Different repopulation profile between erythroid and nonerythroid progenitor cells in genetically anemic W/Wv mice after bone marrow transplantation

Repopulation kinetics of erythrocytes and neutrophils and replacement of hematopoietic progenitors were studied in genetically anemic (WB x C57BL/6)F1-W/Wv (WBB6F1-W/Wv) hosts after bone marrow transplantation from C57BL/6-bgJ/bgJ or C57BL/6-bgJ/bgJ;Pgk-1a/Y mice. Electrophoretic pattern of hemoglobin was used as a marker of donor-type erythrocytes, giant granules of bgJ/bgJ mice as a marker of donor-type neutrophils, and A-type phosphoglycerate kinase-1 (PGK-1) as a marker of hematopoietic colonies produced by donor-derived progenitor cells. Repopulation of donor-type erythrocytes was significantly faster than that of donor-type neutrophils. Moreover, the extent of replacement was greater for erythroid progenitor cells than for nonerythroid progenitor cells. When nonirradiated WBB6F1-W/Wv mice with B-type PGK-1 received 10(5) bone marrow cells from C57BL/6-bgJ/bgJ;Pgk-1a donors, only approximately 20% replacement of erythroid progenitor cells gave rise to total reconstitution of erythrocytes. The present result suggests that normal multipotential stem cells may preferentially differentiate into erythroid lineage cells in anemic WBB6F1-W/Wv hosts and that normal erythroid progenitor cells may suppress the differentiation of erythroid progenitors of WBB6F1-W/Wv hosts.     

Osteogenic growth peptide increases blood and bone marrow cellularity and enhances engraftment of bone marrow transplants in mice

The osteogenic growth peptide (OGP) was characterized recently in regenerating bone marrow (BM) and normal serum. In vitro, the OGP regulates stromal-cell proliferation and differentiated functions. In vivo, an increase in serum OGP accompanies the osteogenic phase of postablation BM regeneration. The present results in normal mice show that OGP induces a balanced increase in WBC counts and overall BM cellularity. In mice receiving myeloablative irradiation and syngeneic or semiallogeneic BM transplants, OGP stimulates hematopoietic reconstruction and doubles the survival rate; these effects are dependent on initiating the OGP administration before irradiation. Chimerism measurements in semiallogeneic graft recipients suggest no preferential effect of OGP on residual host cells. The data implicate OGP in the acceleration of hematopoiesis secondary to expansion of the stromal microenvironment and/or enhancement of stroma-derived signals to stem cells. The low-dose effectiveness of OGP is explained by the demonstration of an autocrine positive feedback loop that together with the OGP-binding protein sustains high serum levels of the peptide. A potential OGP-based treatment in combination with chemoradiotherapy is attractive because of the OGP-induced balanced multi-lineage enhancement of hematopoiesis and possible replacement of expensive recombinant cytokines by a readily synthesized peptide.     

Comparison of haematological recovery times and supportive care requirements of autologous recovery phase peripheral blood stem cell transplants, autologous bone marrow transplants and allogeneic bone marrow transplants.

The haematological recovery time, infection rate and supportive care requirements of patients receiving recovery phase autologous peripheral blood stem cell transplants (APBSCT) (n = 38), autologous bone marrow transplants (autoBMT) (n = 13) and allogeneic bone marrow transplants (alloBMT) (n = 14) were compared with respect to the time post-transplant to reach 0.1, 0.5 and 2.0 x 10(9) neutrophils/l and 50 and 150 x 10(9) platelets/l, the length of hospitalization, fever and antibiotic use, the incidence of documented infection and the number of red cell and platelet transfusions. The APBSCT group had a significantly more rapid recovery of neutrophils and platelets and their supportive care requirements were significantly less than the autoBMT and the alloBMT groups. There was no difference between the latter two groups. The most significant variables contributing to the differences in haematological recovery times were the granulocyte-macrophage progenitor (CFU-GM) dose infused and, to a lesser extent, patient age. The APBSCT group received a higher CFU-GM dose of 87 +/- 12 x 10(4)/kg BW compared with 12 +/- 5 and 17 +/- 3 x 10(4)/kg BW in the autoBMT and the alloBMT groups, respectively (p = 0.0001). Patient age showed a negative correlation with the rate of recovery because the APBSCT group, which recovered faster was also older (48 +/- 2 years, compared with 33 +/- 3 and 31 +/- 2, respectively, p = 0.0001). On multivariate analysis, CFU-GM dose was the only variable to show a significant correlation with all the haematological recovery endpoints studied in these 65 patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations of cell cycle distribution in the bone marrow of aging mice measured by flow cytometry

The effect of aging on the cell cycle distribution in bone marrow cells was measured by flow cytometry with special reference to the lability in bone marrow physiology. Female C3H mice were examined every 3 h during a 24-h period at the age of 16, 21 and 26 months, vs 3-month-old control mice. Considerable circadian fluctuations were found in the different cell cycle phases in young mice. The rhythmicity patterns were different when comparing different months. In aging mice the variations were dampened, while consistent age-related phase shifts were not seen. The maximal 24-h mean numbers of cells in all three cell phases, but especially the S and G2 phases were reached at 21 months. The relative number of S and G2 phases were significantly reduced in 26-month-old mice, indicating an age-related shift of the proliferative capacity. The present findings are discussed in relation to age-related changes in granulopoiesis and the increase of myelotoxic effects during cancer chemotherapy in aging individuals.     

Comparison of hematopoietic and immune recovery after autologous bone marrow or blood stem cell transplants.

We studied hematopoietic and immune recovery in 40 subjects receiving autologous bone marrow (ABMT) or blood stem cell transplants (ABSCT). Supportive care, transplant-related morbidity, duration of hospitalization and cost were also considered. ABSCT was associated with more rapid recovery of all hematopoietic lineages than was ABMT. However, kinetics of immune recovery were similar between the groups. In the ABSCT group, there was a correlation between numbers of blood progenitor cells infused and the rate of hematopoietic recovery. The accelerated hematopoietic recovery following ABSCT correlated with less morbidity, fewer transfusions, briefer hospitalization and lower cost than ABMT.     

Delay in red blood cell recovery after major ABO incompatible bone marrow transplantation

In 6 of 27 patients in whom major ABO incompatible marrow was transplanted, the recovery of red blood cells (reticulocyte count greater than or equal to 1%) required more than 50 days after the transplantation. In 3 of them, more than 100 days were required. High titers of pre-transplant anti-A or anti-B agglutinin correlated with the delay in red blood cell recovery (IgM: p less than 0.01, IgG: p less than 0.05). In all of the patients whose pre-transplant IgM titers were 4 or less, and whose IgG titers were 32 or less, the red blood cells recovered within 50 days. Administration of cyclosporine did not correlate with the delay in red blood cell recovery. All the patients finally developed red cell production within 8 months without any treatment.     

Physicochemical properties of circulating red blood cells of lethally x-irradiated mice treated with rat bone marrow.

1. Two months after injection of rat bone marrow into lethally X-irradiatedmice (950 r-RBM mice), 100% of the circulating RBC were serologically of therat type, indicating that the surface molecular configuration of RBC from theseexperimental mice are of the rat type.

2. The hemoglobin was found to be also very much like the rat type in itsease in crystallization, its alkali denaturation property, its electrophoretic property, and its tendency to form a paracrystalline state at low temperature.

3. These cells possessed dual osmotic properties; the relative hemoglobinconcentration released when cells were lysed in water was more comparable tothe rat type, but its temperature dependency was more comparable to the mousetype.

Submitted on April 9, 1957 Accepted on May 15, 1957     

Flow cytometry analysis of dual red blood cell populations after bone marrow transplantation

Flow cytometry represents an alternative method to agglutination assays for the accurate quantification of mixed field populations of erythrocytes observed after bone marrow transplantation. Murine monoclonal antibodies directed against the blood group ABH antigens were selected and processed in order to prepare ready-to-use fluorescent reagents. Anti-A (NaM87-1F6; IgG3), anti-B (NaM9-2E11; IgG3) and anti-H (NaM19-7E11; IgM) were purified, labelled with fluorescein isothiocyanate, and used in a direct flow cytometry assay. Anti-A1 (NaM1-1C9; IgG3) was no longer active after FITC-labelling and then was used in an indirect assay. The agglutination was prevented by formaldehyde pretreatment of erythrocytes.
Using artificially-made double populations of erythrocytes, measured values with mixtures of 1-100% of cells were very closely related to expected values, showing both the sensitivity and the accuracy of the method. From careful investigation of a series of bone-marrow transplanted patients, we conclude that engraftments could be demonstrated earlier by flow cytometry than by agglutination, because minor populations (1-10%) of cells could be determined accurately only with labelled reagents. In addition, the disappearance of the donor cells on a long-term follow-up of patients enabled an earlier detection of graft failure in one case. The proposed method provides appreciable help to follow engraftment in patients and may have more general applications for the study of other haemopoietic chimaeras.     

Successful bone marrow transplantation in a child with red blood cell pyruvate kinase deficiency

We report the first successful use of BMT for the treatment of RBC pyruvate kinase (PK) deficiency in a boy who developed neonatal jaundice and severe transfusion-dependent hemolytic anemia a few months after birth. He received a BMT at the age of 5 from an HLA-identical sister who has normal PK activity after conditioning with busulfan and cyclophosphamide. The post-transplant course was uneventful. At present, 3 years after transplant, he is 8 years old and has a normal hemoglobin level and normal RBC PK activity without evidence of hemolysis. DNA analysis has confirmed full engraftment.     

Hemolysis of transfused group O red blood cells in minor ABO-incompatible unrelated-donor bone marrow transplants in patients receiving cyclosporine without posttransplant methotrexate.

Hemolysis most commonly occurs following bone marrow transplant when there is "minor" ABO blood group incompatibility between donor and recipient. The hemolysis has been attributed to destruction of the patient's incompatible erythrocytes by donor-derived anti-A and/or anti-B antibody produced from "passenger" immunocompetent donor lymphocytes. Extraordinary transfusion requirements of group O erythrocytes in a series of patients receiving unrelated minor ABO-incompatible marrow grafts led us to investigate whether this mechanism could account for the extent of hemolysis observed. In seven consecutive minor ABO-incompatible unrelated-donor bone marrow transplant recipients receiving cyclosporine without posttransplant methotrexate, we observed excessive hemolysis. For cases in this index group, a strongly reactive donor-derived ABO blood group antibody was identified coincident with development of hemolysis. Transfusion requirements in the first three patients (26 U of group O erythrocytes each) greatly exceeded the recipient's volume of incompatible erythrocytes, indicating that lysis of transfused group O erythrocytes was also occurring. Pretransplant erythrocyte exchange transfusion with group O erythrocytes performed in the four subsequent patients decreased the severity of hemolysis, but did not prevent it. Among minor ABO-incompatible marrow graft recipients, an analysis of variance demonstrated effects on transfusion requirements due to donor-recipient relationship being unrelated (P less than .002) and the use of posttransplant methotrexate (P = .0001), and there was interaction between these two factors (P less than .001). Bone marrow transplants from unrelated donors resulted in an exaggerated immune response to ABO blood group antigens, which was associated with hemolysis of transfused group O erythrocytes, as well as the patient's ABO-incompatible erythrocytes. This serious complication may be prevented by posttransplant immunosuppression with methotrexate.     

Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.     

Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation.

Production of tissue mast cells was evaluated in genetically anemic mice of W/Wv genotype and was found to be abnormal. In the skin of adult W/Wv mice the number of mast cells/cm was less than 1% of the number observed in the congeneic +/+ mice. No mast cells were detectable in other tissues of the W/Wv mice. After transplantation of bone marrow cells from +/+ mice the number of mast cells in the skin, stomach, caecum, and mesentery of the W/Wv mice increased to levels similar to those of the +/+ mice. These results show that the W/Wv mouse is a useful tool for the investigations concerning the physiologic roles and the origin of mast cells.     

The bigger the C-value, the larger the cell: genome size and red blood cell size in vertebrates.

Vertebrate genome sizes vary roughly 350-fold and correlate with a variety of cellular and organismal parameters. Most notable among these is the relationship between genome size ("C-value") and red blood cell (RBC) size, which can be identified within and among each of the five vertebrate classes. This relationship, in turn, leads to important associations between genome size and features such as metabolic rate (at least in homeotherms). The present article describes the correlation between genome size and RBC size in vertebrates and discusses some of the cytological, physiological, and evolutionary implications of this relationship.     

Decreased treatment failure in recipients of HLA-identical bone marrow or peripheral blood stem cell transplants with high CD34 cell doses

We studied the association between CD34 cell dose and transplant outcomes in 359 bone marrow (BM) and 511 peripheral blood stem cell (PBSC) transplant recipients from human leucocyte antigen (HLA)-identical siblings, reported to the International Bone Marrow Transplant Registry (IBMTR). Transplants for leukaemia were performed between 1995 and 1998. Patients were divided into those receiving below or above the median CD34+ dose, for BM (3 x 106/kg) and PBSC (6 x 106/kg) grafts respectively. Cox proportional hazards regression was used to adjust for baseline patient-, disease- and transplant-related characteristics. Analysis of the BM recipients showed that high CD34 cell dose was associated with lower transplant-related mortality [relative risk (RR) = 0.60, P = 0.033] and treatment failure (inverse of leukaemia-free survival, RR = 0.69, P = 0.032). Among PBSC recipients, high CD34 dose was associated with faster recovery of neutrophils to > 0.5 x 109/l (RR = 1.38, P < 0.001) and platelets to > 20 x 109/l (RR = 1.34, P = 0.003), lower risk of relapse (RR = 0.62, P = 0.029) and treatment failure (RR = 0.74, P = 0.03). We conclude that higher CD34 cell doses decrease treatment failure in recipients of HLA-identical sibling BM and PBSC transplants.     

Marrow cell uptake by megakaryocytes in routine bone marrow smears during blood loss.

In studying smears of marrow aspirates, we have encountered the presence of normal appearing haemopoietic cells within megakaryocytes. We have then searched routine marrow smears from 125 patients seen in our service during 3 years. The presence of marrow cells (granulocytic, erythroid, and lymphoid cells) within megakaryocytes was seen in 16 cases of whom 9 had documented bleeding and 5 had carcinoma. 3 patients were suspected of bleeding but this was not documented. 56% of patients with bleeding and 83% of patients with carcinoma seen during this period displayed this pehnomenon. A search for this phenomenon in routine marrow smears may reveal unsuspected blood loss.     

Clinical significance of red blood cell distribution width in the elderly: a potential indicator of bone marrow stimulation by erythropoietin

Summary To investigate the clinical significance of red blood cell distribution width (RDW) and haemoglobin distribution width (HDW) in the elderly and their relationships with erythropoietin (EPO) secretion, we measured red cells parameters using a Technicon HI system and serum EPO using a radioimmunoassay in 247 elderly subjects (normal: n= 150; preanaemic iron deficiency: n= 24; iron deficiency anaemia: n= 8; senile anaemia: n= 65). RDW was slightly higher in the elderly subjects with preanaemic iron deficiency (14.1 ± 1.1%) than in the normal elderly subjects (13.5±0.7%). It was highest in iron deficiency anaemia (16.1 ± 1.3%), while the increase in senile anaemia was limited (13.9 ± 1.2%). The HDW increased only in iron deficiency anaemia. There was a strong positive relationship between EPO and RDW in iron deficiency anaemia (r= 0.817, P<0.01). Moreover, this correlation was also found in preanaemic iron deficiency (r= 0.456, P < 0.05), but not in senile anaemia, suggesting that bone marrow hypoactivity may partly play a role in the pathogenesis of senile anaemia. All the eight subjects with iron deficiency anaemia had a RDW ≥ 14.9% (mean + 2SD of normal subjects), while 55 (85%) of the 65 with senile anaemia had a RDW < 14.9%. Both the RDW and EPO levels of six anaemic subjects with high RDW values (≥ 14.9%) after oral iron therapy for 56–78 days decreased significantly. Our results suggest that RDW is useful to distinguish iron deficiency anaemia from senile anaemia, and may be a potential parameter of bone marrow stimulation by EPO.     

Disseminated infection with Fusarium in recipients of bone marrow transplants.

Clinical data from 10 episodes of disseminated infection with Fusarium among eight recipients of bone marrow transplants and from 31 cases reported previously in the literature were analyzed in an effort to characterize the natural history of this rare infection and its response to therapy. The characteristic signs of fusarial infection--disseminated skin nodules, fungemia, and multiple-organ involvement--are results of its propensity for early spread. From a review of the literature and our own experience, it appears that recovery of phagocytic mechanisms (the primary immunologic defenses against Fusarium) in the form of rising neutrophil counts is mandatory for clinical resolution. Even after a graft begins to function adequately, Fusarium may not be completely eradicated, as evidenced by the high incidence of recurrence among patients with subsequent neutropenic episodes. Fusarium is highly resistant to conventional antifungal drugs in vitro, but its progression may be slowed by intensive antifungal therapy until the recovery of adequate neutrophil levels.