ALBERT

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

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

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

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

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

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

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

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

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

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

Description: Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.

Description: This study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se- methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms.

Description: We have measured the fully carboxylated (native) prothrombin antigen and the undercarboxylated (abnormal) prothrombin antigen in patients treated with sodium warfarin using specific immunoassays to evaluate a new approach for monitoring oral anticoagulant therapy. Plasma and serum samples (391) were assayed for the prothrombin time, native prothrombin antigen, and abnormal prothrombin antigen. The results were correlated with the presence of bleeding or thromboembolic complications at the time of phlebotomy. The native prothrombin antigen correlated with the occurrence of complications in 95% of samples. Of 13 samples from patients with bleeding complications, 13/13 (100%) had a native prothrombin of 12 micrograms/mL or lower. Of seven samples from patients with thromboembolic complications, 6/7 (86%) had a native prothrombin of 24 micrograms/mL or greater. By comparison, a prothrombin time index of 1.5 to 2.5, 1.5 to 2.2, 1.5 to 2.0, or 1.3 to 1.8 identified 6/20 (30%), 9/20 (45%), 11/20 (55%), or 12/20 (60%) patients at risk, respectively. Although the prothrombin time index did correlate with the presence of bleeding complications, the native prothrombin antigen correlated closely with the presence of bleeding and thromboembolic complications. According to these results, the native prothrombin antigen, maintained in a range of 12 to 24 micrograms/mL by regular adjustment of the warfarin dosage, may be associated with a reduced risk of complications due to excessive or insufficient warfarin therapy. On the basis of these preliminary data, we recommend that the native prothrombin antigen be considered to monitor warfarin therapy.

Description: High proliferative potential macrophage progenitor cells (HPP-CFC) in 5- fluorouracil (FU) treated and normal mouse bone marrow (BM) have been shown to be less sensitive to inhibition of proliferation by prostaglandins of the E series (PGE) than low proliferative potential macrophage progenitor cells (LPP-CFC) in normal BM in agar cultures. The growth of large colonies (diameter greater than 0.5 mm) derived from HPP-CFC in FU BM, which require a combination of macrophage colony- stimulating factor (CSF-1) plus a new growth factor called synergistic activity (SA), are inhibited by 50% in the presence of 5.5 X 10(-6) M PGE1. On the other hand, LPP-CFC in normal BM, which form smaller colonies (diameter less than or equal to 0.5 mm) in the presence of CSF- 1 alone, require only 5 X 10(-8) M PGE1 for the same level of inhibition. Addition of appropriate concentrations of PGE1 to the agar culture assay should improve detection of HPP-CFC by inhibiting the proliferation of LPP-CFC. These observations suggest that the apparent negative feedback control of macrophage production by PGE operates largely on the LPP-CFC, which respond to CSF-1 alone, and is probably not involved in the regulation of the more primitive HPP-CFC.

Description: We used both radiolabeled and fluorescein-labeled antiglobulins in assays to detect antibodies against platelets in multiply transfused patients to determine the value of these tests in predicting the outcome of platelet transfusion in such patients. In 15 allosensitized patients, we studied 68 single-donor platelet transfusions, 43 (63%) of which had a poor outcome, defined as a corrected count increment (CCI), less than 10,000. The results obtained with either test were significantly correlated with the CCI following transfusion (p less than 0.001), but the assay using the radiolabeled antiglobulin had slightly better sensitivity, specificity, and predictive value. When the assays were used in combination, there was again significant correlation with the CCI of the transfusion, p less than 0.001. When both assays predicted failure of the transfusions, 31/31 (100%) such transfusions resulted in a CCI of less than 10,000, and when both assays predicted success of the transfusions, 14/15 (93%) such transfusions resulted in a CCI of greater than 10,000. Both assays are useful in predicting the outcome of the platelet transfusions; when the assay results were concordant, almost total predictive accuracy was obtained.

Description: A 42-yr-old woman with systemic lupus erythematosus without bleeding diathesis developed a prolonged activated partial thromboplastin time that was not corrected by normal plasma. An inhibitor that acted rapidly and inactivated 0.5 U/ml plasma thromboplastin antecedent (PTA, factor XI) at a 1:200 plasma dilution was demonstrated. In addition to a low titer of PTA (less than 0.01 U/ml), plasma assayed at 20-fold dilution also showed low titers of Hageman (factor XII, 0.02 U/ml), Fletcher (plasma prekallikrein, 0.02 U/ml), and Fitzgerald (high molecular weight kininogen, less than 0.01 U/ml) factors. The titer of these factors, except PTA, returned to normal upon further plasma dilution or upon removal of the inhibitor by protein A adsorption. Thus, the inhibitor appeared to interfere with these clotting factor assays, possibly by inactivating PTA in the substrate plasmas in the test system. Its specificity was further confirmed. The inhibitor did not interfere with surface-induced proteolytic cleavage of Hageman factor. Surface-induced generation of plasma kallikrein activity (amidolysis of H-D-pro-phe-arg-pNa and cold-promoted factor VII activity enhancement) requires only Hageman, Fletcher, and Fitzgerald factors and was normal. Reactions requiring all 4 contact phase factors, including PTA, such as surface-induced generation of plasmin activity (amidolysis of H-D-val-leu-lys-pNa) and activated Christmas factor (factor IXa) activity, were defective. Furthermore, the inhibitor bound to agarose-protein A inactivated and removed PTA selectively from normal plasma. The inhibitor was an IgG-lambda autoantibody that precipitated PTA. The inactivated activated PTA (factor XIa) without the requirement for an additional cofactor. Furthermore, it inhibited surface-induced activation of PTA by interfering with its proteolytic cleavage upon glass surface exposure and with its binding onto the reactive surfaces.

Description: Because clinical disorders of spontaneous iron overload have no experimental counterpart, we studied iron distribution (atomic absorption analysis) and intestinal absorption (59Fe) in mice with hereditary alpha-thalassemia. Mice heterozygous for a radiation-induced alpha-Hb gene deletion exhibit a mild hemolytic anemia, like the human condition, with microcytosis, reticulocytosis, splenomegaly, and chemical evidence of defective alpha-chain synthesis. Quantitative iron determination showed that total iron content in spleen, liver, and kidney, but not heart or lung, of adult alpha-thalassemic mice was greater (P less than .05) than that in unaffected littermates. Iron concentration was also increased in liver (P less than .001), spleen (P = .025), kidney (P = .058), and heart (P = .010); in general, the greater the iron concentration in liver, the greater that in spleen (r = .39, P = .009), kidney (r = .70, P less than .001), and heart (r = .46, P less than .001). In mice examined 8 months postoperatively, splenectomy, as compared to sham operation, significantly raised iron content in extrasplenic tissues, but did not affect total body iron. At 10–11 weeks of age, but no longer at 12–14 weeks, thalassemic mice showed higher rates of iron absorption than age-matched controls. Thus, alpha-thalassemic mice display an early occurring iron absorption defect, leading to a modest, sustained, nonprogressive iron overload, and thereby represent a valuable model for exploring disorders of iron homeostasis.

Description: The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.

Description: Peripheral blood granulocytes from patients with chronic myelogenous leukemia (CML) were studied for accessibility of membrane sialic acid and galactose residues to sodium borohydride-3H radiolabeling after oxidation with sodium metaperiodate (PI/B3H4) or with galactose oxidase (GO/B3H4). Granulocytes from untreated patients with chronic myelogenous leukemia showed increased radiolabeling with PI/B3H4, and decreased labeling with GO/B3H4 when compared to normal granulocytes. Granulocytes from leukemic patients receiving chemotherapy showed normal labeling patterns. Gel electrophoresis of membrane extracts showed that the changes in PI/B3H4 and GO/B3H4 reactivity of CML cells were distributed over all membrane protein bands. Our data suggest that CML granulocyte membrane proteins are aberrantly sialylated, with decreased accessibility of galactose residues, and that these changes may be reversed by clinical drug treatment.

Description: We present a colony assay system that allows in situ identification of human basophil/mast cell (basophil) colonies. In methylcellulose culture, in the presence of phytohemagglutinin-leukocyte conditioned media (PHA-LCM), human peripheral blood and bone marrow cells form colonies that can be distinguished by their unique morphological characteristics. Pure basophil colonies are diffuse, small colonies containing small, round, highly refractile cells. These characteristics of the constituent cells led us to the observation that a significant number of basophils are found in combination with eosinophils. The mixed eosinophil/basophil colonies have the distinctive elements of pure eosinophil and pure basophil colonies. Usually, these are diffuse colonies with compact clusters of slightly larger, darker-appearing cells. We also found colonies that contained basophils and neutrophils/monocytes, but this type could not be consistently identified by in situ morphology. Cytochemical analysis confirmed the metachromatic nature of the granules in the basophils. The presence of IgE receptors on the cells was documented by indirect immunofluorescent staining after passive sensitization with purified human IgE. Peripheral blood cells from six healthy volunteers formed 5.7 +/- 1.0 (mean +/- SEM) pure colonies in 2 X 10(5) cells. Cultures of bone marrow cells from patients with various types of anemia had 9.0 +/- 1.5 colonies in 10(5) cells. This is the first description of a colony assay system for in situ identification of a pure population of basophilic granulocytes.

Description: Morphological, immunologic, and functional properties of peripheral blood cells from two patients with chronic proliferations of granular lymphocytes are described. Cells from both patients showed a heterogeneous pattern from both a morphological and immunologic standpoint, indicating a polyclonal, rather than a monoclonal, expansion of these cells. In fact, both large and small-to-medium-sized granular lymphocytes were observed, and different percentages of positivity were found in the analysis with a large panel of monoclonal antibodies. Serologic and histologic features support the hypothesis that this lymphocytosis could be secondary to bacterial or viral infections rather than a primary event, suggesting that these patients may have chronic reactive immunoregulatory disorders.

Description: This report describes the experience of the Southeastern Cancer Study Group (SECSG) with a transport medium used for immunologic phenotyping of non-Hodgkin’s lymphomas. In a 2-mo pilot study, portions of 53 specimens of non-Hodgkin's lymphoma from four member institutions of the SECSG and affiliated community hospitals were sent by regular mail to a central laboratory. Immunologic phenotyping was carried out using a frozen section immunoperoxidase technique. In 48 of the cases, a clear-cut immunologic phenotype was obtained. Thirty-four tumors were of B cell origin and 7 had T cell markers. Six of the remaining lymphomas had neither B nor T cell markers, and the seventh had both. In 12 cases, phenotyping was also carried out at the originating institution using conventional cell suspension techniques; agreement between the two methods was excellent. The immunologic results were correlated with histopathologic diagnosis standardized using the Working Formulation for non-Hodgkin’s lymphomas. It was found that the low grade tumors were all B cell, but that the intermediate grade tumors were very heterogeneous immunologically. About one-fourth of the diffuse, intermediate grade or miscellaneous tumors had T cell markers. Our results indicate that immunologic phenotyping may be performed satisfactorily on transported material, making multiinstitution studies on the prognostic significance of immunologic phenotype in non- Hodgkin’s lymphomas feasible.

Description: In the baboon (Papio species), the two nonallelic gamma-genes produce gamma-chains that differ at a minimum at residue 75, where isoleucine (I gamma-chain) or valine (V gamma) may be present. This situation obtains in baboons that are sometimes designated as Papio anubis, Papio hamadryas, and Papio papio. However, in Papio cynocephalus, although the I gamma-chains are identical with those in the above mentioned types, the V gamma-chains have the substitutions ala----gly at residue 9 and ala----val at residue 23. The V gamma-chains of P. cynocephalus are called V gamma C to distinguish them from the V gamma A-chains of P. anubis, etc. A single cynocephalus animal has been found to have only normal I gamma-chains and I gamma C-chains (that is, glycine in residue 9, valine in 23, and isoleucine in 75). When HbF is produced in response to stress with 5-azacytidine, P. anubis baboons respond with greater production than do P. cynocephalus, and hybrids fall between. Minimal data on P. hamadryas and P. papio suggest an even lower response than P. cynocephalus. As HbF increases under stress, the ratio of I gamma to V gamma-chains changes from the value in the adult or juvenile baboon toward the ratio in the newborn baboon. However, it does not attain the newborn value. The V gamma A and V gamma C-genes respond differently to stress. In hybrids, the production of V gamma A- chains exceeds that of V gamma C-chains. A controlling factor in cis apparently is present and may be responsible for the species-related extent of total HbF production. It may be concluded that the more primitive the cell in the erythroid maturation series that has been subjected to 5-azacytidine, the more active is the I gamma-gene.

Description: In the baboon (Papio species), the two nonallelic gamma-genes produce gamma-chains that differ at a minimum at residue 75, where isoleucine (I gamma-chain) or valine (V gamma) may be present. This situation obtains in baboons that are sometimes designated as Papio anubis, Papio hamadryas, and Papio papio. However, in Papio cynocephalus, although the I gamma-chains are identical with those in the above mentioned types, the V gamma-chains have the substitutions ala----gly at residue 9 and ala----val at residue 23. The V gamma-chains of P. cynocephalus are called V gamma C to distinguish them from the V gamma A-chains of P. anubis, etc. A single cynocephalus animal has been found to have only normal I gamma-chains and I gamma C-chains (that is, glycine in residue 9, valine in 23, and isoleucine in 75). When HbF is produced in response to stress with 5-azacytidine, P. anubis baboons respond with greater production than do P. cynocephalus, and hybrids fall between. Minimal data on P. hamadryas and P. papio suggest an even lower response than P. cynocephalus. As HbF increases under stress, the ratio of I gamma to V gamma-chains changes from the value in the adult or juvenile baboon toward the ratio in the newborn baboon. However, it does not attain the newborn value. The V gamma A and V gamma C-genes respond differently to stress. In hybrids, the production of V gamma A- chains exceeds that of V gamma C-chains. A controlling factor in cis apparently is present and may be responsible for the species-related extent of total HbF production. It may be concluded that the more primitive the cell in the erythroid maturation series that has been subjected to 5-azacytidine, the more active is the I gamma-gene.

Description: Fibrin prepared from 15 pathologic thrombi was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the extent and pattern of fibrinolysis that occurs in vivo. Two groups of patients could be distinguished on the basis of the polypeptide chain composition of fibrin in their thrombi. Those patients who presented with acute vascular obstruction, either arterial or venous, showed a minimal degree of fibrin degradation, with a dominance of intact, undegraded crosslinked gamma-gamma dimers. On the other hand, patients with long-standing symptoms associated with chronic aortic aneurysms had thrombi containing extensively degraded fibrin. Thrombi in large aortic aneurysms were dissected into concentric layers that showed different degrees of fibrinolysis. The luminal surface consisted of fresh, red thrombus and contained undegraded crosslinked fibrin similar to that found in patients with acute occlusive disease. Deeper layers of the thrombus showed gamma-gamma chain degradation throughout, indicating that this portion was undergoing active thrombolysis. The findings demonstrate that the variability in the pathophysiologic balance between coagulation and fibrinolysis is reflected in vivo by the polypeptide chain composition of crosslinked fibrin in thrombi. The results support the hypothesis of a dynamic equilibrium between clotting and lysis, but indicate that the balance between these two processes may be distinctly different in separate areas of a single clot.

Description: The fucose-binding lectin from Lotus tetragonolobus ( FBL -L) has been previously shown to bind specifically to normal cells of the myeloid and monocytic lineages. The purpose of this study was to explore the utility of fluoresceinated FBL -L as a leukemia differentiation marker in conjunction with a panel of other frequently used surface markers (Fc receptor, HLA-DR, OKM1, and antimonocyte antibody). FBL -L reacted with leukemic cells in 8/9 cases of clinically recognized acute myeloid leukemia, including myeloid blast crisis of chronic granulocytic leukemia, 3/3 cases of chronic phase chronic myelogenous leukemia, and in 2/7 cases of clinically undifferentiated acute leukemia. Correlations were noted between reactivity with FBL -L, and DR and Fc receptor expression. Among continuous cell lines, FBL -L bound with high intensity to a majority of HL-60 and U937 cells. The less well differentiated myeloblast cell lines, KG-1, KG1a , and HL-60 blast II, exhibited less FBL -L binding than HL-60 and U937. A moderate proportion of K562 cells exhibited low level binding of FBL -L. Several lymphoblastic cell lines exhibited a pattern of low intensity binding that was distinguishable from the high intensity binding pattern of the myeloblastic lines. FBL -L reactivity of U937 was enhanced by induction of differentiation with leukocyte conditioned medium, but not dimethylsulfoxide. Such treatments induced contrasting patterns of change of HL-60 and U937 when labeled with OKM1, alpha-Mono, and HLA- DR. These studies demonstrate the application of FBL -L to analysis and quantitation of myelomonocytic leukemic differentiation.

Description: To investigate the cellular events that accompany erythroid hyperplasia, we studied several effects of erythropoietin (Epo) on marrow CFU-E in sickle cell anemia (SCA). We measured CFU-E number, CFU- E growth as a function both of Epo exposure time and of Epo concentration, and suppression of Epo-induced CFU-E formation by anti- Epo antiserum. With 0.5 U Epo/ml, the number of CFU-E was elevated in SCA (1,087 +/- 520) compared to normal (430 +/- 130). CFU-E were formed even when Epo was immediately neutralized by a 1/150 dilution of anti- Epo. After 40 hr of Epo exposure, only 2% of total CFU-E were expressed in normal marrow, whereas 12%-40% of CFU-E were expressed in SCA. Inhibition of CFU-E growth required at least 1/50 dilution of anti-Epo in SCA and a 1/300 dilution in normal marrow. In contrast to normal, a small number (5%-20%) of CFU-E were expressed in the absence of added Epo in SCA, and this pool required a 1/150 dilution of anti-Epo for inhibition. The Epo dose-response curve in SCA revealed a peak in colony formation around 0.1 U Epo/ml and 0.5 U Epo/ml, whereas only one peak at 0.5 U Epo/ml was seen in normals. These data strongly suggest that, in response to the demands of chronic erythroid hyperplasia in SCA, a pool of CFU-E is present characterized by increased in vitro sensitivity to Epo.

Description: Thirteen patients with acute leukemias that were difficult to classify by the use of cytochemical staining and terminal deoxyribonucleotidyl transferase (TdT) activity are reported. The phenotype of the leukemic cells was characterized by the presence of mature or early monocyte lineage antigens and intense Ia antigen expression detected by monoclonal antibodies, terminal deoxytransferase activity, and cytochemical features, including both Sudan black B and periodic acid- Schiff activity. The mean age of this group of patients was 60 years. Five patients had leukemia occurring after chemotherapy or radiotherapy of a prior malignant disease, and two patients had a refractory anemia prior to development of acute leukemia. These patients had a low response rate to chemotherapy. This series of leukemia appears to form a distinct nosologic entity, representing a leukemic transformation among early cells of the monocyte lineage, resulting in a predominant neoplastic cell that is less mature than either the French-American- British M4 acute myelomonocytic leukemia or M5 acute monoblastic leukemia. The presence of terminal deoxytransferase activity was interpreted as indicating the primitive state of the cells in the differentiation sequence, rather than as implying any significance with respect to lineage.

Description: Levels of platelet-associated IgG (PA-IgG) were studied in 72 patients with Hodgkin–s (HD) and non-Hodgkin–s lymphoma (NHL). Thirty-nine percent of patients with HD and 20% of patients with NHL had elevated PA-IgG levels. There was a positive correlation between disease activity and the presence of PA-IgG in HD and NHL. In patients with HD, PA-IgG strongly correlated with extent of disease and may serve as a marker of disease activity. PA-IgG may have facilitated platelet destruction in 5 of 11 thrombocytopenic patients with HD and increased PA-IgG and in 2 patients with HD and increased PA-IgG who developed severe thrombocytopenia when treated with chemotherapy.

Description: The structure of factor IX gene was analyzed in a hemophilia B patient with inhibitor. Genomic DNA, digested with a variety of restriction endonucleases, was hybridized with the cDNA and various genomic factor IX probes. A large subtotal deletion of the gene was observed. The borders of the deletion span from a approximately 125 nucleotide region within the last exon to an unknown domain at least 7.5 kb upstream from the first exon: it thus involves approximately 33 kb of the factor IX locus. The abnormal gene was inherited by the daughter of the propositus, who showed both the normal and the deleted allele.

Description: Sedimentation analysis of factor VIII complex was performed in the analytical ultracentrifuge using partition cells. This method allowed for the calculation of three different sedimentation coefficients from each run: one based on ristocetin agglutination activity for von Willebrand protein, SWF; one based on coagulant activity for factor VIIIC, SVIIIC; and one based on the schlieren or adsorption data for protein concentration, Sconc. In most cases, there was no agreement between the three values calculated from the same run, indicating a heterogeneous system. The calculated functional sedimentation coefficients give values that require the molecules to be highly asymmetric to be consistent with a glycoprotein of high molecular weight, which is in agreement with results observed in electron microscope studies. The dissociation of VIIIC into a smaller form can be demonstrated by this method. Determination of the three sedimentation coefficients in a series of fractions from gel filtration indicates a uniform size for the VIIIC activity but not for the WF activity. These observations are in agreement with the concept of a copolymer between WF and VIIIC and also with the concept of separate polymers for the two activities.

Description: Two molecular defects involving the spectrin heterodimer (SpD) contact site of the alpha chain (the alpha I domain) were previously identified using limited tryptic digestion followed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both are characterized by atypical peptide maps which reveal a marked decrease of the 80,000-dalton alpha I domain and a formation of new major peptides of either 74,000 (Sp alpha I/74) or 46,000 (Sp alpha I/46) daltons. We now report a third variant of the spectrin alpha chain, designated Sp alpha I/65, in three unrelated black families. In all three probands, the percentage of SpD in the low ionic strength (O degrees C) membrane extracts was increased to 19% to 32%. One- and two- dimensional electrophoretic separations of limited tryptic digests of spectrin from all three probands revealed a decrease of the alpha I domain of spectrin and the concomitant appearance of peptides at 65,000 daltons and isoelectric points ranging from 5.2 to 5.3. The abnormal 65,000-dalton peptides could be stained with an antiserum which had been raised against the alpha I domain, indicating that it was derived from the alpha I domain.

Description: Monoclonal antibodies (MoAbs) to myeloid differentiation antigens have a potential use in purging bone marrow of leukemia cells in autologous bone marrow transplantation (ABMT) therapy for acute myelogenous leukemia (AML). Because the efficiency of purging by MoAb and complement (C) is important to the success of ABMT, we have designed an assay to determine optimal conditions for leukemia cell lysis. In order to mimic the conditions of remission bone marrow, normal buffy coat cells were mixed with cells from the HL-60 promyelocytic leukemia cell line at a concentration that approximated the normal-leukemia cell ratio found in remission marrow. The cell mixture was treated at variable times and temperatures in the presence of C and PM-81, an IgM MoAb that reacts with both normal granulocytes and monocytes as well as with HL-60 cells. PM-81 binds to the majority of cells from 90% of patients with AML yet does not react significantly with normal stem cell populations. Because of the potential use of PM-81 in ABMT, it seemed especially important to show that the antibody was capable of mediating cytotoxicity of HL-60 cells in the presence of an excess of antigen-positive cells. A clonogenic assay that permitted the growth of HL-60 cell colonies but not normal progenitor cells in methylcellulose cultures was used to measure the efficiency of HL-60 cell lysis. We found that under certain conditions, PM-81 was capable of removing the small percentage of HL-60 clonogenic cells admixed with normal buffy coat cells. This information was useful for determining the optimal conditions for purging bone marrow of leukemia cells for ABMT.

Description: The malignant monoclonal population in hairy cell leukemia (HCL) has been variously ascribed to be of myeloid, B, or even T cell origin. Recent data have been interpreted as suggesting that hairy cells (HC) may concomitantly or serially express both B and T surface determinants, a phenomenon which, if verified, would be unique among the lymphoproliferative malignancies. Data described here, however, demonstrate that (1) at least the majority of HCL are phenotypically of B cell derivation, and (2) the initial B cell phenotype is retained and solely expressed on cultured as well as phytohemagglutinin (PHA) activated monoclonal malignant HC.

Description: The expression of Rhesus antigens on hematopoietic progenitor cells was studied using monoclonal antibodies. Because these antibodies are not capable of lysing mature red blood cells in a complement-dependent cytotoxicity assay, fluorescence-activated cell sorting was performed. Using the monoclonal anti-Rh 29 antibody B10, 68% +/- 6% of the mature erythroid progenitor cells (CFU-E) were sorted into the positive fraction, while only 2% +/- 1% of the relatively immature erythroid progenitor cells (BFU-E), and 3% +/- 1% of the granulocyte-macrophage progenitor cells (CFU-GM) were cultured from this same fraction. Thus up to a 15-fold enrichment of CFU-E could be obtained. In two experiments more than 4% of the cells in the positive fraction consisted of CFU-E; in one experiment even more than 7% did. Using fractionated cell sorting, the Rhesus antigens appeared to have a lower density on CFU-E than HLA-DR determinants. Antibodies against the Rhesus antigens can be applied to enrich erythroid-committed stem cells and to separate mature from immature erythroid progenitor cells.

Description: Surface membrane glycoproteins (SMGs) of cells from the parental wild- type HL-60 cell line and from three sublines variably cross-resistant to the granulocyte differentiation-inducing effects of retinoic acid (RA), dimethylsulfoxide (DMSO), and certain purine bases (6-thioguanine [6TG] or hypoxanthine) were studied by one-dimensional and two- dimensional gel electrophoresis. After both oligosaccharide (periodate/borotritide) and peptide (1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycouril) ectolabeling procedures, striking common changes were noted in the gel electrophoretic patterns of the SMGs from the RA- and 6TG-resistant sublines compared to those from the wild-type HL-60 line or the DMSO-resistant subline. Most prominently, this included the presence in the RA- and 6TG-resistant cells of an apparent high molecular weight acidic glycoprotein(s) (mol wt, 200 to 285 kilodaltons [kD]; isoelectric point range [pl], 4.5 to 6.0) not observed in the wild-type or DMSO-resistant cells and, conversely, the presence of a lower molecular weight glycoprotein(s) (mol wt, 120 to 165 kD; pl, 4.2 to 5.9) in the wild-type and DMSO-resistant cells, which was absent or much reduced in the RA- and 6TG-resistant cells. These acidic SMGs did not change as a function of the induction of granulocyte differentiation. However, some other more basic SMGs varied as a function of granulocyte differentiation in both the wild-type and differentiation inducer-resistant sublines, including the loss of the transferrin receptor and the gain of a mol wt 55- to 60-kD neutrophil- associated protein. In the context of previously reported information, these results indicate (1) that the overall pattern of SMG changes in the RA- and 6TG-resistant cells closely resembles that associated with multidrug (pleiotropic) resistance to cytotoxic agents in a variety of mammalian cells; (2) that the RA/6TG resistance-associated SMG changes are not granulocyte differentiation stage-specific; and (3) that either the RA/6TG resistance-associated SMG changes are not related to the resistance mechanism or they are involved in the resistance/cross- resistance mechanism(s) for RA/purine bases but not for DMSO.

Description: The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+- dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.

Description: To study the influence of a biologic environment on cultured human leukemia cells, KG-1, KG-1a, and HL-60 cells were inoculated subcutaneously into newborn nude mice. The cells developed myelosarcomas at the site of inoculation and in lungs and kidneys. KG-1 and HL-60 myelosarcomas were successfully passaged through adult nude mice, whereas KG-1a tumors proliferated only after transplantation into newborn hosts. The human nature of the cells forming myelosarcomas in mice was assessed by chromosomal analyses and detection of cross- reactivity with an antibody to the human leukemia cell line K562. We undertook electron microscopic and cytochemical examinations of the cells proliferating in vitro and in the mice. The granules of KG-1 cells in vivo did not react for acid phosphatase, as observed in vitro, and the HL-60 cells proliferating in mice lost the perinuclear myeloperoxidase (MPO) demonstrated in cultured cells. Although the influence of an in vivo selection of cell subpopulations cannot be ruled out, the enzymatic changes are compatible with induced cell differentiation. Conclusive evidence of differentiation in vivo was observed in the KG-1a cell subline. The undifferentiated KG-1a blasts developed cytoplasmic granules and synthesized MPO during proliferation in vivo. These observations indicate that human leukemia cells from established cell lines proliferate in nude mice and may acquire new differentiated properties in response to the in vivo environment.

Description: The Southeastern Cancer Study Group conducted a post-remission induction randomized trial in adult acute myelogenous leukemia to assess the efficacy of alternate drug therapy during consolidation and of immunotherapy during maintenance. Of 508 evaluable patients entered into the study, 335 (66%) achieved a complete remission treated with a 7-day infusion of cytosine arabinoside at a dose of 100 mg/sq m/day and 3 days of daunorubicin at a dose of 45 mg/sq m/day. Those in remission were randomized to receive 3 courses of 1 of 3 consolidation regimens: (A) a continuous infusion of 5-azacytidine, 150 mg/sq m/day for 5 days; (B) 5-azacytidine plus beta-deoxythioguanosine, 300 mg/sq m/day for 5 days; or (C) cytosine arabinoside, 100 mg/sq m/day intravenously, and thioguanine, 100 mg/sq m orally every 12 hr, plus daunorubicin, 10 mg/sq m every 24 hr daily for 5 days. There was no difference in relapse rate among the 3 arms. Those completing consolidation and remaining in remission were randomized to 1 of 3 maintenance regimens: (D) chemotherapy, 5-day infusion of cytosine arabinoside and 2 days of daunorubicin (same doses as induction) given every 13 wk for 1 yr; (E) BCG given twice weekly for 1 mo and then monthly for 1 yr; or (F) the combination of regimens D and E. The median duration of remission was significantly better on regimen D (17.4 versus 9.4 and 9.5 mo), and median survival was 29 mo compared to 21 mo for the other regimens. Those given different drugs during consolidation than used for induction (regimens A and B) and subsequent chemotherapy for maintenance (regimen D) had the longest remission durations and survival. Immunotherapy was not as good as intensive chemotherapy for maintenance.

Description: The density and size of human erythrocytes has been roughly correlated with cell age, with the denser and smaller cells being older. Observations of this type have led to a hypothesis that the membranes of circulating erythrocytes are dynamic with respect to composition and that material is lost from the membrane during cell maturation and circulation. In this study, flow cytofluorimetry was used to investigate the distribution of the human erythrocyte anion transport protein (protein 3) in heterogeneous samples of circulating red cells. We verified that protein 3 can be specifically and quantitatively labeled in intact human erythrocytes with eosin-5-maleimide, a luminescent probe. Individual cells were accordingly analyzed for size by forward light scattering and for protein 3 content by quantitation of eosin fluorescence. Initial results indicated that the smallest erythrocytes had a protein 3 content equal to that of the largest circulating erythrocytes. This result was independently verified by light scatter-activated cell sorting; direct measurement of cell diameters by microscopy verified that the cell sizes of erythrocytes showing the 10% greatest and 10% smallest light-scattering signal were indeed distinct. Independent analysis of the size-sorted erythrocytes for protein 3 content was accomplished by gel electrophoresis of stroma from 150,000 large and small erythrocytes. Quantitative scanning densitometry of silver-stained gels of prepared stroma showed that protein 3 content of each set of fractionated cells was equal and did not vary as a function of cell size. Taken in combination with the reported correlation between increasing red blood cell age and decreasing cell size, these results indicate that any loss of membranous material during the cell aging process is not random.

Description: A long-term liquid culture system of hemopoietic tissue derived from adult hamster spleen has been described. These primary liquid cultures can maintain stem cell proliferation and differentiation for more than three months without secondary repopulation. A characteristic of the liquid cultures is the formation of clusters of hemopoietic cells around adherent stromal cells. Some islands were composed exclusively of megakaryocytes and adherent cells. Isolation of these clusters of differentiating megakaryocytes and their adherent cellular substrate permitted the analysis of the morphological and ultrastructural features of the interaction between cells of megakaryocytic lineage with the adherent stroma.

Description: We have used immunogold staining to locate thrombospondin (TSP) on thrombin-activated human platelets, and have compared its distribution with that of fibrinogen (or fibrin) on thrombin- and ADP-stimulated platelets. To do this, isolated platelets were incubated with monospecific antibodies to TSP or fibrinogen (fib) and the bound IgG located with a second antibody adsorbed to gold particles. Thrombin- induced secretion in Tyrode-Ca2+ was followed by both anti-TSP and anti- fib binding, with large clusters of gold particles observed on the platelet surface. Little or no labeling was observed on unstimulated platelets with either antibody. When secretion was effected in Tyrode- EDTA, anti-TSP IgG still bound to the activated platelets, but the number of particle clusters was significantly reduced. Little binding of anti-fib IgG now occurred. Platelets activated with ADP in the presence of added fib, and subsequently incubated with anti-fib IgG, showed small particle clusters over the whole platelet surface. Thrombin-stimulated platelets from two patients with thrombasthenia bound anti-TSP IgG similarly to normal platelets activated in Tyrode- EDTA. No anti-fib binding occurred. Our results suggest that fib and TSP bind to specific domains on the stimulated platelet membrane. Such sites may be responsible for the mediation of platelet surface contact interactions.

Description: Samples of leukemic cell DNA from 14 children with acute nonlymphocytic leukemia (ANLL) and 4 human myeloid leukemia cell lines were analyzed for rearrangement in the heavy chain region of the immunoglobulin gene. The diagnosis of ANLL was confirmed in all patients by morphological, cytochemical, and immunologic studies. By restriction endonuclease digestion and hybridization with cloned heavy chain immunoglobulin gene probes for the constant (Cmu) and joining (JH) regions, the DNA of 2 patients and 1 cell line (ML-1) was found to contain rearrangements. The DNA from the remaining 12 patients and 3 cell lines was not rearranged (germline configuration). Both patients with apparent immunoglobulin gene rearrangement achieved complete remission on therapy for ANLL. Immunoglobulin gene rearrangement in phenotypically defined ANLL suggests (1) that such changes may not be limited to lymphoid leukemia of B cell lineage, or (2) that, in some patients, the leukemic transforming event may involve stem cells capable of both B cell and myeloid differentiation.

Description: Protein-heparin complexes, prepared by a water-soluble carbodiimide coupling technique, were used to produce anti-heparin antibodies in rabbits. Antiserums that recognized carbodiimide-treated heparin, but not untreated heparin, were obtained. Carbodiimide-treated heparan sulfate exhibited 10% to 20% cross-reactivity compared with a similarly treated heparin, whereas there was no cross-reactivity with five other carbodiimide-treated mucopolysaccharides. 3H-1-ethyl-3-(3- trimethylammoniumpropyl) carbodiimide iodide was used to demonstrate that carbodiimide forms a stable adduct with heparin and other mucopolysaccharides. Using an antibody fraction that eluted from 1- ethyl-3-(3-trimethylammoniumpropyl) carbodiimide iodide-treated heparin- Sepharose with 2 mol/L KI, it was demonstrated that, for the antibody population studied, the addition of one carbodiimide per heparin molecule resulted in complete epitope expression without loss of anticoagulant activity. The addition of up to eight additional carbodiimide molecules to heparin did not increase the extent of epitope formation, although anticoagulant activity was lost. Except for heparan sulfate, the addition of radiolabeled carbodiimide to other mucopolysaccharides did not result in epitope formation. These data demonstrate that antibodies to an epitope derived from heparin can be formed, that the epitope is fully expressed while anticoagulant activity is present, and that the antibody is specifically directed against an altered portion of the polysaccharide.

Description: We tested conditioned media from 12 patients with T lymphocyte neoplasms and four T cell lines for their ability to stimulate the in vitro growth of erythroid-burst-forming units (BFU-E) from bone marrow mononuclear cells in a methylcellulose culture system. Nine patients suffered from acute lymphocytic leukemia, two from chronic lymphocytic leukemia, and one from non-Hodgkin's lymphoma. The T lymphocytes were characterized by a series of monoclonal antibodies and their stage of development was correlated with their ability to produce burst- promoting activity (BPA). Conditioned media from cells classified as prothymocytes (three cases), common thymocytes (one case), mature thymocytes (three cases), and mature lymphocytes of the helper subtype (two cases) increased BFU-E proliferation four- to 19-fold over control values using normal bone marrow as target cells. Conditioned media from OKT8+ malignant T lymphocytes (three cases) did not enhance BFU-E proliferation. Conditioned media from cells classified as immature T cells stimulated CFU-GM proliferation in only one of seven cases even though they secreted BPA. Conditioned media from three of the four cell lines stimulated by phytohemagglutinin, enhanced BFU-E growth. Our results indicate that malignant cells that have characteristics of immature T cells are able to produce BPA. Studies using techniques to isolate homogeneous populations of normal T cell subsets are required to determine whether normal immature T lymphocytes have the same capability.

Description: Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well- characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross- reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin- binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.

Description: Patients with multiple myeloma (MM) are at an increased risk for infections with bacteria that require opsonization with complement. Because Streptococcus pneumoniae is the most frequently encountered pathogen in these patients, we investigated the ability of serum from patients with MM to mediate the binding of C3b, the major opsonin of the complement system, to S. pneumoniae. S. pneumoniae types 3, 14, and 25 were chosen for study, since S. pneumoniae type 3 activates primarily the classical complement pathway (CCP), type 25 primarily the alternative complement pathway (ACP), and type 14 both pathways. S. pneumoniae were treated with normal serum or serum from 17 patients with MM, and the bound C3b was quantified with fluorescein-conjugated anti-C3 in a spectrophotofluorometric assay. Despite normal or elevated serum concentrations of C3, total hemolytic complement, and C-reactive protein in all of the MM sera, factor B in 16/17 such sera, and C4 in 14/17 MM sera studied, all 17 sera demonstrated a defect in C3b binding to type 3 (32.7% +/- 6% of normal). In addition, serum from 15/17 patients bound decreased amounts of C3b to types 14 (39.6% +/- 8%) and 25 (52.2% +/- 8%). Mixing normal serum with MM serum restored MM C3b binding activity to all three S. pneumoniae types, suggesting that the defect was related to a deficiency rather than an inhibitor of C3 activation. Although MM patients are unable to produce specific antibodies to bacterial antigens, the addition of anti-S. pneumoniae antibodies to MM serum did not enhance C3b binding to any of the S. pneumoniae types. However, when S. pneumoniae were opsonized in a mixture of MM serum and C3-depleted normal serum, C3b binding was restored to all three S. pneumoniae types, demonstrating that MM C3 functions normally in the presence of other normal serum factors. In the present studies, the MM C3b binding defect appeared to correlate with the incidence of S. pneumoniae infections. Serum from patients with a history of an S. pneumoniae infection bound significantly less C3 (20.5% +/- 4%) than those study patients without a history of an S. pneumoniae infection (55.8% +/- 8%) (p less than 0.0025). Thus, MM serum has a defect in the activation of C3, and this may contribute to the increased susceptibility of MM patients to S. pneumoniae infections.

Description: The inhibition of delta-aminolevulinic acid (ALA) synthase activity by heme is commonly thought to regulate the overall rate of heme synthesis in erythroid cells. However, since heme inhibits erythroid cell uptake of iron from transferrin, we have tested the hypothesis that in reticulocytes heme regulates its own synthesis by controlling the cellular acquisition of iron from transferrin rather than by controlling the synthesis of ALA. We found that hemin added to reticulocytes in vitro inhibits not only the total cell incorporation of 59Fe from transferrin but also the incorporation of [2–14C]-glycine and transferrin-bound 59Fe into heme. However, hemin did not inhibit [2 –14C]-glycine incorporation into protoporphyrin. Furthermore, cycloheximide, which increases the level of non-hemoglobin heme in reticulocytes, also inhibited [2–14C]-glycine into heme but not into protoporphyrin. With high concentrations of ferric pyridoxal benzoylhydrazone (Fe-PBH), which, independent of transferrin and transferrin receptors, can be used as a source of iron for heme synthesis in reticulocytes, significantly more iron is incorporated into heme than from saturating concentrations of Fe-transferrin. This suggests that some step (or steps) in the pathway of iron from extracellular transferrin to protoporphyrin limits the overall rate of heme synthesis in reticulocytes. In addition, hemin in concentrations that inhibit the utilization of transferrin-bound iron for heme synthesis has no effect on the incorporation of iron from Fe-PBH into heme. Our results indicate that in reticulocytes heme inhibits and controls the utilization of iron from transferrin but has no effect on the enzymes of porphyrin biosynthesis and ferrochelatase. This mode of regulation of heme synthesis may be a specific characteristic of the hemoglobin biosynthetic pathway.

Description: Leukemic blasts from 774 children with newly diagnosed acute lymphocytic leukemia (ALL) have been phenotyped by microcytotoxicity testing with a panel of monoclonal antibodies and heteroantisera as part of a Pediatric Oncology Group classification study of acute leukemia. One hundred twenty-two cases, or 16% were designated as T cell leukemia based on the reactivity of blast cells with previously well-characterized antisera (PT) against a T lymphocyte-associated antigen. Using this antisera-based definition as a standard, we looked for a monoclonal antibody combination that would be a suitable substitute. An algorithm calling for reactivity with either monoclonal antibody 3A1 or Leu-1 was a 92% sensitive and 97% specific predictor of PT reactivity. Only 27 of 755 cases of leukemia were incorrectly classified using this algorithm. Subsequently, Ficoll-Hypaque-separated bone marrow cells from 118 additional patients with ALL (21 of whom had T cell ALL) were stained by immunofluorescence using a combination of directly fluoresceinated 3A1 and Leu-1. Reactivity of 20% or more of the cells with this antibody combination was a 100% sensitive and 94% specific indicator of T cell ALL defined by PT positivity; with a higher cutoff value for positive values, or the use of supplemental tests, even this small number of false-positives could be eliminated. We conclude that this monoclonal antibody combination is a satisfactory replacement for our heteroantisera definition of T cell ALL.

Description: The effect of a zinc metalloprotease from Serratia marcescens on platelet surface glycoproteins (GP) Ib and V was analyzed. Increasing protease treatments caused progressive loss of GP Ib with appearance of the major fragment, glycocalicin, in the supernatant solution. No GP V was detected in the supernatant solution, and protease-pretreated platelets had the same capacity as control platelets to release fragment 1 of GP V in response to thrombin. The Serratia protease- pretreated platelets did show the lag before thrombin-induced dense granule secretion, characteristic of platelets modified by pretreatment with other nonstimulating proteases. Treatment with Serratia protease gives the only demonstrated selective loss of GP Ib without apparent effect on GP V. It suggests that GP V (1) does not depend on GP Ib for its association with platelets and (2) is not the substrate for protease modification of platelet function.

Description: The blood platelet is the only human cell known to have a circumferential band of microtubules. However, the mechanisms involved in assembly of the multi-looped coil, its interaction with the cell membrane to support discoid shape, and constriction into tight rings around centrally concentrated organelles in activated platelets are unknown. Separation of the microtubule rings from intact platelets would permit new approaches to solution of these questions. The present study has used simultaneous detergent extraction and fixation to isolate intact microtubule coils in significant numbers from suspended platelets for the first time. Isolated coils closely resembled the circumferential band observed in thin sections of plastic embedded platelets and in platelets prepared by the negative-stain whole-mount method. Enough microtubule coils could be recovered from suspensions of concentrated platelets to permit counting and quantitation on microscope grids. Results of this study will permit new approaches to clarification of the structural physiology of platelet microtubule coils.

Description: Twenty-two patients with hairy cell leukemia were treated with biosynthetic (recombinant) alpha-2-interferon in an open-label, single- arm efficacy study. Patients received 2 X 10(6) U/m2 recombinant alpha- 2-interferon three times weekly. Therapy was well tolerated subjectively with minimal short-term hematologic toxicity. Two patients had bacterial infections during the period of study, and one patient experienced a short-lived readily reversible rejection of a corneal transplant. Statistical comparison of the mean hematologic indices at study entry and after three to six months of therapy with recombinant alpha-2-interferon indicates a significant improvement in hemoglobin, granulocyte, and platelet counts. Bone marrow biopsies in six of 14 patients after six months of therapy showed a greater than 50% decrease in the infiltration of leukemia cells. We conclude that recombinant alpha-2-interferon is highly effective therapy for hairy cell leukemia.

Description: Mitogen-stimulated murine spleen cells produce humoral substances capable of supporting murine hematopoiesis and pluripotent stem cell proliferation in vitro. Thus, we evaluated conditioned media generated by human spleen cells (SCM) in the presence or absence of mitogens for factors stimulatory for human pluripotent (CFU-GEMM), erythroid (BFU- E), and myeloid (CFU-GM) precursors. Two and one half percent to 10% SCM stimulated proliferation of all three types of precursor cells from nonadherent buoyant human marrow target cells. Mitogen-stimulated SCM augmented CFU-GM (175% to 225%), whereas CFU-GEMM and BFU-E growth was essentially unchanged. Cell separation procedures used to determine which cells provided these microenvironmental stimuli indicated that nonadherent mononuclear spleen cells provided the bulk of the CSF-GM, whereas adherent cells (95% nonspecific esterase + monocyte- macrophages) and nonadherent cells provided similar proportions of CSF- mix and erythroid burst-promoting activity (BPA). The nonadherent cells generating high levels of CSF-mix, BPA, and CSF-GM were predominantly Leu-1-negative, ie, non-T, cells. In the presence or absence of mitogens, SCM was a more potent source (1.3- to 3.8-fold) than peripheral leukocyte CM of the growth factors for the three progenitor cell types. Specific in situ cytochemical stains for analyzing morphology of myeloid colonies demonstrated that SCM stimulated the proliferation of the same types and proportions of colonies as human placental CM, suggesting that these CMs may contain similar CSF-GMs. These data show the contribution of spleen cell subsets to the generation of hematopoietic growth factors and the responsiveness of these cells to various mitogenic stimuli.

Description: Pregnancy in female carriers of abnormal hemoglobins with great avidity for oxygen provides a unique opportunity to assess the importance of the usual difference in oxygen affinity between fetal and maternal blood. Outcome of pregnancy was recorded for carriers of hemoglobins Bethesda, Osler, and Yakima, whose p50s (9.5, 9.1, and 12 mm Hg at pH 7.4) were far lower than that of a normal fetus (23 mm Hg at pH 7.3). Neither spontaneous abortions nor intrauterine growth retardation could be attributed to the presence of high oxygen affinity in the mothers. In vitro simulations suggested that neither maternal or fetal polycythemia alone was sufficient to adjust for perturbation of the normal situation, and increased uterine and/or fetal blood flow probably provided additional compensation.

Description: Neutrophil function was studied in a patient with polymorphonuclear leukocyte (PMN) glycoprotein-180 deficiency and in her parents. PMNs of the patient had abnormal chemotaxis, phagocytosis, adherence, surface charge, and membrane-associated events of activation. Selective defects to C3b, immunoglobulin G (IgG), phorbol myristate acetate (PMA) and N- formyl-methionyl-leucyl-phenylalanine (FMLP) are described, although C3b receptor density was normal. The parents were found to have abnormal adherence to nylon-wool fibers, abnormal transmembrane potential depolarization with PMA, and reduced amounts of glycoprotein- 180 in their PMNs. These studies provide further evidence that the oxidative burst has several different pathways for activation. They demonstrate that the absence of a single PMN surface glycoprotein is associated with a broad spectrum of PMN functional abnormalities. Finally, the observations made in the parents support an autosomal recessive mode of inheritance.

Description: Ecto-5′nucleotidase (5′NT) activity of peripheral blood (PB) lymphocytes was determined in 31 patients with serum monoclonal gammopathies (MG). Twenty-one patients had a diagnosis of multiple myeloma (MM), and ten patients had monoclonal gammopathy of undetermined significance (MGUS). The proliferative activity of the bone marrow plasma cells (LI%) was investigated in 28 of these MG patients by means of tritiated thymidine uptake evaluated by simultaneous autoradiography and cytoplasmic immunofluorescence. 5′NT activity was significantly lower in MG patients as compared with normal controls. MM patients had lower 5′NT activity than MGUS patients, but the difference was not significant. By contrast, MM had significantly higher LI% than MGUS patients. There was a linear regression of 5′NT on LI% which was statistically significant: the higher the LI%, the lower the 5′NT. Because the LI% is an accurate prognostic and monitoring factor in MG, this correlation indicates that 5′NT may be of assistance in predicting the clinical progress of MG patients. In seven MGs, the PB T and B lymphocytes were studied separately. The T cell subpopulation was 5′NT deficient compared to the normal controls, shown as a significant linear regression of T cell 5′NT on the LI%. This suggests that in MG there may be an alteration of nonneoplastic T lymphocytes correlated with tumor growth. The OKT8+ lymphocytes were mainly responsible for the 5′NT deficiency of unseparated T lymphocytes.

Description: Peripheral blood leukemia cells from patients with acute monoblastic leukemia (AMoL) were tested for killer cell activity against target cells that detected natural killer cell-mediated or monocyte-mediated spontaneous cytotoxicity. The fibrosarcoma cell line Wehi 164, pretreated with actinomycin D to induce susceptibility to lysis, specifically detects the activity of unstimulated human monocytes. In four of six cases of AMoL, high killer cell activity could be measured against this target. In three of these four cases, the killer cell activity could be assigned exclusively to the leukemic clone, based on the high leukocyte counts and the resultant dilution of normal cells, as evidenced by marker and by functional analysis. While leukemic cells with killer cell activity against Wehi 164 contained 34% to 45% cells that were positive for binding of the 63D3 monoclonal antibody, the two leukemic samples without killer cell activity contained only 1% and 12% 63D3-positive cells. Cell sorting of 63D3-positive and -negative cells from two leukemias with killer cell activity demonstrated that the killer cell activity was restricted to the 63D3-positive fraction of AMoL cells. These data demonstrate that monoblastic leukemia cells can be potent killer cells and that killing activity is linked to the 63D3- defined cell surface molecule.

Description: The Belgrade laboratory rat (b/b rat) has hereditary, hypochromic, microcytic anemia with a variety of red cell abnormalities. Although this anemic syndrome has been recently ascribed to the defective delivery of iron to the developing red cell, the basic hematopoietic defect is still unknown. In this article we present evidence that the b/b rat has an additional hematologic defect. We have found that the megakaryocyte number in the marrow of the b/b rat is decreased to one half that of the normal rat, but the maturation rate of recognizable megakaryocytes is accelerated and the size is increased. The platelet count is moderately reduced. These findings indicate that megakaryocytopoiesis in the anemic b/b rat is abnormal and suggest that the genetic defect may involve the progenitors of the megakaryocyte cell lineage. Alternatively, the megakaryocytic abnormalities may be secondary to the severe anemia.

Description: DNA from mononuclear blood and tumor cells from 33 patients undergoing bone marrow transplantation for leukemia was examined for the presence of Epstein-Barr virus (EBV) genomes by blot hybridization. Four groups of patients were studied soon after engraftment, during long-term remission, after relapse of the original leukemia, and after development of secondary B cell neoplasms. Only the cells of patients with secondary neoplasms demonstrated EBV genomes, where all five adequately studied samples were positive. Samples from all other patient categories were negative for EBV genomes. We conclude that EBV genomes do not frequently persist in normal engrafted lymphocytes or in mononuclear cells of patients suffering recurrent leukemia. These results are consistent with EBV playing a role in the genesis of secondary B cell neoplasms following bone marrow transplantation.

Description: The effects of hemin on the conversion of pyruvate kinase (PK) isozymes from the M2-type to the L-type in K562 cells were investigated. Immunofluorescence, ion exchange chromatography, and electrophoretic studies showed that the untreated K562 cells contained only the M2-type PK, while eight to 20 days after induction with hemin, concomitant with hemoglobin F synthesis, L-type PK levels increased while M2-type PK levels decreased. Electrophoretic study revealed three hybrid isozymes of the L-type and M2-type PK. We conclude that the conversion of PK isozymes from the M2-type to the L-type in erythroid precursor cells occurs in the early stage of maturation.

Description: In this study, the effects of 5-aza-2′-deoxycytidine on differentiation of human leukemic cells in primary suspension culture are reported for the first time. Morphological and functional differentiation was induced in cells from two acute monoblastic leukemias and two of three acute myeloid leukemias following repeated exposures to 1 mumol/L 5-aza- 2′-deoxycytidine. The observation that nontoxic concentrations of the drug are able to induce the in vitro differentiation of both monoblastic and myeloblastic leukemic cells into mature elements may encourage the exploitation of the differentiating properties of 5-aza- 2′-deoxycytidine in chemotherapy protocols for acute non-lymphoblastic leukemias.

Description: Fibrinogen from plasma was compared with fibrinogen from platelets using two-dimensional electrophoresis. The source of platelet fibrinogen was isolated alpha-granules, thrombin- and collagen-released platelet material. The B beta- and gamma-chains from the different sources showed similar two-dimensional patterns, while gamma'-chains were not observed in platelet fibrinogen preparations. Furthermore, the A alpha-chain could hardly be identified in platelet preparations. When individual fibrinogen was studied in persons heterozygous for genetic B beta- and gamma-chain variants, the two-dimensional variant pattern could be demonstrated in plasma fibrinogen as well as in platelet fibrinogen. This observation strongly indicates that the structural genes for plasma and platelet fibrinogen B beta- and gamma-chains are identical.

Description: We describe four patients with impaired platelet aggregation and 14C- serotonin secretion during stimulation with adenosine diphosphate (ADP), epinephrine, collagen, and platelet-activating factor. The response to arachidonic acid was normal in all patients with regard to aggregation and in three of the four with regard to 14C-serotonin secretion. The total platelet adenosine triphosphate (ATP) and ADP content and the ATP to ADP ratio was normal in all patients, thereby excluding storage pool deficiency as the cause of the secretion defect. Studies with 3H-arachidonic acid-labeled platelets revealed that the thrombin-induced liberation of arachidonic acid from membrane-bound phospholipids was impaired in these patients. Further, platelet thromboxane B2 production, measured using a radioimmunoassay, was diminished during stimulation with ADP and thrombin, but was normal with arachidonic acid, indicating that the oxygenation of arachidonic acid was normal and that the diminished thromboxane production was due to a defect in the liberation of arachidonic acid. Release of arachidonic acid is mediated by phospholipases that are Ca++ dependent. To examine whether these patients may have a defect in making intracellular Ca++ available, another Ca++-dependent process, myosin light chain phosphorylation, was studied during thrombin stimulation. Platelets from three of the patients were found to behave the same as normal ones, suggesting that the deficiency in phospholipase activity may not be due to impaired Ca++ mobilization. Our studies demonstrate a novel group of patients with platelet secretion defects associated with impaired liberation of arachidonic acid from phospholipids. These patients exemplify a congenital defect, other than deficiencies of cyclooxygenase and thromboxane synthetase, by which thromboxane production may be impaired in platelets.

Description: Restriction endonuclease mapping defined a partial deletion of about 1.35 kb in the beta-globin gene of a black American patient with hemoglobin S-beta zero-thalassemia and in his uncle with a beta zero- thalassemia trait. The 5′ endpoint of the deletion is about 600 bases upstream from the cap site, and the 3′ endpoint lies within about 500 bases from the 5 splice junction of the second intervening sequence. The deletion is different from that of a previously reported Indian beta zero-thalassemia allele, where 0.6 kb is deleted at the 3′ end of the beta-globin gene.

Description: Hereditary persistence of fetal hemoglobin (HPFH) is a genetically heterogeneous and clinically benign condition characterized by persistent expression of fetal hemoglobin (Hb F) into adulthood. In the G gamma beta + type, no major deletions in the globin gene cluster occur; adult heterozygotes produce approximately 20% Hb F, which results from overproduction of G gamma chains, with no apparent increase in production from the adjacent A gamma gene. We have recently described a point mutation 202 base pairs 5′ to the cap site of the G gamma gene in an individual with G gamma beta + HPFH. This mutation abolishes a normal ApaI restriction endonuclease site, and thus can be detected by blotting of genomic DNA. We present here further data on the ApaI mutation: (1) It occurs in six of seven families with G gamma beta + HPFH. (2) In three families, detailed haplotype analysis using 11 polymorphic restriction sites in the beta globin cluster has been done. The two that carry the missing ApaI site are identical but the third, which has a normal ApaI pattern, differs from the other two in at least two sites, one of which is a new polymorphic Nco I site between the delta and beta globin genes. This suggests the possibility of a different HPFH mutation in the third family. (3) The haplotype of the G gamma beta + HPFH chromosome carrying the ApaI mutation is different from that of 108 beta A chromosomes of black individuals that have been tested. (4) The G gamma ApaI site is normal in 61 beta A and 109 beta S alleles from non-HPFH black individuals, including 22 who share the same haplotype for the intragenic G gamma, A gamma HindIII polymorphisms. These data add support to the possibility that the -202 mutation is actually causative of the G gamma beta + HPFH phenotype.

Description: Potential limitations of prenatal diagnosis of hemophilia B, as compared to hemophilia A, include (1) occurrence of far more frequent defects with abnormal circulating antigen, (2) lower levels of factor IX in fetal plasma at 16 to 20 weeks gestation, and (3) the presence of factor IX antigen in amniotic fluid. In addition, proteolysis could occur, especially with amniotic fluid contamination of fetal plasma. A sensitive polyclonal immunoradiometric assay for factor IX antigen was used to characterize the range of levels in amniotic fluids and fetal plasma samples. To assess for altered forms, factor IX species were compared to those of a homologous clotting factor, prothrombin. Fourteen postmortem abortus blood samples from fetuses of 14 to 23 weeks gestation had factor IX antigen levels that averaged 5.1 U/dL and ranged from 1.7 to 15 U/dL. Amniotic fluid factor IX antigen averaged 2.9 U/dL, with a range from 1.4 to 8.5 U/dL in 19 separate amniocentesis samples. Thus, in a male fetus at risk of hemophilia B and with a low circulating level of gene product, mixture of fetal plasma with amniotic fluid could severely limit prenatal diagnosis, assuming that the amniotic fluid factor IX is of maternal origin. Despite rapid processing of amniotic fluid samples, the prothrombin was extensively cleaved, suggesting that it had been activated in vivo. On gel electrophoresis of amniotic fluid samples, however, factor IX was only minimally cleaved. In the postmortem fetal blood specimens, prothrombin was partially cleaved. On crossed-immunoelectrophoresis, fetal plasma prothrombin showed decreased migration in calcium, compared to EDTA, indicative of mature gamma-glutamyl carboxylation. The latter presumably resulted from fetal hepatic synthesis.

Description: Colony-forming cells in ten cases of acute myeloid leukemia (AML) were studied with six cytotoxic monoclonal antibodies that react with antigens expressed at discrete stages of differentiation of normal and leukemic hematopoietic cells. The reactivity of the whole leukemic population was measured by indirect immunofluorescence, and the reactivity of the colony-forming cells was established by complement- mediated cytotoxicity and by fluorescence activated cell sorting. Comparison of the immunofluorescent reactivity with cytotoxicity and cell sorting showed that colony-forming cells were found within a fraction of the leukemic subpopulations that expresses these antigens. This finding implies that immunofluorescence reactivity of the total leukemic population does not necessarily predict the phenotype of the clonogenic cells. When the surface phenotype of the clonogenic leukemic cells was compared to that previously established for normal marrow hemopoietic clonogenic cells, several patterns were seen: (1) in four of ten cases, the clonogenic cells expressed a phenotype like that of relatively mature normal granulocyte-macrophage colony-forming cells (late CFU-GM) or, (2) in two cases, a phenotype similar to the less mature colony-forming cells (early CFU-GM or CFU-GEMM), and (3) in four cases, a composite phenotype of early and late CFU-GM. Thus, the level of impairment of differentiation in AML may vary from case to case. In those cases phenotypically similar to the late CFU-GM, it may be possible to separate leukemic clonogenic cells from less mature normal clonogenic cells using monoclonal antibodies selectively cytotoxic for the late CFU-GM.

Description: We evaluated 37 patients with moderate or severe hemophilia A and six patients with severe factor IX deficiency for clinical or laboratory evidence of immune abnormalities. Patients were assigned to one of four groups according to the type of clotting factor replacement. Twenty patients had received only cryoprecipitate during the two years preceding the evaluation (group I); 11 additional patients were treated predominantly with cryoprecipitate but had also received up to nine bottles of factor VIII concentrate (group II); six patients received factor VIII concentrate (group III); six patients received factor IX concentrate (group IV). There was no clinical or laboratory evidence of immunodeficiency among the 43 patients. The mean absolute number of Th cells was normal in all patient groups, but the mean absolute number of Ts cells was increased compared with controls, both in patients treated with cryoprecipitate and in patients treated with factor VIII or factor IX concentrate. There was no correlation between the Th/Ts ratio and patient age, alanine aminotransferase level, hepatitis serology, in vitro lymphocyte function, or amount of clotting factor administered. Our observations demonstrate that the volunteer or commercial origin of clotting factor replacement cannot fully explain the alterations in lymphocyte subset distribution previously described in patients with hemophilia A.

Description: Human monocytes generate the procoagulant tissue factor (MTF) following exposure to a variety of immune stimuli in vitro. The generation of MTF is modified by T cells, lymphokines, and immunoregulatory lipoproteins, and recent studies have shown that MTF can be activated in an immune- specific manner following exposure to antigen. We have examined the role of serum factors in the regulation of MTF generation. Low concentrations (less than 1%) of heat-inactivated normal human serum greatly enhanced MTF generation in cultures of normal peripheral blood mononuclear cells. The stimulatory effect was observed in cultures of both unstimulated cells and cells exposed to bacterial lipopolysaccharide. Stimulation was not observed at high serum concentrations (greater than 10%) and could not be explained by endotoxin contamination or activation of the assay system. Stimulatory activity was present in plasma and BaSO4-adsorbed plasma as well as autologous and allogeneic serum, was not abolished by removal of serum lipoproteins, and did not require the presence of T cells for its expression. Sera from 28 different normal volunteers were screened for stimulatory activity and demonstrated a wide variation in potency. These results suggest that a potent factor is present in sera that enhances the expression of MTF activity in vitro. This factor is distinct from previously described lipoprotein regulators and may play a role in the initiation of coagulation in both normal hemostasis and pathologic states.

Description: Non-Hodgkin's lymphoma (NHL) is a very rare complication of acute lymphoblastic leukemia (ALL). We present the pathologic, clinical, immunologic, and ultrastructural features of the third reported example of NHL following successfully treated ALL. This white girl developed ALL with predominantly L1 cells at 3.5 yr of age. The lymphoblasts were terminal deoxynucleotidyl transferase (TdT) positive and were non-B, non-T cells. She achieved a complete remission with standard induction therapy and has remained in continuous complete remission. Four and one- third years after the onset of ALL, she developed multifocal, pleomorphic large cell lymphoma of the small bowel, which resulted in episodes of intussusception and obstruction. These pleomorphic and frequently multinucleated lymphoma cells lacked TdT, common ALL antigen, and all tested markers of B cell, T cell, and histiocyte differentiation. Following three small bowel resections, systemic multiagent chemotherapy, and abdominal irradiation, she is currently free of disease.

Description: Leukemia in the newborn is an infrequent disease that has not been well defined using modern laboratory techniques. We describe two infants, one at birth and one at four weeks, with acute lymphoblastic leukemia. The blasts from each patient were studied in great detail, using a battery of cytochemical and immunologic procedures in addition to ultrastructural studies. Immunologic cell marker studies, not previously reported in congenital leukemia, showed the lymphoblasts from each infant to be of the pre-B cell phenotype. Each infant relapsed, one after a 17-week clinical remission and the other after a 44-week remission. The former has died while the latter is in a second remission. The subtype of pre-B cell acute lymphoblastic leukemia (ALL) which in childhood appears to confer an unfavorable prognosis, may have the same significance in neonatal ALL.

Description: The proliferation and differentiation of granulocyte and monocyte progenitor cells (CFU-C) in vitro is dependent on the presence of a group of closely related glycoproteins termed colony-stimulating factors (CSF). In order to investigate the interaction of these factors with CFU-C, we purified CFU-C from the peripheral blood of chronic myeloid leukemia patients with an immune rosette technique using specific monoclonal antibodies (mean 74-fold enrichment, 45% cloning efficiency). Colony formation by purified CFU-C demonstrated an absolute dependence on an exogenous source of CSF. Liquid culture of small aliquots of enriched CFU-C with CSF-containing medium resulted in a rapid, time- and concentration-dependent induction of DNA synthesis as measured by 3H-thymidine incorporation. This specific CSF induction of DNA synthesis by enriched CFU-C was used to develop a microassay system for CSF activity. CSF activity could be reproducibly quantitated in 24–48 hr. The proliferating cells in this assay system were shown to be myeloid progenitor cells by examining the morphology of their progeny and by determining the surface antigen phenotype of the responding cells (Ia+, T3-, B1-, Mo1-). This microassay provides a quantitative assessment of CSF activity that may be useful in the purification of human CSF and in the generation of monoclonal antibodies to CFU-C surface structures.

Description: Colony-forming cells in ten cases of acute myeloid leukemia (AML) were studied with six cytotoxic monoclonal antibodies that react with antigens expressed at discrete stages of differentiation of normal and leukemic hematopoietic cells. The reactivity of the whole leukemic population was measured by indirect immunofluorescence, and the reactivity of the colony-forming cells was established by complement- mediated cytotoxicity and by fluorescence activated cell sorting. Comparison of the immunofluorescent reactivity with cytotoxicity and cell sorting showed that colony-forming cells were found within a fraction of the leukemic subpopulations that expresses these antigens. This finding implies that immunofluorescence reactivity of the total leukemic population does not necessarily predict the phenotype of the clonogenic cells. When the surface phenotype of the clonogenic leukemic cells was compared to that previously established for normal marrow hemopoietic clonogenic cells, several patterns were seen: (1) in four of ten cases, the clonogenic cells expressed a phenotype like that of relatively mature normal granulocyte-macrophage colony-forming cells (late CFU-GM) or, (2) in two cases, a phenotype similar to the less mature colony-forming cells (early CFU-GM or CFU-GEMM), and (3) in four cases, a composite phenotype of early and late CFU-GM. Thus, the level of impairment of differentiation in AML may vary from case to case. In those cases phenotypically similar to the late CFU-GM, it may be possible to separate leukemic clonogenic cells from less mature normal clonogenic cells using monoclonal antibodies selectively cytotoxic for the late CFU-GM.

Description: A complement (C)-dependent antiglobulin assay was utilized to determine the reactivity of non-C-fixing monoclonal antibodies (MoAb) with human granulocyte-macrophage progenitor cells (CFU-GM). The variables of the assay were analyzed with non-C-fixing MoAb against Ia antigens, including CR11–462, which recognizes the same (or spatially close) determinant identified by the C-fixing anti-Ia MoAb Q5/13. The sensitivity of the antiglobulin assay was influenced by dilutions of anti-mouse Ig xenoantiserum and of rabbit C. Five non-C-fixing MoAb to Ia antigens, seven non-C-fixing MoAb to HLA-A,B antigens, and one non-C- fixing MoAb to beta 2-microglobulin induced marked inhibition of human CFU-GM in the antiglobulin assay. The activity of non-C-fixing MoAb in the antiglobulin assay was comparable to that of C-fixing anti-Ia and anti-HLA-A,B MoAb in the standard cytotoxicity assay. In addition, the cytotoxic effect of dilute C-fixing anti-Ia MoAb was enhanced when the antiglobulin technique was employed. The results of this study indicate that the antiglobulin assay is a rapid and simple technique for the characterization of antigens on human hematopoietic progenitors. Our data also indicate that Ia antigens are expressed on most CFU-GM and that the conflicting results in the literature (that is, those suggesting that Ia antigens are expressed on a smaller proportion of CFU-GM) may reflect differences in the cytolytic activity of the MoAb and rabbit C used.

Description: Serum cobalamin (vitamin B12) and unsaturated B12 binding capacity (UBBC) have been measured in 24 cases of hypereosinophilia: 16 were cases of hypereosinophilic syndrome (HES) and 8 of secondary eosinophilia. The two groups were similar with respect to absolute eosinophil counts. Serum cobalamin and UBBC were found to be markedly increased in most cases of HES and normal in secondary eosinophilia. This elevation of UBBC was mainly related to the increase of R binders (transcobalamins I and III). The elevated serum cobalamin and R binders in HES were due neither to a higher intracellular content of R binders nor to an increased release of these binders from eosinophils of HES. Pure fractions of eosinophils obtained from HES and secondary eosinophilia did not exhibit any difference in vitamin B12 binders. On the other hand, neutrophil-rich fractions from the same patients showed a higher content of intracellular B12 binding proteins than pure eosinophil fractions, irrespective of the cause of eosinophilia. These findings suggest that the increased serum vitamin B12 and UBBC could reflect an expanded pool of both eosinophils and neutrophils in HES and, thus, provide an additional argument for the inclusion of this syndrome in the group of myeloproliferative disorders.

Description: The present studies report erythropoietin (Ep) production in primary cultures of a human renal carcinoma from a patient with erythrocytosis that has been serially transplanted to BALB/c nude mice. The levels of erythropoietin in the culture media were estimated using the exhypoxic polycythemic mouse assay (EHPCMA), fetal mouse liver erythroid colony- forming technique (FMLC), and a radioimmunoassay (RIA). The spent culture media of the exponentially growing cells contained less than 10 mU/ml of Ep measured by RIA. However, after the cells became confluent, Ep levels (RIA) in the spent media showed a marked increase to approximately 300 mU/ml. Ep levels estimated using the FMLC and EHPCMA were approximately 2/3 and 1/10, respectively, of those measured by RIA. Rabbit antiserum to highly purified human urinary Ep (70,400 U/mg protein) was utilized for immunocytochemical (peroxidase-antiperoxidase method) localization of Ep in the cultured cells. Very few of the cells in exponential growth exhibited Ep-like immunoreactivity, whereas intense Ep-like immunoreactivity was observed in the cytoplasm of the cells maintained in culture for a prolonged period after reaching confluency. The most intense staining was observed in some of the cells forming domes. The domes developed after the cells reached confluency, and their numbers increased with increasing time in confluent culture, in parallel with the increase in Ep levels in the spent media. This primary cell culture system of a renal cell carcinoma maintained in nude mice, which produces immunologically and biologically active Ep, may provide a useful model for studies of the mechanism of Ep production.

Description: We assessed the number of Langerhans cells (LC) before and after bone marrow transplantation (BMT) in 27 patients in order to study the fate and behavior of these dendritic antigen-presenting cells following allogeneic BMT. LC were identified using monoclonal antibody OKT6 on skin biopsies performed on days - 10, 0, 11, 25, 39, 120, and 365. In a control group composed of 15 healthy adults aged 20–37 yr, the mean number of LC (+/- SEM) was 25.6 +/- 1.17/0.1 sq mm of epidermal surface. Our study shows that pretransplant, the number of LC in patients with aplastic anemia or leukemia was lower than that of controls. The finding of low numbers of LC in patients with untreated aplastic anemia is suggestive of a medullary origin of LC in man. Moreover, during the early posttransplant period, nearly all patients present a severe deficit in LC. This deficit may delay the maturation of their immune system. The number of LC reaches nearly normal levels 4– 12 mo after BMT. Finally, we have noted a significant impairment of LC reconstitution in patients with acute graft-versus-host disease (GVHD), providing evidence that this defect may be an important mechanism involved in acute GVHD-related immunodeficiency.

Description: The acute effects of a single intravenous dose of L-asparaginase on protein synthesis were studied in normal rabbits and in animals that had received turpentine to stimulate fibrinogen production. Male New Zealand rabbits received L-asparaginase (500 U/kg) 16 hr before the injection of the radiolabeled amino acid [75Se]selenomethionine (75SeM). Incorporation of 75SeM into fibrinogen and serum proteins in the L-asparaginase-treated rabbits was the same as for saline-treated controls, with fibrinogen representing approximately 5% of the labeled plasma proteins. In turpentine-treated rabbits, the maximal incorporation of 75SeM into serum proteins remained unchanged, whereas 75SeM-fibrinogen increased sixfold and accounted for 25% of the labeled proteins. Animals that received L-asparaginase at the same time as turpentine or 14 hr later showed significant decreases in synthesis of both serum proteins and fibrinogen. 75SeM-fibrinogen that was purified from L-asparaginase-treated rabbits underwent normal catabolism when injected into normal recipient rabbits. These data indicate that L- asparaginase can acutely cause partial inhibition of both serum protein and fibrinogen synthesis when administered to rabbits shortly before or during a period of increased fibrinogen production. Fibrinogen that is synthesized in the presence of L-asparaginase does not have an abnormal rate of catabolism.

Description: 1,(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and other chloroethylnitrosourea anticancer agents in clinical use produce severe and cumulative bone marrow toxicity. Chlorozotocin, a glucose analogue, has demonstrated reduced hematologic toxicity while retaining full antitumor activity. The biochemical-pharmacologic properties of chlorozotocin and CCNU were compared in human bone marrow. After a 2-hr incubation with a 0.1-mM drug concentration, total cellular uptake of chlorozotocin in whole marrow was 2.47 +/- 0.80 pmole/10(4) cells and was not significantly different compared to the uptake of 1.94 +/- 0.53 pmole/10(4) cells with CCNU. The quantitative alkylation of bone marrow DNA by chlorozotocin, 22.8 +/- 1.2 pmole/mg DNA, was equivalent to that produced by CCNU, 22.9 +/- 0.5 pmole/mg DNA. Bone marrow was separated into 14 fractions by centrifugal elutriation. CCNU uptake was found to be greater than that of chlorozotocin in 3 fractions that were primarily composed of lymphocytes, monocytes, and normoblasts. Chlorozotocin uptake was greater than CCNU in 6 fractions that contained primarily mature and immature myeloid cells as well as the highest CFU-GM activity. The two drugs produced a comparable degree of DNA strand breakage and DNA-protein cross-linking as measured by alkaline elution of pooled fractions of elutriated bone marrow. DNA interstrand crosslinking was not found with either drug. The most significant finding of this study is the differences in the site of drug alkylation by chlorozotocin and CCNU in bone marrow chromatin. Endonuclease digestions with MCN, DNase I, and DNase II showed nonrandom alkylation of specific regions of chromatin by the two drugs: CCNU demonstrated a preferential binding to the transcriptionally active regions of chromatin, whereas chlorozotocin predominantly alkylated the transcriptionally inactive regions. These data suggest that the lethal damage of nitrosourea alkylation in human bone marrow is principally expressed in transcriptionally active regions of chromatin.

Description: Tumor burden in adult patients with acute leukemia is assessed using the percentage of blast cells in the bone marrow or blood. It is clear, however, that not all blast cells are leukemic cells, especially during rapid marrow regeneration. Similarly, some leukemia cell lines have been shown to differentiate in vitro, and the same process also occurs in vivo. Therefore, the leukemic burden may be due to more differentiated cells as well as to blast cells. The purpose of this study was to investigate whether the human malignancy-associated nucleolar antigen (HMNA) could be used as a marker for leukemic cells and to examine its potential as a diagnostic tool. The proportion of HMNA-positive cells in the bone marrow of patients with acute leukemia was determined by indirect immunofluorescence with antibodies to HMNA and was compared with the differential counts routinely made in the clinic laboratory. The percentages of HMNA-positive cells among the nucleated cells in the marrow of 72 patients with clinical evidence of leukemia were significantly higher (range 9%-98%, median 83%) than those observed for nonleukemic individuals (range less than 0.05%-2.5%, median 1%) or for fractions of marrow cells from normal volunteers enriched for normal early progenitor cells (less than or equal to 2%). Patients with leukemia in remission had a lower percentage of HMNA- positive cells (range 0%-83%, median 3%). The percentage of HMNA- positive cells increased as patients approached relapse. Although the percentage of HMNA-positive cells was related to the percentage of blast cells in the bone marrow of the patients with leukemia, some partially differentiated cells were also HMNA-positive in some specimens, and some blastic cells were HMNA-negative in other specimens. These studies indicate the potential usefulness of HMNA as a marker for leukemic cells.

Description: Kidney allografting was performed in a group of ten beagles, and viable leukocytes infiltrating the transplanted organs were isolated during episodes of acute rejection 5 or 6 days postoperatively. These infiltrate populations, consisting predominantly of lymphocytes and monocytes/macrophages, were found to have significantly increased amounts of procoagulant activity relative to control leukocytes isolated from circulating blood and lymph. Using nonspecific esterase staining in an agar microclot assay, procoagulant activity in the infiltrate leukocytes was found to reside in monocytes/macrophages rather than other coisolated cell types. By contrast, control monocytes from blood had no activity in this microclot assay. Procoagulant activity in the infiltrate cells was characterized as tissue factor. Increased amounts of this activator of the extrinsic pathway, as found in infiltrate monocytes/macrophages, may initiate clotting reactions and fibrin deposition within allografts.

Description: A case of transcobalamin II deficiency with several unique features is described. The clinical presentation was typical, except for a slightly delayed age at presentation and the occurrence of apparent neurologic dysfunction from the beginning. The unusual biochemical feature was a low serum cobalamin level (97 pg/ml). Several cobalamin-binding protein abnormalities coexisted and antedated cobalamin therapy. Chief among these was the complexing of all serum R binder (transcobalamin I), leaving the patient with no detectable R binder. This defect appeared to be transient. Noteworthy, too, was a prominent binder of 70,000 mol wt that also carried the bulk of his serum cobalamin after therapy; it was prominent in his presumably heterozygous relatives too. The interrelationship between all these abnormalities is intriguing but unclear. The abnormality in transcobalamin II deficiency is clearly not limited solely to deficiency of transcobalamin II. It is also evident that this entity must now be considered in the differential diagnosis of low serum cobalamin levels in infancy.

Description: We have shown previously that the cause of anemia in healthy elderly subjects can usually not be identified. In this study, hematopoiesis was examined in 18 healthy elderly subjects with unexplained anemia and in 15 young and 15 healthy elderly individuals without anemia. No reduction in circulating testosterone was noted, making decreased androgen levels as a cause for the anemia unlikely. The 2,3 diphospho- glycerate (2,3DPG) levels in the anemic subjects were significantly higher than their corresponding controls, suggesting that the anemia was pathologic, as no increase would be expected if the low hemoglobin was a physiologic adjustment to age. The anemia was associated with a reduction in marrow normoblast and CFU-E number, but no decrease in BFU- E levels was seen. This suggests that the mechanism of the anemia is a decrease in stem cell proliferation. This could be caused by a reduction in circulating erythropoietin or a defect in end organ response. A second possibility is that a basic cellular abnormality exists. The presence of an overall reduction in hematopoiesis in anemic elderly (decreased peripheral blood counts, reduced marrow myeloid precursors, and CFU-C levels) makes this especially likely. The abnormality may be caused by a mechanism unrelated to the aging process. The fact that nonanemic elderly also have reductions in hematopoiesis suggests that age contributes to the defect.

Description: A simple technique using an aggregometer and fixed washed human platelets (FWP) and fibrillar collagen has been used to evaluate the contribution of the two components of the factor VIII (FVIII) complex to platelet-collagen interactions. FWP bound individually to collagen fibrils in suspension, and both the total number of FWP bound and the rate of adhesion increased with increasing collagen concentration. Von Willebrand's disease (vWD) type I or normal plasma immunoadsorbed with anti-factor VIII-related antigen (anti-FVIIIR:Ag) antiserum gave 20% and vWD type IIa gave 50% of the rate of adhesion obtained with normal, hemophilia A, or hemophilia A with inhibitor plasma, but the same percent adhesion was found with all plasmas. The rate of adhesion of both vWD type I and type IIa was corrected by the addition of purified FVIII complex. These results indicated that the FVIIIR:Ag and not the factor VIII coagulant activity (FVIII:C) in normal plasma or purified FVIII complex caused an accelerating effect on the rate at which FWP bound to collagen. Collagen fibrils not only bound FWP, but also adsorbed the FVIII complex with preferential adsorption of the forms of FVIIIR:Ag with the greatest ristocetin cofactor (FVIIIR:RCoF) activity. Saturation of collagen with FWP did not change the adsorption pattern of the FVIII complex. Also anti-FVIIIR:Ag blocked the accelerating effect of the FVIII complex but not the adhesion of FWP. Thus, FWP and FVIIIR:Ag appeared to bind to separate sites on collagen.

Description: The infusion of 1-deamino-(8-D-arginine)-vasopressin (DDAVP) causes not only an elevation in factor VIII-related antigen (FVIIIR:Ag), but also a marked elevation of plasma von Willebrand antigen II (vWAgII). vWAgII reaches a peak concentration at 60 min and is elevated 3–8-fold over basal levels in normal individuals and individuals with type I, IIA, and IIB von Willebrand's disease. As the mechanism of hemostatic alteration brought about by DDAVP might be due to release of endothelial cell proteins, endothelial cell cultures were performed. The cultures demonstrated synthesis and secretion of vWAgII, as evidenced by the incorporation of 35S-methionine into the vWAgII molecule. Thus, vWAgII, like FVIIIR:Ag, is an endothelial cell protein.

Description: The level of assimilation of dietary iron is believed to have an important influence on iron status. To examine the effect of enhancing the availability of dietary iron on iron balance, 17 adult volunteer subjects were given 2 g of ascorbic acid daily with meals for 16 weeks. Serum ferritin levels before and after the study averaged 46 and 43 micrograms/L, respectively, indicating a negligible effect on iron stores. When vitamin C supplementation was continued for an additional 20 months in five iron-replete and four iron-deficient subjects, serum ferritin determinations again failed to indicate any significant effect of the vitamin C on iron reserves. These findings were not explained by intestinal adaptation to the enhancing effect of the vitamin, because radioisotopic measurements of nonheme iron absorption showed no reduction in the enhancing effect of 1 g of ascorbic acid after four months of megadoses of vitamin C. It is concluded that altering the availability of nonheme dietary iron has little effect on iron status when the diet contains substantial amounts of meat.

Description: Potential limitations of prenatal diagnosis of hemophilia B, as compared to hemophilia A, include (1) occurrence of far more frequent defects with abnormal circulating antigen, (2) lower levels of factor IX in fetal plasma at 16 to 20 weeks gestation, and (3) the presence of factor IX antigen in amniotic fluid. In addition, proteolysis could occur, especially with amniotic fluid contamination of fetal plasma. A sensitive polyclonal immunoradiometric assay for factor IX antigen was used to characterize the range of levels in amniotic fluids and fetal plasma samples. To assess for altered forms, factor IX species were compared to those of a homologous clotting factor, prothrombin. Fourteen postmortem abortus blood samples from fetuses of 14 to 23 weeks gestation had factor IX antigen levels that averaged 5.1 U/dL and ranged from 1.7 to 15 U/dL. Amniotic fluid factor IX antigen averaged 2.9 U/dL, with a range from 1.4 to 8.5 U/dL in 19 separate amniocentesis samples. Thus, in a male fetus at risk of hemophilia B and with a low circulating level of gene product, mixture of fetal plasma with amniotic fluid could severely limit prenatal diagnosis, assuming that the amniotic fluid factor IX is of maternal origin. Despite rapid processing of amniotic fluid samples, the prothrombin was extensively cleaved, suggesting that it had been activated in vivo. On gel electrophoresis of amniotic fluid samples, however, factor IX was only minimally cleaved. In the postmortem fetal blood specimens, prothrombin was partially cleaved. On crossed-immunoelectrophoresis, fetal plasma prothrombin showed decreased migration in calcium, compared to EDTA, indicative of mature gamma-glutamyl carboxylation. The latter presumably resulted from fetal hepatic synthesis.