-60 populations studied, including the earliest available passage, 9, both elaborated and responded to HL-60 CM. HL-60 CM did not stimulate normal human or mouse granulocyte-monocyte colony-forming cell (CFU-GM) growth. Conditioned media from other human cell lines varied in the ability to stimulate HL-60 cell and CFU-GM proliferation. Some, such as GCT CM, stimulated both HL-60 cells and normal CFU-GM, whereas others, like HL-60 CM, stimulated only HL-60 growth. The majority of cell line CMs tested did not stimulate either HL-60 or CFU-GM. Chromatography of HL-60 CM on Ultrogel AcA54 showed a single peak of HL-60 stimulating activity of apparent molecular weight 13,000. The ability of HL-60 cells to elaborate this activity provides a possible explanation for their proliferation at higher cell densities. Autostimulation may prove to be important in the high growth potential of other cell populations that undergo unrepressed proliferation.

Description: In long-term marrow cultures, proliferation and differentiation of hemopoietic stem cells occurs for several months. Normally, only the most primitive erythroid progenitor cells are produced (the BFU-E). Following treatment with anemic mouse serum (AMS) or normal mouse serum plus erythropoietin, the BFU-E mature into CFU-E, which then go to produce mature nonnucleated red cells. This development is associated with the production of adult type hemoglobin. Furthermore, erythropoiesis and granulopoiesis occur in association with discrete cellular elements of the adherent cell layer in the long-term culture. Following treatment with AMS, erythropoiesis is enhanced while granulopoiesis is depressed, with no apparent competition at the stem cell or progenitor cell level.

Description: The histologic features of thymuses from three patients who underwent thymectomy for acute lymphoblastic leukemia or lymphoblastic lymphoma in complete clinical remission are described. The thymuses from all three patients were fibrotic with a variability in the appearance of the lobules. Some of the lobules consisted predominantly of epithelial cells with small numbers of mature appearing lymphocytes, while other lobules were expanded and composed predominantly of cells having morphological features of immature lymphoid cells consistent with residual or recurrent disease.

Description: A pathway for the synthesis of membrane phosphatidylcholine involving the N-methylation of phosphatidylethanolamine has been detected in several types of mammalian cells. Furthermore, it has been implicated in the coupling of agonist binding to cell response. We examined whether human platelets exhibit this synthetic pathway and whether platelet agonists influence its activity. When washed platelets were incubated with 0.15 microM L-[methyl-3H]methionine at 37 degrees C, they incorporated methyl-3H into their phospholipids linearly at the rate of 1 pmole/10(9) platelets/hr. When incubated with 20 microM radiolabeled methionine, they incorporated about 15 pmole/10(9) platelets/hr. The radioactivity was found predominantly in phosphatidyl- N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine. Thrombin caused an immediate (within 15 sec) and sustained (up to 30 min) decrease in the rate and extent of N- methylation of platelet phospholipids. This was accounted for by a decrease in synthesis of methylated phospholipids rather than an increase in their degradation. This thrombin effect correlated with serotonin release and could be dissociated from platelet aggregation and prostaglandin synthesis. Thrombin also decreased the synthesis of phosphatidylcholine when choline was used as the radiolabeled substrate. Other agonists such as epinephrine, adenosine diphosphate (ADP), or A23187 also decreased phospholipid methylation under conditions in which they stimulated serotonin release. These data demonstrate that platelets are capable of synthesizing phosphatidylcholine from phosphatidylethanolamine by N-methylation and that agonists perturb this pathway as they induce platelet secretion. The precise role of phospholipid methylation in either resting or stimulated platelets remains to be established.

Description: Neuraminidase treatment of human fetal liver or adult marrow cells prior to culture results in an increased number of erythroid colonies and bursts. No increase occurs in the number of nonerythroid colonies. The number of bursts having more than eight subunits is increased preferentially. Individual burst subunits are also enlarged. Neuraminidase-treated cells yield erythroid bursts when cultured in concentrations of erythropoietin insufficient to produce bursts from untreated cells. It is proposed that (1) neuraminidase treatment of adult and fetal cell mixtures specifically stimulates differentiation of erythroid precursors, (2) the preferential stimulation of erythroid bursts having many subunits suggests a preferential susceptibility of more primitive BFU-Es, and (3) neuraminidase treatment enhances the response of erythroid precursors to erythropoietin.