Kinetics of haemopoietic progenitor cells during a Trypanosoma congolense rechallenge infection in Boran cattle

In a preliminary study, using clonogenic assays, the in vitro kinetics of committed haemopoietic progenitors were monitored during a Trypanosoma congolense rechallenge infection in five trypanosusceptible Boran cattle. Early in the infection (week 2), in the absence of any detectable parasitaemia, a drop in the number of nucleated marrow cells was recorded. This was accompanied by a marked but transient decrease in the levels of the colony-forming units-erythroid (CFU-E) followed by a partial recovery by weeks 3–4 after infection. The burst-forming units-erythroid (BFU-E) and the colony-forming units-granulocyte macrophage (CFU-GM) also significantly decreased between weeks 2 and 4. After a transient rise at weeks 3–5 postinfection, the CFU-GM steadily declined and remained below preinfection levels throughout the infection. The BFU-E remained below preinfection levels until the end of the experiment. The drop in nucleated marrow cells associated with the decreased numbers of CFU-E, BFU-E and CFU-GM was suggestive of a defect at the pluripotential stem cell level early in the infection (week 2). The erythrocyte indices, i.e. mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC), were unchanged until week 10 postinfection. Two animals became severely anaemic; one was euthanised at week 8 and one treated at week 9. The three remaining animals developed chronic anaemia with mean packed cell volume (PCV) fluctuating around 18%–19% between weeks 11 and 14. Low parasitaemia levels were recorded during that period. A CFU-E peak above preinfection levels was noted at week 12 and BFU-E appeared in the peripheral blood culture of two animals between weeks 11 and 14. A progressive rise in MCV associated with a gradual decrease in MCHC also characterised that period. A return to near preinfection levels was recorded for the numbers of all three progenitors three weeks after trypanocidal treatment followed by a full recovery five months after treatment. Although ineffective haemopoiesis has been suggested to contribute to the anaemia of bovine trypanosomiasis, this is the first demonstration of a negative effect on erythroid development in cultures of bone marrow of trypanosome-infected cattle.     

Effect of the Aldehyde Dehydrogenase Inhibitor,Cyanamide, on the Ex Vivo Sensitivity of Murine Multipotent and Committed Hematopoietic Progenitor Cells to Mafosfamide (Asta Z 7557)

Abstract

The ex vivo sensitivity of murine multipotent (CFU-GEMM) and committed (CFU-Mk, CFU-GM, BFU-E and CFU-E) hematopoietic progenitor cells to mafosfamide was quantified with and without concurrent exposure to cyanamide, an inhibitor of aldehyde dehydrogenase activity. In the absence of cyanamide, CFU-GEMM, CFU-Mk and CFU-GM were approximately equisensitive to mafosfamide while the erythroid progenitors were more sensitive to the drug. Cyanamide potentiated the cytotoxicity of mafosfamide toward CFU-GEMM and CFU-Mk, but not toward CFU-GM, BFU-E and CFU-E. Cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophos-phamide/aldophosphamide, the major intermediate in cyclophosphamide and mafosfamide activation, to the relatively nontoxic acid, carboxyphosphamide. Thus, our findings indicate that 1) murine CFU-GEMM contain the relevant aldehyde dehydrogenase activity, and 2) the relevant aldehyde dehydrogenase activity is retained upon differentiation to progenitors committed to the megakaryocytoid lineage, but lost upon differentiation to progenitors committed to the granulocytoid/monocytoid and erythroid lineages. The relative insensitivity of CFU-GM to mafosfamide is apparently due to a cellular determinant that influences their sensitivity to all cross-Unking agents since CFU-GM were found to be relatively insensitive to non-oxazaphosphorine cross-linking agents as well.     

Recipient-mediated effect of a traditional chinese herbal medicine,Ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to), on hematopoietic recovery following lethal irradiation and syngeneic bone marrow transplantation

Ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to, NYT), a traditional Chinese herbal medicine, was evaluated for recipient-mediated effect on hematopoietic recovery in a murine model of syngeneic bone marrow transplantation (BMT). BALB/c recipient mice were preconditioned with a lethal total body irradiation (TBI) at a dose of 6.5 Gy and transplanted with syngeneic bone marrow (BM) cells. NYT treatments, given intraperitoneally (i.p.) once per day for 3 consecutive days in a dose of 0.625 mg, were performed either before or after TBI and BMT to assess any recipient-mediated effect of this compound. NYT pretreatment was as effective as NYT posttreatment in enhancing the total number of colony-forming unit erythroid (CFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) per marrow and spleen after TBI and BMT. NYT pretreatment caused a significant increase in marrow and splenic CFU-E and CFU-GM numbers over a prolonged period following TBI and BMT, and affected late-stage erythropoiesis (CFU-E) more profoundly than early-stage erythropoiesis (burst-forming unit erythroid, BFU-E). NYT pretreatment significantly accelerate recovery of not only erythrocyte and leukocyte counts but also platelet counts after transplantation with a limited number (1 × 10⁵) of BM cells. The same treatment, however, was significantly less effective in hematopoietic recovery after transplantation with a minimal number (1 × 10⁴) of BM cells, indicating that NYT accelerates recovery of donor-derived rather than recipient-derived cells. The data are consistent with the idea that NYT has an enhancing effect on hematopoiesis via the recipient microenvironment, and suggest that NYT may have an important role in the acceleration of hematopoietic recovery of donor-derived cells following BMT.     

Clonogenic haematopoietic progenitor assays from clinically normal horses

Clonal assays for haematopoietic progenitors were performed on mononuclear cells isolated from bone marrow samples collected from the sternebrae of normal horses. Colony-forming units-erythroid (CFUE), burst-forming units-erythroid (BFU-E), colony-forming units-granulocyte/monocyte (CFU-GM) and colony-forming units-fibroblastic (CFU-F) were assayed, and reference values for clinically normal horses were established. The mean numbers of CFU-E, BFU-E and CFU-GM per 5 × 10⁴ cells were 329 ± 48, 30 ± 5 and 131 ± 13, respective. The mean number of CFU-F was 49 ± 6 per 2 ± 10 cells. The numbers of CFU-E and BFU-E were linearly related to the concentrations of fetal bovine serum (FBS), bovine serum albumin (BSA) and the cell number plated. The number of CFU-E were increased in the presence of erythropoietin (EPO) but its presence was not required for colony growth. BFU-E had an absolute requirement for EPO but the number of BFU-E was not linearly related to dose of EPO. Methods for clonal assays of equine hematopoietic progenitors are described.     

Effect of the Aldehyde Dehydrogenase Inhibitor, Cyanamide, on the Ex Vivo Sensitivity of Murine Multipotent and Committed Hematopoietic Progenitor Cells to Mafosfamide (Asta Z 7557)

The ex vivo sensitivity of murine multipotent (CFU-GEMM) and committed (CFU-Mk, CFU-GM, BFU-E and CFU-E) hematopoietic progenitor cells to mafosfamide was quantified with and without concurrent exposure to cyanamide, an inhibitor of aldehyde dehydrogenase activity. In the absence of cyanamide, CFU-GEMM, CFU-Mk and CFU-GM were approximately equisensitive to mafosfamide while the erythroid progenitors were more sensitive to the drug. Cyanamide potentiated the cytotoxicity of mafosfamide toward CFU-GEMM and CFU-Mk, but not toward CFU-GM, BFU-E and CFU-E. Cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophos-phamide/aldophosphamide, the major intermediate in cyclophosphamide and mafosfamide activation, to the relatively nontoxic acid, carboxyphosphamide. Thus, our findings indicate that 1) murine CFU-GEMM contain the relevant aldehyde dehydrogenase activity, and 2) the relevant aldehyde dehydrogenase activity is retained upon differentiation to progenitors committed to the megakaryocytoid lineage, but lost upon differentiation to progenitors committed to the granulocytoid/monocytoid and erythroid lineages. The relative insensitivity of CFU-GM to mafosfamide is apparently due to a cellular determinant that influences their sensitivity to all cross-Unking agents since CFU-GM were found to be relatively insensitive to non-oxazaphosphorine cross-linking agents as well.     

Effect of the aldehyde dehydrogenase inhibitor, cyanamide, on the ex vivo sensitivity of murine multipotent and committed hematopoietic progenitor cells to mafosfamide (ASTA Z 7557)

The ex vivo sensitivity of murine multipotent (CFU-GEMM) and committed (CFU-Mk, CFU-GM, BFU-E and CFU-E) hematopoietic progenitor cells to mafosfamide was quantified with and without concurrent exposure to cyanamide, an inhibitor of aldehyde dehydrogenase activity. In the absence of cyanamide, CFU-GEMM, CFU-Mk and CFU-GM were approximately equisensitive to mafosfamide while the erythroid progenitors were more sensitive to the drug. Cyanamide potentiated the cytotoxicity of mafosfamide toward CFU-GEMM and CFU-Mk, but not toward CFU-GM, BFU-E and CFU-E. Cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophosphamide/aldophosphamide, the major intermediate in cyclophosphamide and mafosfamide activation, to the relatively nontoxic acid, carboxyphosphamide. Thus, our findings indicate that 1) murine CFU-GEMM contain the relevant aldehyde dehydrogenase activity, and 2) the relevant aldehyde dehydrogenase activity is retained upon differentiation to progenitors committed to the megakaryocytoid lineage, but lost upon differentiation to progenitors committed to the granulocytoid/monocytoid and erythroid lineages. The relative insensitivity of CFU-GM to mafosfamide is apparently due to a cellular determinant that influences their sensitivity to all cross-linking agents since CFU-GM were found to be relatively insensitive to non-oxazaphosphorine cross-linking agents as well.     

Letter to the EditorActivin A: A Possible Marker for Liver Transplant Outcome

Abstract

The influence of recombinant human IL-17 on granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) progenitors and the release of IL-1α/β, IL-6 and erythropoietin (EPO) was estimated in the bone marrow cells obtained from normal and sub-lethally irradiated mice. In normal mice IL-17 increased CFU-GM and BFU-E and reduced CFU-E derived colonies numbers and augmented release of IL-6 and EPO. In irradiated mice the effects of IL-17 on hematopoietic progenitors were lineage-dependent, as well as dependent on their stage of differentiation and the time after the irradiation. IL-17 had no major effects on CFU-GM on day 1 and 3, but decreased their number on day 2, while enhanced both BFU-E and CFU-E on day 1 and 2 after irradiation, whereas on day 3 its effect on erythroid progenitors was again as observed in normal mice. After irradiation, IL-17 increased the release of IL-1α, IL-6 and EPO. The observed effects suggested the involvement of IL-17 in the regulation of hematopoiesis and indicated that its effects on both hematopoietic progenitors and cytokine release are dependent on the physiological/ pathological status of the organism.     

Comparison of dideoxynucleoside drugs (DDI and zidovudine) and induction of hematopoietic toxicity using normal human bone marrow cells in vitro

The drug zidovudine (AZT), a synthetic thymidine analog, has been used in the treatment of acquired immunodeficiency syndrome (AIDS). Clinical use of zidovudine has induced hematopoietic toxicity manifested by anemia, neutropenia and on occasion thrombocytopenia. Such toxicity has stimulated the development of alternative dideoxynucleoside drugs capable of exerting anti-viral potency while minimizing the risk for inducing organ toxicities. One such alternative dideoxynucleoside drug is 2′,3′-dideoxyinosine (ddI). Recent therapeutic anti-viral strategy, now undergoing clinical trial, is the evaluation of combined zidovudine ddI treatment. Unfortunately a complete assessment of their potential toxicity using this drug regimen has not been thoroughly examined. We report here the results of studies comparing the toxicity profile of zidovudine versus ddI on their ability to influence several classes of hematopoietic progenitor stem cells, e.g. granulocyte-macrophage (CFU-GM), megakaryocyte (CFU-Meg) and erythroid (CFU-E/BFU-E) following in vitro co-culture with normal human bone marrow. Since the main clinical toxicity associated with zidovudine in vitro is the development of anemia, additional in vitro studies compared the dose-escalation effect of erythropoietin in the presence of combined zidovudine and ddI. CFU-GM, CFU-Meg, CFU-E and BFU-E were all reduced (P<0.05) following incubation with either zidovudine or ddI thus determining their ID₅₀ concentrations for these classes of hematopoietic progenitors; however, the extent of toxicity associated with ddI was lower than what was observed with zidovudine. More importantly, dose-escalation escalation of erythropoietin was effective in reversing the inhibition observed for ddI on erythroid progenitors CFU-E and BFU-E (P<0.05), an effect not reported with zidovudine in vitro. Furthermore, in combination, ddI plus zidovudine significantly reduced erythroid colony formation which was not influenced by dose-escalation of erythropoietin. These results indicate ddI plus zidovudine may produce synergistic hematopoietic toxicity; however, with ddI, amelioration of the potential to develop anemia may respond to erythropoietin treatment.     

阵发性睡眠性血红蛋白尿病人骨髓红系和粒－单系祖细胞的特点

本文采用造血祖细胞体外培养技术观察了阵发性睡眠性血红蛋白尿（ＰＮＨ）病人骨髓红系祖细胞（ＢＦＵ一Ｅ和ＣＦＵ一Ｅ）和粒一单系祖细胞（ＣＦＵ一ＧＭ）的增殖能力；骨髓细胞经酸化ＡＢ型血清处理后的ＢＦＵ一Ｅ，ＣＦＵ一Ｅ和ＣＦＵ一ＧＭ的增殖能力；以及ＢＦＵ一Ｅ、ＣＦＵ一Ｅ对红细胞生成素（Ｅｐｏ）和（ＣＦＵ一ＧＭ对粒一单系集落刺激因子（ＧＭ一ＣＳＦ）的反应能力，发现ＰＮＨ病人骨髓ＢＦＵ一Ｅ，ＣＦＵ一Ｅ和ＣＦＵ一ＧＭ集落数明显低于正常；骨髓细胞经新鲜酸化ＡＢ型血清处理后培养的ＢＦＵ一Ｅ、ＣＦＵ一Ｅ和ＣＦＵ一ＧＭ集落数明显低于经热灭活酸化ＡＢ型血清处理后培养的集落数；以及ＢＦＵ一Ｅ，ＣＦＵ一Ｅ对Ｅｐｏ和ＣＦＵ一ＧＭ对ＧＭ一ＣＳＦ的剂量反应曲线低平。因此认为ＰＮＨ病人骨髓红系和粒一单系祖细胞有以下特点：１．增殖能力降低：２．在酸性条件下对补体的敏感性增加；３．对造血因子的敏感性降低。     

Sustained zidovudine treatment on hematopoiesis in immunodeficient mice

Zidovudine (AZT) has been the drug of choice in the treatment of human AIDS; however, associated with the use of zidovudine has been the development of hematopoietic toxicity, the mechanism of which is not clearly defined. We report here studies designed to evaluate dose-escalation of zidovudine, i.e. 0.1 and 1.0 mg/ml placed in the drinking water on hematopoiesis in C57BL/6 normal and LP-BM5 immunodeficiency virus-infected mice. Over a 6-week evaluation period, compared to normal, non-virus-infected controls, murine immunodeficiency (MAIDS) infection was associated with reduced hematopoietic progenitors, i.e. CFU-E, BFU-E, CFU-GM, and CFU-Meg from bone marrow and spleen. Following zidovudine treatment, further suppression of marrow-derived progenitors was observed, while increased numbers of progenitors were obtained from the spleen. Spleen-derived erythroid progenitors, i.e. CFU-E, were increased by 950% (P<0.001) from MAIDS-infected animals receiving 1.0 mg/ml of drug following 4-weeks exposure compared to non-drug-treated MAIDS control animals. Splenic BFU-E were increased 654% following 6-weeks exposure compared to non-drug-treated MAIDS-infected mice. This study suggests that the bone marrow is particularly sensitive to zidovudine toxicity which, at least early in exposure, appears to be compensated by splenic-derived hematopoiesis, in particular, erythropoiesis. Overt toxicity develops when, at least in this immunodeficiency model, the spleen is unable to provide progenitors is response to continued zidovudine exposure in vivo.     

Increased sensitivity to complement or erythroid and myeloid progenitors in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria is an acquired hemolytic anemia characterized by a membrane defect leading to increased sensitivity of erythrocytes, granulocytes, platelets, and bone-marrow erythroid and myeloid cells to complement-mediated lysis. To determine whether the phenotype of paroxysmal nocturnal hemoglobinuria is also expressed on erythroid and myeloid progenitors, marrow cells from five patients with the disease were exposed to a sucrose hemolytic system and then assayed for colony-forming units-erythroid (CFU-E), burst-forming units-erythroid (BFU-E), and colony-forming units-granulocyte/macrophage (CFU-GM). A 50 percent or greater decrease in the numbers of erythroid and myeloid colonies was noted when marrow cells from the patients with paroxysmal nocturnal hemoglobinuria were exposed to a sucrose solution of low ionic strength in the presence of complement but not in its absence. Such a decrease was not noted in similarly treated normal marrow cells or in marrow cells from a patient with the disease in remission. These results suggest that in paroxysmal nocturnal hemoglobinuria, CFU-E, BFU-E, and CFU-GM express a membrane abnormality similar to that on erythrocytes, and that the disease is the result of a change occurring at the level of the pluripotent hematopoietic stem cell.     

Suppression of erythroid progenitors in ponies infected with equine infectious anaemia virus

Six clinically normal ponies were infected intravenously with equine infectious anaemia virus (EIAV) to determine the effect of EIAV on numbers of erythroid, granulocyte/monocyte and fibroblastic progenitors in the bone marrow. Bone marrow progenitor assays were performed at weeks 1, 2, 3, 4 and 8 postinfection. The late erythroid progenitors, colony-forming units-erythroid (CFU-E), were suppressed at each time point postinfection with the maximal suppression occurring at week 2 postinfection. The maximal suppression corresponded to the peak of plasma virus concentration. The maximal suppression of the early erythroid progenitors, burst-forming units-erythroid (BFU-E), also occurred at week 2 postinfection. Removal of adherent cells from the bone marrow mononuclear cells (BMMC) abrogated the suppression. Neither granulocyte/monocyte progenitors (CFU-GM) nor fibroblastic progenitors (CFU-F) were affected by EIAV infection.     

Antithymocyte globulin treatment of retrovirus-induced feline erythroid aplasia: in vivo and in vitro studies

The Kawakami-Theilen strain of feline leukemia virus (FeLV-KT) was used experimentally to produce erythroid aplasia in cats. The in vivo effects of goat anti-feline-thymocyte globulin (ATG) on hematopoiesis were investigated in FeLV-negative normal and FeLV-positive anemic cats. Treatment was initiated in anemic cats between 4 and 6 weeks postinoculation (PI) when erythroid progenitors were reduced to 10% of normal levels. During the first 2 weeks of treatment, ATG significantly increased the numbers of erythroid precursors in bone marrow from 15 to 35% in anemic cats and from 28 to 43% in normal cats. ATG stimulated a twofold increase of CFU-E and a threefold increase of CFU-GM in normal cats between 2 and 4 weeks after initiation of treatment but had no effect on CFU-E or CFU-GM in anemic cats. The in vivo effects of ATG were transient despite weekly treatment. Cats treated with normal globulin were not significantly different from untreated anemic control cats. In vitro treatment of low density bone marrow mononuclear cells with ATG plus complement increased CFU-E and BFU-E of bone marrow from cats prior to inoculation but not from viremic cats. These results indicate that, although ATG stimulates erythropoiesis and granulopoiesis in normal cats, it does not reverse retrovirus-induced erythroid aplasia.     

Characterization of FPL-52694 [5-(2-hydroxypropoxyl)-8-propyl-4-oxo-4H-benzopyran-2-carboxylic acid Na] on histamine release from rat peritoneal mast cells induced by antigen,compound 48/80 and A 23187

We studied thein vitro effects of FPL-52694 [5-(2-hydroxypropoxyl)-8-propyl-4-oxo-4H-benzopyran-2-carboxylic acid Na] on histamine release from rat peritoneal mast cells. These cells exposed to ascaris antigen, compound 48/80 or the ionophore A 23187 concentration-dependently released histamine. About a 30–40% histamine release was obtained by 1×10^–4 g/ml of antigen, 1×10^–7 g/ml of compound 48/80 and A 23187. FPL-52694 (10^–9–10^–4 g/ml) concentration-dependently inhibited the histamine release from mast cells in response to antigen (1×10^–4 g/ml) and compound 48/80 (1×10^–7 g/ml), but only slightly inhibited the histamine release induced by A 23187 (1×10^–7 g/ml). Similar results were obtained with disodium cromoglycate (DSCG), in the same dose ranges. However, the inhibitory activity of FPL-52694 on histamine release by antigen and compound 48/80 was approximately 10 times more potent than that of DSCG at certain coincentrations. Tachyphylaxis was observed when these two agents were preincubated with mast cells for 10 min. These results show FPL-52694 to be a novel mast cell stabilizer.     

Pluripotent and committed haemopoietic progenitor cells in rat peripheral blood

The assay system for determination of haemopoietic progenitors in peripheral blood of rats is essen tial for potential studies on mobilisation and transplantation of circulating progenitor cells in a rat experimental model. This paper demonstrates the possibility of detection and quantification of pluripotent progenitors (Colony Forming Units-Spleen day 8-CFU-Sd8) and committed progenitors (Colony Forming Units Granulocyte Macrophage-CFU-GM and Burst Forming Units-Erythroid-BFU-E) in peripheral blood of rats in a steady state. For determination of CFU-Sd8 the rat to mouse in vivo assay was used, and for committed progenitors in vitro assays on methylcellulose were employed. The CFU-Sd8 incidence ranged from 7.3 to 11.6/ml of rat blood, similar to that reported in literature for mice. The incidence of CFU-GM was found to be 59.7 ± 9.4/ml which is in the range of the literature data for mice, rabbits, dogs and humans. The incidence of BFU-E in rat peripheral blood was 4.3 ± 1/ml, which was relatively low, but could be also considered as comparable with some literature data for dogs and humans. The CFU-E were not detected by the technique used. These results confirmed the existence of circulatory blood pluripotent progenitors (CFU-Sd8) and committed (CFU-GM and BFU-E) progenitors in rat, as has been established for some other mammalian species.     

Combined effect of IL-17 and blockade of nitric oxide biosynthesis on haematopoiesis in mice

Aim: The study was undertaken to extend our investigation concerning both the in vivo activity of interleukin (IL)-17 and the specific role of nitric oxide (NO) in IL-17-induced effects in the process of haematopoiesis. Methods: CBA mice were simultaneously treated with IL-17 and/or nitric oxide synthase (NOS) inhibitor, l -NAME, for 5 days and changes within various haematopoietic cell lineages in bone marrow, spleen and peripheral blood were analysed. Results: Findings showed that administration of both IL-17 and l -NAME stimulated increase in net haematopoiesis in normal mice. IL-17-enhanced myelopoiesis was characterized by stimulation of both femoral and splenic haematopoietic progenitor cells and morphologically recognizable granulocytes. Additionally, IL-17 induced alterations in the frequency of erythroid progenitor cells in both bone marrow and spleen, accompanied with their mobilization to the peripheral blood. As a consequence of these changes in the erythroid cell compartments, significant reticulocytosis was observed, which evidenced that in IL-17-treated mice effective erythropoiesis occurred. Exposure of mice to NOS inhibitor also increased the number of both granulocyte-macrophage and erythroid progenitors in bone marrow and spleens, and these alterations were followed by the mobilization of erythroid progenitors and elevated content of reticulocytes in peripheral blood. The specific role of NO in IL-17-induced haematopoiesis was demonstrated only in the IL-17-reducing effect on bone marrow late stage erythroid progenitors, CFU-E. Conclusion: The results demonstrated the involvement of both IL-17 and NO in the regulation of haematopoietic cell activity in various haematopoietic compartments. They further suggest that IL-17 effects are differentially mediated depending on the haematopoietic microenvironments.     

Kinetics of haemopoietic progenitor cells during a Trypanosoma congolense rechallenge infection in Boran cattle

In a preliminary study, using clonogenic assays, the in vitro kinetics of committed haemopoietic progenitors were monitored during a Trypanosoma congolense rechallenge infection in five trypanosusceptible Boran cattle. Early in the infection (week 2), in the absence of any detectable parasitaemia, a drop in the number of nucleated marrow cells was recorded. This was accompanied by a marked but transient decrease in the levels of the colony-forming units-erythroid (CFU-E) followed by a partial recovery by weeks 3–4 after infection. The burst-forming units-erythroid (BFU-E) and the colony-forming units-granulocyte macrophage (CFU-GM) also significantly decreased between weeks 2 and 4. After a transient rise at weeks 3–5 postinfection, the CFU-GM steadily declined and remained below preinfection levels throughout the infection. The BFU-E remained below preinfection levels until the end of the experiment. The drop in nucleated marrow cells associated with the decreased numbers of CFU-E, BFU-E and CFU-GM was suggestive of a defect at the pluripotential stem cell level early in the infection (week 2). The erythrocyte indices, i.e. mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC), were unchanged until week 10 postinfection. Two animals became severely anaemic; one was euthanised at week 8 and one treated at week 9. The three remaining animals developed chronic anaemia with mean packed cell volume (PCV) fluctuating around 18%–19% between weeks 11 and 14. Low parasitaemia levels were recorded during that period. A CFU-E peak above preinfection levels was noted at week 12 and BFU-E appeared in the peripheral blood culture of two animals between weeks 11 and 14. A progressive rise in MCV associated with a gradual decrease in MCHC also characterised that period. A return to near preinfection levels was recorded for the numbers of all three progenitors three weeks after trypanocidal treatment followed by a full recovery five months after treatment. Although ineffective haemopoiesis has been suggested to contribute to the anaemia of bovine trypanosomiasis, this is the first demonstration of a negative effect on erythroid development in cultures of bone marrow of trypanosome-infected cattle.     

Superoxide Dismutase Specifically Inhibits Erythroid Cell DNA Synthesis and Proliferation

Abstract

The antioxidant enzyme superoxide dismutase (SOD) was previously shown to inhibit both the proliferation of murine erythroid DA-1 cells growing in the presence of Interleukin-3 (IL-3) and the DNA synthesis of marrow erythroid progenitor cells (BFU-E) in vitro. We show here that the inhibition of marrow cell DNA synthesis by SOD is specific for BFU-E and erythroid precursors (CFU-E), with other myeloid progenitors (CFU-GM) and stem cells (CFU-S) being unaffected, and IL-3 blocks the inhibitory effects of SOD on BFU-E in a dose-dependent manner. Extending earlier observations on the effects of SOD on cell proliferation, it was found that SOD was capable of inhibiting DA-1 cell proliferation supported by either IL-3 or erythropoietin (epo), but had no effect on IL-3 dependent FDCP-1 cells, nor on epo-dependent HCD-57 cells. Of several murine erythroleukemia cell lines tested, only those transformed with Friend SFFVa virus were inhibited by SOD, while those transformed with Friend SFFVp or MuLV virus were not affected. These results show that the effects of SOD are not antagonistic to particular growth factors but rather the inhibition is specific for eryrthroid cells, and cells of the proper stage can be inhibited even if they have been transformed to factor independence.     

Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

Transforming growth factor β (TGFβ) can inhibit the in vitro proliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1^-/- mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1^- pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1⁺ pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell development in vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.     

Identification of a massive reserve of hematopoietic progenitors in mice

Previous studies have demonstrated that mice null (-/-) for either CD34 or c-mpl are viable and have greatly decreased numbers of multipotential (CFU-Mix), erythroid (BFU-E), and granulocytemacrophage (CFU-GM) progenitor cells in the bone marrow (BM), spleen (Spl) and peripheral blood (PB), without noticeable decreases in the nucleated cellularity of these organs. To evaluate the significance of these two proteins further, mice null for both CD34 and c-mpl were assessed for hematopoietic progenitor cells (HPC) and nucleated cellularity and compared with these cells in CD34-/- and c-mpl-/- mice. The following progenitors were assessed: CFU-GM, BFU-E, CFU-Mix with an erythroid component, CFU-Mix with erythroid and megakaryocyte components, nonerythroid CFU with a megakaryocyte (Meg) component and pure CFU-Meg. Results demonstrated significant decreases in progenitors in the BM of dual CD34/c-mpl-/- mice compared to decreases from CD34-/- or c-mpl-/- mice; progenitor numbers in CD34/c-mpl-/- mice were decreased by 83-99.3% compared to that in wild-type littermate control mice. Decreases in progenitors in spleens of c-mpl-/- mice (89-96%) were more drastic than those of CD34-/- mice (50-78%) whereas those of dual CD34/c-mpl-/- mice were equal to or lower than that of c-mpl-/- mice (93-98%). Decreases in PB progenitors were seen in the c-mpl-/- and dual CD34/c-mpl-/- mice (75-90%). Whereas progenitor cells in BM, Spl and PB were drastically reduced in dual CD34/c-mpl-/- mice compared to controls, absolute numbers of nucleated cells in these organs were essentially not reduced. These studies demonstrate that CD34 and c-mpl have non-redundant effects on maintenance of steady-state hematopoiesis and highlight how few progenitor cells are required in steady-state conditions to populate and maintain the BM, Spl, and PB with nucleated cells.