ALBERT

Description: Levels of fetal hemoglobin (HbF) bearing reticulocytes (F reticulocytes) range from 2% to 50% in patients with sickle cell (SS) anemia. To learn whether any portion of such variation in F cell production is regulated by loci genetically separable from the beta- globin gene cluster, percentages of F reticulocytes were compared in 59 sib pairs composed solely of SS members, including 40 pairs from Jamaica and 19 from the United States. We reasoned that differences in F reticulocyte levels might arise (1) from any of several kinds of artifact, (2) via half-sib status, or (3) because one or more genes regulating F cell production segregate separately from beta S. We minimized the role of artifact by assay of fresh samples from 84 SS individuals, including both members of 38 sib pairs. In 78 of the 84 subjects, serial values for percent F reticulocytes fell within 99.9% confidence limits or were alike by t test (P greater than or equal to .05). This left 32 sib pairs for which F reticulocyte levels in each member were reproducible. When sib-sib comparisons were limited to these 32 pairs, percentages of F reticulocytes were grossly dissimilar within 12 Jamaican and 3 American sibships. Within them, the probability that sibs were alike was always less than or equal to .005 and usually less than or equal to 10(-4). We next minimized the contribution of half-sibs among Jamaicans by a combination of paternity testing and sib-sib comparison of beta-globin region DNA restriction fragment length polymorphisms, especially among discordant pairs. We thereafter concluded that at least seven to eight Jamaican pairs were composed of reproducibly discordant full sibs. There is thus little doubt that there are genes regulating between-patient differences in F cell production that are separate from the beta-globin gene cluster. Still unanswered is (1) whether or not these genes are actually linked to beta S, (2) why F reticulocyte levels in Americans tend to be lower than in Jamaicans, and (3) whether or not differences in F cell production among SS patients are regulated by several major loci or by only one.

Description: Levels of fetal hemoglobin (HbF) bearing reticulocytes (F reticulocytes) range from 2% to 50% in patients with sickle cell (SS) anemia. To learn whether any portion of such variation in F cell production is regulated by loci genetically separable from the beta- globin gene cluster, percentages of F reticulocytes were compared in 59 sib pairs composed solely of SS members, including 40 pairs from Jamaica and 19 from the United States. We reasoned that differences in F reticulocyte levels might arise (1) from any of several kinds of artifact, (2) via half-sib status, or (3) because one or more genes regulating F cell production segregate separately from beta S. We minimized the role of artifact by assay of fresh samples from 84 SS individuals, including both members of 38 sib pairs. In 78 of the 84 subjects, serial values for percent F reticulocytes fell within 99.9% confidence limits or were alike by t test (P greater than or equal to .05). This left 32 sib pairs for which F reticulocyte levels in each member were reproducible. When sib-sib comparisons were limited to these 32 pairs, percentages of F reticulocytes were grossly dissimilar within 12 Jamaican and 3 American sibships. Within them, the probability that sibs were alike was always less than or equal to .005 and usually less than or equal to 10(-4). We next minimized the contribution of half-sibs among Jamaicans by a combination of paternity testing and sib-sib comparison of beta-globin region DNA restriction fragment length polymorphisms, especially among discordant pairs. We thereafter concluded that at least seven to eight Jamaican pairs were composed of reproducibly discordant full sibs. There is thus little doubt that there are genes regulating between-patient differences in F cell production that are separate from the beta-globin gene cluster. Still unanswered is (1) whether or not these genes are actually linked to beta S, (2) why F reticulocyte levels in Americans tend to be lower than in Jamaicans, and (3) whether or not differences in F cell production among SS patients are regulated by several major loci or by only one.

Description: Red cell lysis in isotonic solutions containing NH4Cl, NH4HCO3, and a carbonic anhydrase enzyme inhibitor (acetazolamide) is a function of erythrocyte enzyme activity and permeability of cells to the inhibitor. Erythrocyte carbonic anhydrase activity is at least fivefold greater and acetazolamide permeability about tenfold less for adults than for newborns. In this setting, greater than 99.9% of red cells from adults can be hemolyzed at a time when greater than 25% of those from newborns remain intact. This easily applied method may be useful when antenatal diagnosis of hemoglobinopathies is otherwise precluded by contaimination with maternal erythrocytes. The feasibility of differential hemolysis via NH4Cl--HCO3-mediated, acetazolamide- modulated reactions is shown by the successful isolation of the few fetal-origin erythrocytes present in grossly nonbloody amniotic fluids and, in one instance, by approximately 3300-fold enrichment of apparently authentic fetal-origin red cells from the arm blood of a woman in her 18th wk of pregnancy.

Description: Non-Hodgkin's lymphoma (NHL) is the most common human immunodeficiency virus (HIV)-associated malignancy in hemophiliacs. We studied the incidence and clinicopathologic features of NHL in 3,041 hemophiliacs followed at 18 US Hemophilia Centers between 1978 and 1989. Of the 1,295 (56.6%) who were HIV(+), 253 (19.5%) developed acquired immunodeficiency syndrome (AIDS), of whom 14 (5.5%) developed NHL. Three NHL occurred in HIV(-) hemophiliacs, for a 36.5-fold greater risk in HIV(+) than HIV(-) hemophiliacs (P 〈 .001). The NHL incidence rate was 29-fold greater than in the US population by Surveillance, Epidemiology, and End Results (SEER) estimates (P 〈 .001). Between 0 and 4 lymphomas have been observed per year between 1978 and 1989. At presentation 13 (92.9%) of the HIV(+) NHL were extranodal. Ten were stage IV, 1 stage II, and 3 stage IE. Ten (71.4%) were high-grade, 3 (21.4%) intermediate-grade, and 1 (7.1%) was a low-grade B-cell lymphoma. Epstein-Barr virus (EBV) DNA was detected in 36% by in situ hybridization, including one central nervous system (CNS) lymphoma. The mean CD4 cell count at NHL diagnosis was 64/mm3, the mean latency from initial HIV infection was estimated to be 59 months, and the median survival was 7 months. The incidence of basal cell carcinoma in HIV(+) hemophiliacs was 18.3-fold greater than in HIV(-) hemophiliacs (P 〈 .001) and 11.4-fold greater than in the US population (P 〈 .001). In conclusion, incidence rates of NHL and basal cell carcinoma in HIV(+) hemophiliacs are significantly increased over rates in HIV(-) hemophiliacs and over rates in the US population. Clinicopathologic presentation of NHL in HIV(+) hemophiliacs is similar to that in HIV(+) homosexual men.

Description: We have used 75% to 90% pure murine erythroid colony-forming units (CFU- E) to delineate the processes and factors underlying their maturation. These CFU-E form 32 cell colonies and are drawn from what we term generation I of a six-generation long maturation sequence (Landschulz et al, Blood 79:2749, 1992). Applying assays of 59Fe-heme biosynthesis and colony numbers as measures of maturation and analyses of DNA degradation as an index of programmed cell death, we find that (1) erythropoietin (Epo) enhances maturation throughout most of its course; (2) Epo first seems able to forestall DNA degradation when CFU-E reach generation II; (3) the processes that Epo elicits thereafter start to persist when Epo is withdrawn; (4) insulin-like growth factor I (IGF-I) also forestalls DNA breakdown, but later loses effectiveness; (5) IGF-I adds little to maturation when Epo levels are high, but when Epo levels are low, enhances it substantially; and (6) for maturation to be entirely optimal, an unidentified serum factor(s) is probably required when Epo levels are high and is certainly needed when Epo levels are like those in normal animals. Quantitatively, about 40% of optimal in vitro erythropoiesis at normal Epo levels depends on Epo alone, another 30% or less on the addition of IGF-I, and the remaining 30% or more on the addition of unidentified serum factor(s). Applied together, these three or more factors lead to two-thirds of the maximum maturation realized with saturating Epo levels. Because we also find that heme accumulated in CFU-E culture can closely approach levels in red blood cells, we suppose that our conclusions apply as well to CFU-E maturation in vivo.

Description: Recombinant human stem cell factor (SCF), the ligand for c-kit, has been shown to stimulate increased numbers of hematopoietic progenitor cells of multiple types to circulate in the blood of baboons, but it was not known if the cells stimulated to circulate by SCF contained cells capable of engrafting and rescuing lethally irradiated baboons. Peripheral blood mononuclear cells (PBMNC) were collected by leukapheresis from four untreated control baboons and from three baboons on the 10th or 11th day of treatment with SCF (200 micrograms/kg/d). All animals were transplanted with 1.00 to 1.04 x 10(8)/kg of cryopreserved autologous PBMNC after treatment with a single dose of 1,020 cGy total body irradiation (TBI). Three animals were transplanted with PBMNC that had been collected during SCF treatment, 24 to 38 days after the last dose of SCF. Rapid trilineage engraftment was documented by bone marrow biopsy in all three. The mean time to a total white blood cell count (WBC) 〉 or = 500/microL, WBC 〉 or = 1,000/microL, and an absolute neutrophil count (ANC) 〉 or = 500/microL was 15 +/- 3 (mean +/- SD), 19 +/- 1, and 19 +/- 2 days, respectively. Two animals remain alive with stable engraftment more than 180 and 245 days posttransplant. The third died of sepsis 32 days posttransplant with a hypercellular marrow showing trilineage engraftment. The surviving animals were transfusion independent by 10 and 59 days posttransplant. Four control animals were transplanted with PBMNC collected in the absence of SCF stimulation. One was treated for 11 days with SCF (200 micrograms/kg/d) after PBMNC were collected. This animal was transplanted 25 days after the last dose of SCF. None of the four control animals engrafted and they died 13, 16, 28, and 38 days posttransplant with marrow aplasia. Treatment with SCF stimulates the circulation of cells that engraft and rescue lethally irradiated baboons. The characteristics of the transplantable cells present in the circulation are now amenable to direct study.

Description: The ligand for the human c-kit, recombinant human stem cell factor (SCF), was administered to baboons at doses of 200, 100, 50, 25, and 10 micrograms/kg/d. SCF induced a dose-dependent expansion of hematopoietic colony-forming cells (CFC) of multiple types in both blood and marrow, including colony-forming unit (CFU) granulocyte- monocyte, burst-forming unit-erythroid, CFU-MIX, and high proliferative potential-CFC. These changes were associated with a dose-dependent leukocytosis, involving all leukocyte lineages, a reticulocytosis, and increases in marrow cellularity. At 200 micrograms/kg/d of SCF, CFC in blood were increased 10-fold to greater than 100-fold. This correlated with an increased frequency of CD34+ cells in blood. The frequency of CFC in blood approached that of marrow in some animals. These changes were reversed within 7 to 14 days of stopping SCF. The results of these studies suggest a role for the c-kit ligand in stimulating the expansion of multiple CFC types in blood and marrow for potential therapeutic purposes.

Description: CD34+ cells devoid of detectable mature and immature T and B lymphocytes, expressing the CD2, CD10, and CD20 antigens, were isolated from marrows of three pairs of sex-mismatched, mixed lymphocyte culture (MLC) nonreactive, sibling baboons. Reciprocal transplants were performed between members of each pair, using the sex chromosomes, identified by standard cytogenetic techniques, as markers of the transplanted cells. Five animals from these three pairs were transplanted with 0.6 to 2.1 x 10(6)/kg of isolated cryopreserved and/or fresh isolated cells that were greater than 95% to 97% CD34+. Before transplantation, animals were treated with either single (920 or 1,020 cGy) or split (700 cGy x 2) dose total body irradiation. All animals engrafted with donor cells, as demonstrated by cytogenetic analysis of bone marrow metaphase cells 4 weeks after transplantation, with days to white blood cell count (WBC) greater than 500 being 19 +/- 2, to WBC greater than 1,000 23 +/- 2, to absolute neutrophil count greater than 500 24 +/- 3, and to platelets greater than 20,000 30 +/- 7. Three animals died of infectious-related complications at 34, 42, and 109 days after transplantation with evidence of host and donor cells (mixed chimerism) in marrow. Two animals remain alive and healthy more than 545 and 455 days after transplantation with stable mixed chimerism in marrow and blood. For these two animals, cytogenetic analysis of granulocyte/macrophage and erythroid colonies derived from marrow precursors between weeks 25 and 42 posttransplant showed evidence of mixed chimerism. Cytogenetic studies of CD2+ T cells and CD20+ B cells isolated from blood of these two animals between weeks 21 and 51 posttransplant showed the presence of mixed chimerism in both lymphocyte populations. Thus, isolated allogeneic CD34+ marrow cells devoid of detectable mature and immature T and B lymphocytes can engraft and reconstitute stable long-term myelopoiesis and lymphopoiesis in lethally irradiated baboons. These results are consistent with the hypothesis that CD34+ marrow cells contain pluripotent hematopoietic stem cells capable of fully reconstituting lymphohematopoiesis in the transplanted host.

Description: Hematopoietic growth factors are being administered to patients with acute myeloid leukemia (AML) both to shorten the duration of chemotherapy-induced neutropenia and in an attempt to increase cytotoxicity of cell cycle-specific agents. However, limited information is available concerning the effects of growth factors in AML patients. To examine the in vivo effects of recombinant human granulocyte colony-stimulating factor (G-CSF) on AML cells, laboratory studies were performed before and after a 72-hour intravenous infusion of G-CSF (10 micrograms/kg/d) administered to 28 untreated AML patients. Twenty-seven patients (96%) showed increases in at least one of the following parameters after G-CSF: blood blasts, bone marrow (BM) blasts, leukemia cells in S phase or interphase cells with leukemia- specific markers shown by fluorescence in situ hybridization. The median paired change in absolute blast count was +2.7 x 10(9)/L (P = .0001) after G-CSF, as compared with 0.0 during the 72 hours before initiation of G-CSF. The median percentage of BM leukemia cells in S phase increased from 6.0% to 10.7% after G-CSF (median change, %5.9%; P = .009). Interphase BM cells with trisomy 8 or monosomy 7 increased in 6 of 6 patients with these abnormalities (P = .02) with a median percent increase of 47%. Blood neutrophil counts also increased during G-CSF (median paired change, +2.8 x 10(9)/L; P 〈 .0001). Trisomy 8 or monosomy 7 was shown by fluorescence in situ hybridization in post-G- CSF blood neutrophils from 4 of 6 patients but was also present in neutrophils before G-CSF. We conclude that the percentage of leukemia cells in S phase increases and that leukemia cell populations undergo expansion during short-term administration of G-CSF in almost all AML patients.

Description: Cell populations highly enriched for the different stages of erythroid cell maturation were obtained by three sequential operations: harvesting of erythroid cells after induction of erythroid hyperplasia in the spleens of mice, elimination of the more mature erythrocytes by immunologic techniques, and separation of the residual nucleated erythroid cells as a function of size by the velocity sedimentation technique. The resulting cell fractions were studied both directly and after overnight incubation in the presence or absence of erythropoietin. In short-term culture, erythropoietin stimulated proliferation of pronormoblasts and basophilic normoblasts but probably not cells at later stages of differentiation. Erythropoietin also appeared to recruit increased numbers of pronormoblasts. In this experimental system, erythroid cell differentiation was able to proceed in the absence of erythropoietin, but without proliferation of these early erythroid cells. These techniques have provided a model system for the study of erythroid cells at different stages of maturation isolated from a uniform source at one point in time. The morphologic observations indicated that erythropoietin stimulates erythroid cell proliferation at several early stages of the maturation pathway.