Bone Marrow Hyperplasia Research Articles

Hair-on-End

A 25-year-old man was admitted with a long history of weakness and wasting. In fact, he said he “was born—and fell ill.” He claimed to be still unmarried as a result of his illness. One year earlier he had been admitted with pain in his left hip. Despite several hospital admissions, an explanation for his illness had not been found. He was wasted, pale, and slightly jaundiced. His chest, abdomen, and extremities appeared normal. His forehead and maxillae were prominent, and this prompted us to take an x-ray of his skull. The x-ray (Figure 1A) showed a hair-on-end pattern compatible with a hemoglobinopathy—the skull changes reflect bone marrow hyperplasia. A peripheral blood slide revealed sickle cell disease. Several complications were noted: anemia, hyposplenism (Howell-Jolly bodies, Figure 1B), nephropathy (protein 2+ on dipstick). And, in hindsight, the pain in his left hip was caused by femoral head necrosis (Figure 1C) during a sickle cell crisis. Figure 1. (A) Skull x-ray with a hair-on-end pattern, reflecting bone marrow hyperplasia. (B) Peripheral blood slide with a Howell-Jolly body in an erythrocyte (arrow). (C) X-ray of pelvis showing left femoral head necrosis. Sickle cell disease is a relatively common condition in Malawi and many other African countries: in one study 2.5% of infants in southern Malawi were found to have the HbSS genotype.1 The mortality among children with sickle cell disease is very high, primarily because the disease is under-recognized.2 Indeed, in our patient sickle cell disease was diagnosed some 20 years too late, after the onset of several severe complications. Early recognition coupled to relatively simple interventions such as vaccinations, prophylactic antibiotics, and disease education has proven to reduce the morbidity and mortality of sickle cell disease patients.

A high-fat diet increases interleukin-3 and granulocyte colony-stimulating factor production by bone marrow cells and triggers bone marrow hyperplasia and neutrophilia in wistar rats

It is well established that the excessive consumption of a high-fat diet (HFD) results in overweight, obesity and an increase in leptin concentrations, which triggers a chronic inflammatory condition that is associated with a high white blood cell count. Two-month-old male Wistar rats were fed a control (CON) diet or an HFD for 12 weeks. After this period, hemogram, myelogram and biochemical parameters were evaluated along with the cell cycle and the percentage of CD34(+) cells in the bone marrow as well as cell proliferation and differentiation assays and the production of stem cell factor, interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF). The HFD animals exhibited leukocytosis and neutrophilia with increased C-reactive protein, leptin, cholesterol and triglyceride concentrations. In the HFD group, the bone marrow revealed myeloid hyperplasia, especially of the granulocytic compartment with a higher percentage of CD34(+) cells and a higher percentage of cells in the G2/S/M cell cycle phases. In addition, the HFD bone marrow cells had a higher capacity to proliferate and differentiate into granulocytic cells in an in vitro system and a higher capacity to produce IL-3 and G-CSF. These data led us to infer that the HFD induces leukocytosis and neutrophilia suggesting alterations in hematopoiesis system modulation.

Age and Sex Determinants of Bone Phenotype in a Transgenic Mouse Model of Sickle Cell Disease

Abstract 3249Variations in the amino acid sequence of the beta globin gene (HBB) chain can result in the production of variant hemoglobins. Sickle cell anemia is caused by homozygosity for a single nucleotide mutation in codon 6 of HBB, GAG>GTG, resulting in the substitution of valine for glutamic acid (glu6val). Bone pathologies are among the myriad complications of sickle cell disease and are a common cause of pain and morbidity. These pathologies include osteonecrosis, osteoarthritis and vaso-occlusive infarct. Chronic vaso-occlusion in sickle cell disease patients leads to ischemia and necrotic lesions while bone marrow hyperplasia contributes to the softening of trabecular bone, and overall bone loss. The mechanism underlying these structural changes in sickle bone are not fully understood.Bone constantly undergoes a self-renewal process and adapts to damage through remodeling. The remodeling process is highly regulated and carried out by bone forming cells (osteoblast) and bone resorption cells (osteoclast). Disease states can alter the remodeling capacity of bone and compromise its structure and quality. To determine the effect of sickle cell disease on bone, we used microcomputed tomographic (microCT) analysis, real time PCR and immunohistochemistry to characterize the bone phenotype in a transgenic mouse model (Townes) of sickle cell disease.Osteoclasts are characterized by their expression of tartrate-resistant acidic phosphatase (TRAP), while proliferating osteoblasts show alkaline phosphatase (AP) activity. Relative levels of TRAP and AP mRNA were measured in RNA derived from the bone of 10 week and 21 week old mice. TRAP and AP were elevated (P<0.05) in (heterozygous) trait mice compared to (homozygote) sickle mice at 10weeks (Figure 1a), suggesting reduced bone formation in sickle mice. At 21 weeks, TRAP and AP mRNA in trait stayed relatively the same, while there was a 6 fold decrease in AP expression (P<0.05) in sickle mice and TRAP remained the same as 10 weeks (Figure 1b). The reduction in AP expression in sickle mice shows a balance shift favoring bone resorption in 21 week old mice. Taken together, sickle mice show reduced bone formation, and in an age dependent manner show bone loss.MicroCT analysis of the femoral trabecular morphology of the epiphysis and metaphysis also showed an age-dependent effect on bone. In addition, there were significant differences observed on the basis of sex. At 10 weeks, there were no significant differences observed between trait and sickle mice, however, there was a trend towards lower values in sickle mice. Although these differences were not statistically significant, an increase in the sample size might lead to more significant findings. Comparison of the 10 week old mice to the 21 week old mice showed a general decrease in trabecular number, and an increase in trabecular spacing, which may be indicative of bone loss. This difference was significant in the female trait mice. Although the trend in the female sickle mice was noted, a full statistical analysis was not possible due to the small sample size of the 21week old female sickle mice.Sickle cell disease and its impact on bone are highly variable. Our preliminary result suggest that age and sex may play a role and form the basis for further study. [Display omitted] (A) AP and TRAP show relative and elevated expression in 10 week and 21 weeks old respectively trait mice compared to sickle mice. Results are shown as mean +/− SD (n=3 for each). (B). Alkaline phosphatase significantly reduces in sickle mice at 21weeks. (*) denotes statistical significance. [Display omitted] Representative 3D thickness maps for femoral metaphyseal trabecular volumes of interest. Top panel shows trait mice at 10 and 21 weeks respectively. Bottom panel shows sickle mice at 10 and 21 weeks respectively. Measurements are depicted in pseudocolor scale - thicker = red, thinner = blue/green. Disclosures:No relevant conflicts of interest to declare.

A High-Fat Diet Increases IL-1, IL-6, and TNF-α Production by Increasing NF-κB and Attenuating PPAR-γ Expression in Bone Marrow Mesenchymal Stem Cells

It is well established that a high-fat diet (HFD) can lead to overweight and ultimately to obesity, as well as promoting low-grade chronic inflammation associated with increased levels of such mediators as TNF-α, IL-1, and IL-6. Bone marrow mesenchymal stem cells (MSCs), which are involved in hematopoietic niches and microenvironments, can be affected by these cytokines, resulting in induction of NF-κB and inhibition of PPAR-γ. Because this phenomenon could ultimately lead to suppression of bone marrow adipogenesis, we set out to investigate the effect of an HFD on the expression of PPAR-γ and NF-κB, as well as the production of IL-1, IL-6, and TNF-α in MSCs. Two-month-old male Wistar rats were fed a HFD diet and evaluated by means of leukograms and myelograms along with blood total cholesterol, triglyceride, and C-reactive protein levels. MSCs were isolated, and PPAR-γ and NF-κB were quantified, as well as IL-1, IL-6, and TNF-α production. Animals that were fed a HFD showed higher levels of blood total cholesterol, triglycerides, and C-reactive protein with leukocytosis and bone marrow hyperplasia. MSCs from HFD animals showed increased production of IL-1, IL-6, and TNF-α and increased NF-κB and reduced PPAR-γ expression. Therefore, ingestion of an HFD induces alterations in MSCs that may influence modulation of hematopoiesis.

Clinical aspects and pathogenesis of congenital dyserythropoietic anemias: from morphology to molecular approach

Congenital dyserythropoietic anemias belong to a group of inherited conditions characterized by a maturation arrest during erythropoiesis with a reduced reticulocyte production in contrast with erythroid hyperplasia in bone marrow. The latter shows specific morphological abnormalities that allowed for a morphological classification of these conditions mainly represented by congenital dyserythropoietic anemias types I and II. The identification of their causative genes provided evidence that these conditions have different molecular mechanisms that induce abnormal cell maturation and division. Some altered proteins seem to be involved in the chromatin assembly, such as codanin-1 in congenital dyserythropoietic anemia I. The gene involved in congenital dyserythropoietic anemia II, the most frequent form, is SEC23B. This condition seems to belong to a group of diseases attributable to defects in the transport of newly synthesized proteins from endoplasmic reticulum to the Golgi. This review will analyze recent insights in congenital dyserythropoietic anemias types I and II. It will also attempt to clarify the relationship between mutations in causative genes and the clinical phenotype of these conditions.

Alterations in Bone and Erythropoiesis in Hemolytic Anemia: Comparative Study in Bled, Phenylhydrazine-Treated and Plasmodium-Infected Mice

Sustained erythropoiesis and concurrent bone marrow hyperplasia are proposed to be responsible for low bone mass density (BMD) in chronic hemolytic pathologies. As impaired erythropoiesis is also frequent in these conditions, we hypothesized that free heme may alter marrow and bone physiology in these disorders. Bone status and bone marrow erythropoiesis were studied in mice with hemolytic anemia (HA) induced by phenylhydrazine (PHZ) or Plasmodium infection and in bled mice. All treatments resulted in lower hemoglobin concentrations, enhanced erythropoiesis in the spleen and reticulocytosis. The anemia was severe in mice with acute hemolysis, which also had elevated levels of free heme and ROS. No major changes in cellularity and erythroid cell numbers occurred in the bone marrow of bled mice, which generated higher numbers of erythroid blast forming units (BFU-E) in response to erythropoietin. In contrast, low numbers of bone marrow erythroid precursors and BFU-E and low concentrations of bone remodelling markers were measured in mice with HA, which also had blunted osteoclastogenesis, in opposition to its enhancement in bled mice. The alterations in bone metabolism were accompanied by reduced trabecular bone volume, enhanced trabecular spacing and lower trabecular numbers in mice with HA. Taken together our data suggests that hemolysis exerts distinct effects to bleeding in the marrow and bone and may contribute to osteoporosis through a mechanism independent of the erythropoietic stress.

THE EFFECTS OF TUNICATE EXTRACT ON HEMOPOIESIS UNDER MYELOSUPRESSION CONDITIONS IN MICE

The effects of EA (extract of marine hydrobiont tunicate) on hemopoietic processes under bone marrow hypoplasia were investigated. Model of myelosupression induced by injection CBA mice an alkylating drug cyclophosphane (CF 200 mg/kg). EA was administrated into stomach 30 mg/kg daily, since 0 day and up to the end of experiment (10 day). CF caused the myeloid cells depression, there was shown, that bone marrow cells damage by the cytostatic agent was significantly higher in granulocytic population on 3 day. The count of cells in bone marrow increased on 5 day, but the intact index restoration didn’t registration until 10 day. The application of EA in mice after cyclophosphane action was promoted the increase of cells level in bone marrow faster than in CF control group. The quantity of bone marrow cells increased on 5 day and restored the initial level to 7 day under EA action, most of them were granulocytic neutrophils. The loss of hemopoietic cells under the cyclophoshpane action is result of short-liver mature cells reduction. The regeneration of hemopoiesis was ensured at the expense of the hemopoietic cells, which have stayed in intact state. The application of EA initiated the proliferative processes in bone marrow more early, then in CF group, and restored disturbance functions of hemopoiesis. There was shown, that hemopoiesis recovery activated by EA is manifested by the granulocyte lineage hyperplasia in bone marrow, rapid restoration of the polymorphonuclear leukocyte and monocyte count in blood. The effect of EA on the bone marrow was related to accelerate of proliferation and differentiation of the hemopoietic precursor cells and may be associated with hemopoietic microenvironment cells activation too. DOI: http://dx.doi.org/10.12731/2227-930X-2012-1-15

Long-term toxicology and carcinogenicity of 2,4,6-trichlorophenol

Carcinogenesis bioassays were conducted by giving 2,4,6-trichlorophenol [2,4,6-TCP] in feed to groups of 50 male and female Fischer rats and male B6C3F1 mice for two years. Dietary concentrations were 0 [20/group], 5000 [0.5%], or 10,000 [1%] ppm. Female mice began with 10,000 and 20,000ppm but after 38weeks were lowered due to reduced body weights to 2500 and 5000ppm for 67weeks; exposures averaged 5200 and 10,400ppm. Adverse effects at two years were leukocytosis and monocytosis of peripheral blood and hyperplasia of bone marrow in both sexes of rats. In mice, liver toxicity, including individual liver cell abnormalities, focal areas of cellular alteration, and focal and nodular areas of hyperplasia were commonly present. Regarding carcinogenic activity, TCP caused leukemias/lymphomas in male rats, and possibly in female rats and female mice as well, and induced liver tumors in male and female mice. Using NTP categories of evidence indicates ‘clear evidence of carcinogenicity’ for male rats [hematopoietic system tumors]; ‘equivocal evidence of carcinogenicity’ for female rats [hematopoietic system tumors]; ‘clear evidence of carcinogenicity’ for male and female mice [liver tumors].

Identification of a novel partner gene, TPR, fused to FGFR1 in 8p11 myeloproliferative syndrome

The 8p11 myeloproliferative syndrome (EMS) is an aggressive neoplasm caused by the fusion of various partner genes to fibroblast growth factor receptor 1 (FGFR1). Various FGFR1 fusions are associated with subtly distinct disease phenotypes. Here, we report a new translocation at the FGFR1 locus in a patient who carried t(1;8)(q25;p11.2) and presented with myeloproliferative neoplasm-like symptoms. The patient was characterized by myeloid hyperplasia of bone marrow, markedly elevated numbers of monocytes, and normal to mildly elevated eosinophils. Initial fluorescent in situ hybridization analysis confirmed that FGFR1 in this patient was disrupted. Subsequent analysis led to the identification of a novel translocation, in which exon 23 of the translocated promoter region (TPR) gene at chromosome band 1q25 was fused to exon 13 of FGFR1 (RefSeq NM_0231102.2). The TPR portion of the fusion protein contains putative functional motifs including an N-terminal TprMet domain, nuclear pore complexes associating domain, and multiple coiled-coil domains. It is likely that one or more of the motifs from TPR contribute to dimerization, resulting in constitutive activation of the FGFR1 kinase domain. Our results further support a critical role of FGFR1 in the pathogenesis of EMS and may lead to more accurate diagnosis and potential targeted therapy.

Magnetic resonance imaging and spectroscopy of the bone marrow in children with common hematological diseases

To evaluate the magnetic resonance (MR) imaging and proton magnetic resonance spectroscopy ((1)H-MRS) in the diagnoses of pediatric hematological diseases. A total of 35 cases with pediatric hematological diseases were confirmed by bone marrow puncturing. There were acute leukemia (n = 26), aplastic anemia (n = 6), thalassemia (n = 2) and autoimmune hemolytic anemia (n = 1). Thirty age-marched healthy children underwent MR imaging (T(1)WI, T(2)WI, STIR) and (1)H-MRS of lumber spine and ilium. The lumber spines and iliums were studied by observation of MR imaging and calculation of fat fraction (FF%). Two patterns were classified by MR imaging and (1)H-MRS in lumber spines and iliums of all cases. Pattern 1: hyperplasia of bone marrow (BM). There were acute leukemia (n = 26), thalassemia (n = 2) and autoimmune hemolytic anemia (n = 1). The manifestations included homogeneous low signal intensity (SI) on T(1)WI, homogeneous low SI on T(2)WI, high SI on STIR and high water peak and low fat peak on (1)H-MRS. The FF%s of Regions of Interest (ROI) in lumber 4s and left iliums of 26 cases with acute leukemia were 0%, of 3 cases with thalassemia or autoimmune hemolytic anemia were 5.02% and 3.70%. Pattern 2: inhibition of BM. There were 6 cases of aplastic anemia. The manifestations included homogeneous or inhomogeneous high SI on T(1)WI and T(2)WI, homogeneous or inhomogeneous low SI on STIR, and low water peak and high fat peak on (1)H-MRS. FF%s of ROI in lumber 4s and left iliums for 6 cases of aplastic anemia were 74.69% and 91.51%. FF% in all groups had significant differences according to the Mann-Whitney test (P < 0.05). MR imaging and (1)H-MRS may serve as a noninvasive method for checking hematopoietic status of bone marrow in pediatric hematological diseases.

Effect and mechanism of Cantharides vesiculation moxibustion on hematopoietic function in mice treated with cyclophosphamide

To observe the effects of Cantharides vesiculation moxibustion on hematopoietic function and granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by peritoneal macrophages in mice treated with chemotherapy. The mice treated with Cyclophosphamide (CTX) were randomly divided into 6 groups. Ten mice in the control group were not treated. The mice in the vesiculation I–IV groups received moxibustion at Dazhui (GV 14), Shenshu (BL 23) and Zusanli (ST 36) with different concentrations of Cantharides tincture. The mice in the grain moxibustion group were treated with moxa cone of wheat-grain size at these points. The other 10 normal mice were adopted as control. The peripheral blood leukocytes count, bone marrow nucleated cells count, spleen index and the ability of peritoneal-macrophages-induced GM-CSF were detected at different stages. CTX could cause significant bone marrow suppression, significant decrease of bone marrow nucleated cell counts, peripheral blood leukocytes and spleen index, and reduction of peritoneal macrophages induced GM-CSF in the mice. Vesiculation moxibustion at Dazhui (GV 14), Shenshu (BL 23) and Zusanli (ST 36), not only reduced the bone marrow suppression, promoted bone marrow hyperplasia, increased bone marrow nucleated cell count, enhanced the number of the peripheral blood white blood cell (WBC) and shorted low sustained period of WBC chemotherapy-induced, but also enhanced the ability of macrophage induced GM-CSF. Cantharides vesiculation moxibustion has good resistance to the bone marrow suppression in mice treated with chemotherapy, and enhance the ability of the mouse peritoneal macrophages induced GM-CSF.

Analysis on bone marrow feature of 56 clinic cases of benzene poisoning

To explore the bone marrow feature of hemopoietic system injured by benzene through analyzing 56 benzolism cases. The 56 benzolism cases were divided into mild poisoning group, midrange poisoning group, aplastic anemia group, pancytopenia group and leukemia group. All cases progressed bone marrow aspiration and smear, and counted hundred karyocytes by Wright-Giemsa tinct bone marrow smear to classification and observe the cells' feature. The megakaryocytes and the extent of bone marrow hyperplasia were decreased by turns of mild poisoning group, midrange poisoning group and aplastic anemia group. The archaeocytes and juvenile cells proliferation in mild poisoning group and midrange poisoning group were inhibited and occurred cell paramorphia which related to intoxication. Comparing with the other groups and normal reference value, the pancytopenia group's percentage of bone marrow cells in karyocytes was significantly decreased (P < 0.01, P < 0.05) and the leukemia group's percentage of bone marrow cells in karyocytes was significantly increased (P < 0.01). The proportion of cell paramorphia and nucleus malformation of granulocytes and red blood cells in pancytopenia group and leukemia group were increased, especially in leukemia group. We saw the inhibition of archaeocytes and juvenile cells proliferation and some cell paramorphia appearances in mild poisoning and midrange poisoning cases of chronic benzolism. The abnormality changes which can be seen in bone marrow of severe benzolism cases were corresponding with the clinical classification.

NS-018, a Potent Novel JAK2 Inhibitor, Effectively Treats Murine MPN Induced by the Janus Kinase 2 (JAK2) V617F Mutant

AbstractAbstract 4106An activating mutation in the Janus kinase 2 gene (JAK2) (G1849T, which produces JAK2 V617F) occurs at a high frequency in Bcr-Abl-negative myeloproliferative neoplasms (MPNs). JAK2 V617F induces cytokine-independent growth in cell lines and, in murine models, recapitulates much of the pathobiology observed in MPN patients, suggesting that small-molecule inhibitors targeting JAK2 may be therapeutically useful. Some orally bioavailable inhibitors of JAK2 are already in clinical trials.NS-018 is a novel JAK2 inhibitor that inhibits JAK2 enzyme activity with an IC50 value of less than 1 nM. NS-018 shows 30–50-fold selectivity for JAK2 over other JAK-family kinases such as JAK1, JAK3 and TYK2.We tested NS-018 in a murine model of MPN induced by JAK2 V617F. Mice expressing JAK2 V617F controlled by the H2Kb promoter (V617F-TG mice) show an MPN phenotype: leukocytosis, thrombocytosis, progressive anemia, hepatosplenomegaly with extramedullary hematopoiesis, megakaryocyte hyperplasia and bone marrow fibrosis. They also exhibit body weight loss and high mortality compared to wild-type controls. Bone-marrow cells show constitutive activation of STAT5 and cytokine-independent growth of erythroid colony-forming units (CFU-E).NS-018 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in V617F-TG cells in vitro. For in vivo experiments, V617F-TG mice were divided into treatment and vehicle control groups after disease was established at 12 weeks after birth. NS-018 was administered for 24 weeks by oral gavage at doses of 25 mg/kg or 50 mg/kg bid, and the control groups received vehicle only. Mice were monitored by blood counts, and a subset of mice was euthanized for detailed histopathology and fluorescence activated cell sorting analysis. During the study, 12 of 34 mice died in the vehicle group, whereas 1 of 36 mice died in the 50 mg/kg group. There was a statistically significant prolongation of survival in the 50 mg/kg group (p<0.01). Mice treated with NS-018 gained more weight than vehicle-treated mice, and were comparable to wild-type mice. V617F-TG at 12 weeks old showed severe leukocytosis with average white blood cell counts of 24 × 1010/L. After two weeks of NS-018 treatment, the leukocyte count was reduced to 59% in 25 mg/kg group and 39% in the 50 mg/kg group compared to vehicle group, and the effect was maintained until the end of the study. The inhibitory effect of NS-018 on T or B lymphocytes was much less than on myeloid cells. The 50 mg/kg group showed no progression of anemia. NS-018 treatment also improved hepatosplenomegaly in a dose-dependent manner. In the spleen, Mac-1/Gr-1+ myeloid cells associated with extramedullary hematopoiesis were significantly decreased, and B220+ B cells were increased by NS-018 treatment. In correlation with reduction of organ weights and infiltrating myeloid cells, there was also clear evidence of a dose-dependent reduction in the histopathology of extramedullary hematopoiesis in the spleen, liver, and lungs of NS-018-treated mice. In contrast to the improvement in the pathology of these organs, NS-018 had little impact on the progression of fibrosis and megakaryocyte hyperplasia in bone marrow. No significant toxicity was observed in treated mice.In conclusion, NS-018 demonstrated therapeutic efficacy in a murine model of MPN induced by JAK2 V617F. In V617F-TG, which closely mimics human MPN, NS-018 significantly improved survival, body weight loss, hepatosplenomegaly, leukocytosis and anemia progression, thus confirming the viability of a targeted-therapy approach in managing JAK2 V617F positive MPNs. On the basis of these preclinical experiments, NS-018 appears to be an excellent candidate for phase I/II studies in patients with Bcr-Abl-negative MPNs. Disclosures:Nakaya:Nippon Shinyaku Co., Ltd: Employment. Homan:Nippon Shinyaku Co., Ltd: Employment. Kotera:Nippon Shinyaku Co., Ltd: Employment. Shibayama:Nippon Shinyaku Co., Ltd: Employment. Naito:Nippon Shinyaku Co., Ltd: Employment. Shimoda:Nippon Shinyaku Co., Ltd: Research Funding.

Leukemic Hierarchy and Systemic Factors Dictating Disease Evolution In Chronic Myelogenous Leukemia

AbstractAbstract 886Chronic myelogenous leukemia (CML) is a clonal myeloproliferative neoplasm (MPN) characterized by the t(9;22)(q34;q11) reciprocal translocation, which leads to the expression of the BCR/ABL fusion protein. CML is clinically characterized by the abnormal expansion of the myeloid lineage leading to progressive peripheral neutrophilia. This indolent disease has the propensity to evolve into an acute leukemia-like blastic phase involving either myeloid or B lymphoid cells. As such, CML constitutes a paradigm for understanding cellular and molecular events controlling chronic diseases and their evolution toward frank leukemia. CML arise from a small pool of leukemic stem cells (LSC) that can be operationally defined by their ability to sustain tumor growth over time and to transplant leukemia to recipient mice. Multiple lines of evidence indicate that LSCs originate from abnormally behaving hematopoietic stem cells (HSCs), although it is still largely unknown how BCR/ABL affects the biology of HSCs and the fate of downstream progenitor cells. To study the impact of BCR/ABL expression on these compartments, we used a transgenic mouse model that allows for inducible expression of BCR/ABL in HSCs and downstream progeny (Blood 105: 324, 2005). Induction of BCR/ABL expression in this model recapitulated many features of human CML such as myeloid bone marrow (BM) hyperplasia, myelofibrosis, splenomegaly and myeloid cell infiltration in non-hematopoietic organs. Disease development also correlated with a dramatic reorganization of the stem and progenitor compartments in the BM and their relocation/expansion in the spleen. In the BM, we observed a marked reduction in the number of Lin– Sca1+ c-Kit+ Flk2– CD48– CD150+ long-term (LT) and Lin– Sca1+ c-Kit+ Flk2– CD48– CD150– short-term (ST) HSCs associated with an expansion of several types of non-self-renewing multipotent progenitors (MPP) and myeloid committed progenitors. We confirmed by limited dilution transplantation experiments that BCR/ABL+ LT-HSCs were true LSCs as they were the only cells capable of transferring sustained CML disease in recipient mice with as few as 50 cells injected. We also found that BCR/ABL expression impacts on the biology of the ST-HSC and Lin– Sca1+ c-Kit+ Flk2+ MPP compartments. Transplantation of both populations induced dramatic but transient hyperplasia, which could eventually mimic a leukemic phenotype with high doses of cell injected (4,000 cells per mouse). However, while transplantation of BCR/ABL+ ST-HSCs led to the expected myeloid hyperplasia, transplantation of BCR/ABL+ MPPs led to a massive accumulation of B-cell progenitors in the BM that resembles lymphoid blast crisis. Strikingly, co-transplantation of 4,000 BCR/ABL+ LT- or ST-HSCs with 4,000 BCR/ABL+ MPPs almost always resulted in myeloid hyperplasia suggesting an active inhibition of MPP-derived lymphoid progeny by the leukemic myeloid compartment. We reasoned that the molecular effectors for this lymphoid inhibitory effect could be extracellular signaling molecules that will be detectable in the serum of CML-developing BCR/ABL mice. Using antibody arrays and enzyme-linked immunosorbent assays (ELISA), we found that the serum concentration of the proinflammatory cytokine interleukine-6 (IL-6) correlates with CML progression both in primary and transplanted mice. Moreover, we showed in vitro that IL-6 controls lineage fate decision of leukemic progenitors by promoting myeloid differentiation from MPPs at the expense of the B lymphoid lineage differentiation. As such, IL-6 targets both normal and malignant MPPs thereby providing a positive feedback loops that promote CML development. In summary, our results identify and functionally characterize a pathological hierarchy in CML that includes LSC and immature leukemic progenitors. They uncover a novel fate-regulatory mechanism at the systemic level that controls the differentiation outcome of the leukemic progenitors and can have key implication for disease progression. Taken together, they demonstrate that CML evolution is the result of a balance between BCR/ABL cell intrinsic effects and environmental cues and provide a rational for the paradoxical myeloid-lymphoid conversion that can be observed during lymphoid blast crisis. Disclosures:No relevant conflicts of interest to declare.

The Effects of a Vitamin D–deficient Diet on Chronic Cadmium Exposure in Rats

Itai-itai disease (IID) of humans is one of the most severe forms of chronic cadmium (Cd) intoxication. Itai-itai disease occurs mainly in post-menopausal women and is characterized by osteoporosis with osteomalacia, renal tubular disorder, and renal anemia. Some researchers insist the major cause of IID is not Cd, but rather malnutrition, especially hypovitaminosis D. We administrated a low concentration of Cd chloride intravenously to ovariectomized female rats that were fed a vitamin D-deficient diet or a normal diet for fifty weeks. The vitamin D-deficient diet decreased serum concentration of vitamin D, but it did not affect the metabolism of the kidney or bone. Cadmium treatment alone induced a decrease in serum concentration of vitamin D, as well as renal dysfunction, renal anemia, and abnormal bone metabolism. Osteoporosis with osteomalacia, tubular nephropathy, fibrous osteodystrophy, and bone marrow hyperplasia occurred following Cd treatment. In rats treated with Cd and administered a vitamin D-deficient diet, the toxic effects of Cd on kidney, bone, and hematopoiesis were enhanced in comparison to rats treated with Cd and a normal diet. The present experiment demonstrated that hypovitaminosis D did not evoke morphologic features of IID in humans but did enhance Cd-induced toxicity in the rat model of this disease.

Clinical and Radiologic Manifestations of Sickle Cell Disease in the Head and Neck

Sickle cell disease is a common inherited blood disorder that is characterized by the presence of sickle-shaped red blood cells. The clinical manifestations of sickle cell disease vary, but they may be attributed to three mechanisms: vaso-occlusion, chronic hemolytic anemia, and infection. The imaging appearances of central nervous system and musculoskeletal involvement by sickle cell disease have been well documented; however, involvement in the head and neck often is underappreciated, although it is not uncommon. In the head and neck, sickle cell disease can involve the inner ears, orbits, paranasal sinuses, bones, lymph nodes, and vessels. Manifestations of inner ear involvement include labyrinthine hemorrhage and labyrinthitis ossificans. In the orbits, they include lacrimal gland swelling, orbital wall infarction, and subperiosteal hemorrhage or fluid. In the paranasal sinuses, extramedullary hematopoiesis is seen. When bone is involved, infarction, osteomyelitis, bone marrow hyperplasia, and deposition of iron in bone marrow are seen in the maxillofacial bone and skull base. When lymph nodes are involved, lymphadenopathy is seen, and when blood vessels are involved, arterial stenosis and ectasia are seen. An understanding of the pathophysiology of sickle cell disease and knowledge of the various clinical and radiologic manifestations are crucial for prompt diagnosis and appropriate treatment.

Erythrocytes anion transport and oxidative change in β‐thalassaemias

beta-Thalassaemia is characterized by a decrease in globin beta-chain synthesis and an excess in free alpha-globin chains. This induces alterations in membrane lipids and proteins resulting from a reduction in spectrin/band 3 ratio, partial oxidation of band 4.1 and clustering of band 3. The membrane injury provokes hyperhaemolysis and bone marrow hyperplasia. The pathophysiology of thalassaemia is associated with iron overload that generates oxygen free radicals and oxidative tissue injury with ocular vessel alterations. The aim of this research is to investigate the influence of oxidative stress on band 3 efficiency, which is an integral membrane protein of RBCs (red blood cells). Band 3 protein, of which there are more than 1 million copies per cell, is the most abundant membrane protein in human RBCs. It mediates the anion exchange and acid-base equilibrium through the RBC membrane. Some experiments were performed on thalassaemic cells and beta-thalassaemia-like cells and tested for sulfate uptake. To test the antioxidant effect of Mg(2+), other experiments were performed using normal and pathological cells in the presence of Mg(2+). The oxidant status in thalassaemic cells was verified by increased K(+) efflux, by lower GSH levels and by increased G6PDH (glucose-6-phosphate dehydrogenase) activity. The rate constant of SO(4)(2-) uptake decreases in thalassaemic cells as well as in beta-thalassaemia-like cells when compared with normal cells. It increases when both cells are incubated with Mg(2+). Our data show that oxidative stress plays a relevant role in band 3 function of thalassaemic cells and that antioxidant treatment with Mg(2+) could reduce oxidative damage to the RBC membrane and improve the anion transport efficiency regulated by band 3 protein.

Erythrocytic transglutaminase inhibition hemolysis at presentation of celiac disease

Celiac disease (CD) is a common autoimmune condition. Previously it was considered to be a rare childhood disorder, but is actually considered a relatively common condition, present at any age, which may have multiple complications and manifestations. Hematological disorders of the disease are not uncommon. Among these disorders, the most frequently reported are anemias as a result of iron deficiency, often associated with folate and/or B12 deficiency. Anemias caused by hemolysis are very rarely reported in celiac patients. An 11-year-old girl with a previous uneventful medical history presented with severe hemolytic anemia. Hemolysis was Coombs negative, accompanied by inappropriate low reticulocyte count, despite exaggerated bone marrow hyperplasia of the erythroid precursors which showed normal maturation. Serology for recent infections, including Epstein-Barr virus, parvovirus B19, cytomegalovirus and mycoplasma, were all negative. Levels of serum IgA, IgG and IgM, were all within normal ranges for age. Screening for anti-DNA, antinuclear, antineutrophil cytoplasmic, antimicrosomal, antithyroglobulin, and antimitochondrial antibodies and lupus anticoagulants, was negative. She was also negative for human immunodeficiency virus. Conventional therapy with corticosteroids and intravenous immunoglobulin failed. CD was serendipitously discovered upon screening for anti-tissue transglutaminase autoantibodies. The disease was confirmed by biopsy of the small intestine mucosa. The patient recovered with gluten-free diet. A unique case of CD is presented. CD should be serologically screened in each patient with Coombs negative "immune" hemolytic anemia, particularly if accompanied by "reticulocytopenia". A new hemolytic mechanism and very speculative explanation for "reticulocytopenia" are discussed.

A subchronic murine intravenous pharmacokinetic and toxicity study of Hematide™, a PEGylated peptidic erythropoiesis-stimulating agent

The subchronic toxicity of Hematide™, a synthetic PEGylated peptidic erythropoiesis-stimulating agent (ESA), was evaluated in CD-1 mice at intravenous doses of 0, 1, 5, 25, and 125 mg/kg administered once every 3 weeks for 3 months. Hematide displayed sustained plasma levels with reduced clearance and prolonged half-lives up to 59.4 hours that translated into sustained, pronounced polycythemia, bone marrow hyperplasia, and splenic and liver extramedullary hematopoiesis. Toxicological findings were considered to be secondary to exaggerated pharmacology, rather than a direct drug effect, and included mortality at ≥25 mg/kg/dose. The no-observed-adverse-effect-level was determined to be 5 mg/kg.

Pattern of Bone Mineral Density in Sickle Cell Disease Patients with the High-Hb F Phenotype

Low bone mineral density (BMD) is common in sickle cell disease (SCD) patients of all ages due to erythropoietic stress and consequent bone marrow hyperplasia. Kuwaiti SCD patients have a mild clinical phenotype because of their high Hb F level. There has been no previous documentation of BMD in this population of patients. SCD patients (n = 81) and controls (n = 67) were recruited from hematology clinics of Mubarak Hospital, Kuwait. BMD was measured using dual X-ray absorptiometry (Hologic) in the lumbar spine and the hip (left femoral neck). Among the 53 adult patients, the prevalence of low BMD was 67.4% in the spine and 33.3% in the hip while among controls, the figures were 23.1 and 11.3%, respectively. These differences are significant (p < 0.01). In SCD children, the figures were 17.9% (in the lumbar spine) and 3.6% (in the hip), while in controls the figures were 13.3 and 0%, respectively. The differences are not significant (p > 0.05). Patients with frequent vaso-occlusive crisis had significantly lower mean BMD, but those with MRI evidence of avascular necrosis of the femoral head were more likely to have normal or osteosclerotic BMD. Our study showed that osteopenia/osteoporosis is uncommon among Kuwaiti children with SCD but quite common in adults.