Hematopoietic Marrow Research Articles

Bone Infarct Mimicking Infection in a Young Adult Sickler

A 23-year-old known sickle cell patient presented with left knee swelling and pain of 10 days duration. He was treated several times in the past for painful crises. Currently, at rest the knee pain was 8/10 and upon palpation or movement the pain was 10/10. Moving the knee at all aggravated his symptoms. His temperature was 101. The laboratory investigations demonstrated high white blood cell count at 19,000/μL (normal 4,000 – 11,000/μL) and a hemoglobin level of 8.5 g/dL. Initial plain x-ray was normal. An arthrocentesis yielded 30 cc of yellow dark fluid and synovial fluid analysis revealed 3,000 WBC (80% PMN) with no crystals. Final culture was unrevealing. A magnetic resonance imaging (MRI) showed increased T2 signal in the distal femur and proximal tibia (Fig. 1). The fatty yellow marrow in the distal femur and proximal tibia was replaced with hematopoietic red marrow. These findings indicated bone edema and acute bone infarction. Patients with sickle cell anemia are prone to both infarctive and infective crises. Clinical differentiation between both can be difficult. Initial radiographs are usually normal with an acute infaction [1]. As the condition becomes more chronic sclerosis develops [1]. MRI has an important role in demonstrating the area of infarct in the earlier stages [2]. Bone infarcts of the knees are not as common as in the hips and shoulders [2]. Thus, it is important to consider infarctions in such atypical sites as a differential diagnosis of painful knee pain in sicklers.

The Clinicopathological Characteristics of the Kidney in Acute Graft-versus-Host Disease after Dark Agouti-to-Lewis Rat Bone Marrow Transplantation

Allogeneic hematopoietic and bone marrow cell transplantation causes acute and chronic graft-versus-host disease (GVHD), which is considered a severe side effect. The liver, gut, and skin are well-known primary target sites of acute and chronic GVHD. However, whether the kidney is affected by GVHD remains uncertain. In the present study, we examined the clinical and pathological characteristics of the kidney in acute GVHD in Dark Agouti-to-Lewis rat bone marrow transplantation.

Late Onset Neutropenia Develops Selectively in Only a Subset of Patients with T Large Granular Lymphocyte Proliferation After Rituximab Treatment for Lymphoma,

Abstract 3675Emerging evidence suggests that B cells can actively modulate T cell immune responses by presenting antigen, providing co-stimulation and secreting cytokines. This has prompted investigation whether B cell depletion by monoclonal antibodies, including Rituximab, can alter the subset composition, activation or function of T cells. Rituximab-associated late onset neutropenia (R-LON) is increasingly recognized as a long-term adverse event of Rituximab. Although the etiology of R-LON is not fully elucidated, the syndrome seems to be multifactorial and likely caused by immune-mediated mechanisms. We have previously shown that at least a proportion of R-LON may develop in a setting of expanded cytotoxic T cell populations in peripheral blood (PB) with a large granular lymphocyte (LGL) phenotype (CD3+CD8+CD57+). Here, we extend our observations regarding PB lymphocyte subset composition in a cohort of 107 Rituximab-treated patients with available results from PB flow cytometry analysis performed at roughly similar intervals after the initial Rituximab administration. The present cohort included 107 patients, aged 16–83 (median 60), who received Rituximab for the treatment of chronic lymphocytic leukemia (CLL) (29), diffuse large B cell lymhoma (DLBCL) (20), marginal zone lymphomas (15), follicular lymhoma (FL) (15), mantle cell lymphoma (MCL) (8) and auto-immune cytopenias (20). Overall, we found: (i) increased (>1.0×109/l) CD8+ cells in 45/107 (42%) cases; (ii) CD4+/CD8+ cell ratios <0.7 in 56/107 (52%) cases; and, (iii) T-LGLs >20% in 66/107 (63%) cases. Within this cohort, 33 cases (group A) developed R-LON, whereas the remainder (group B, n=74) did not develop this syndrome over a comparable observation period. Importantly, no patient with autoimmune cytopenia developed R-LON. R-LON was significantly more frequent in patients with lymphoma subtypes treated with intensive chemotherapy (CLL, DLBCL, MCL), as well as patients who underwent autologous transplantation (p\\q0.001 for all comparisons). No significant differences were noted between groups A or B regarding PB lymphocyte subset composition. We next evaluated the findings from the histopathological study of bone marrow biopsy samples, available in 17 Group A and 19 Group B cases, all with a diagnosis of lymphoma. The morphological and immunohistochemical examination revealed a series of features common in both groups, summarized as follows: (i) mild-to-moderate small lymphocytic infiltration by CD20-CD79a-CD3+CD45RO+CD43+ (CD3>CD45RO) cells, predominantly nodular and/or interstitial (non intrasinusoidal); (ii) pronounced hyperplasia of the erythroid and megakaryocytic series with prominent dyserythropoiesis and dysmegakaryopoiesis, respectively, including abnormal paratrabecular localization, suggestive of myelodysplasia (MDS); and (iii) remarkable shift-to-the-left of the granulocytic series, often with abnormal localization of immature progenitors (ALIP), always with \\q2% CD34+ cells. A proportion of cases showed hyperplasia of the granulocytic series. However, a major difference between the two Groups concerned hypoplasia of the granulocytic series, which was noted almost exclusively in group A. We conclude that lymphoma patients treated with Rituximab often develop cytotoxic T cell expansions than can have a variable impact on hematopoiesis, with R-LON perhaps representing the end of a spectrum of T-LGL-mediated autoimmune myelopathy/myelodyplasia. The selective development of R-LON in only a proportion of cases with expanded cytotoxic T cells associated with prominent hypoplasia of the granulocytic series and MDS-like changes of the hematopoietic marrow post Rituximab raises several questions regarding the underlying (genetically determined?) immunopathogenetic mechanisms. Disclosures:No relevant conflicts of interest to declare.

Functional Inhibition of Osteoblastic Cells in An In Vivo Mouse Model of Myeloid Leukemia

Abstract 243Disruption of normal hematopoietic development is a major problem in acute myeloid leukemia (AML). Osteoblastic cells have been shown to support hematopoiesis. We hypothesized that myeloid leukemia inhibits osteoblastic cells contributing to the loss of normal hematopoiesis. To define effects of leukemia on osteoblastic cells, we employed a previously described syngeneic murine model of AML where transplant of leukemic cells does not require irradiation of recipients (Neering et al. Blood, 2007). Leukemic mice had decreased serum levels of the bone formation marker osteocalcin (OC) (92.6 ± 11.6 vs 25.9 ± 4.4 ng/ml Normal (N) vs Leukemic (L) p<0.0001 n=8–12 mice/group). OC mRNA in CD45-marrow cells was reduced in leukemic mice 6 days after transplantation of leukemia cells when disease burden was low, and severely diminished 11 days after transplant when disease burden was high (1290 ± 124.7 vs 440.1 ± 34 and 3.3 ± 0.8 relative expression N vs L day6 p<0.001 and N vs L day11 p<0.001 n=5 mice/group). In situ hybridization revealed almost complete loss of OC and collagen 1 expression at the endosteal bone surface suggesting a severe disruption of bone formation. Immunohistochemical analysis showed a reduction in endosteal-lining osteopontin+ cells in leukemic animals further confirming a phenotype of osteoblastic dysfunction. Osteoprogenitor cells were also reduced, determined by CFU-OB assays (1.45 ± 0.31 vs 0.21 ± 0.09 CFU-OBs/1×106 marrow cells N vs L p<0.01 n=7–10 mice/group). In leukemic mice TRAP+ osteoclastic cells were transiently increased (N: 50.8 ± 3.6 vs L day6: 64.4 ± 3.2; L day10: 33.8 ± 4.7 cells/ histological section N vs L p<0.05 n=5 mice/group) and the bone resorption marker serum CTX did not change. Despite the lack of a robust increase in osteoclast number or bone resorption, leukemic mice exhibited trabecular and cortical bone loss by micro-CT analysis (0.23 ± 0.01 vs 0.13 ± 0.01 tibia trabecular BV/TV, N vs L p<0.001 n=5 mice/group). Treatment with the bisphosphonate zoledronic acid (ZA) inhibited bone resorption in leukemic animals (21.79 ± 0.97 vs 8.28 ± 1.41 serum CTX ng/ml, Vehicle vs ZA n=5 mice/group), and rescued trabecular bone loss, but failed to rescue cortical bone loss (0.24 ± 0.017 vs 0.10 ± 0.011 cortical BV/TV, N vs L p<0.001 n=4 mice/group). ZA treatment did not affect the course of disease. Thus, we suggest that in our model leukemic cells functionally inhibit osteoblastic cells and uncouple bone formation from bone resorption. To determine if osteoblastic inhibition was mediated by cell contact dependent or secreted factors we performed cultures with osteoblastic cells from normal mice co-cultured with leukemic cells or treated with conditioned media from leukemic cell cultures. Osteoblastic differentiation and ability to form alkaline phosphatase positive colonies were absent in both culture conditions suggesting that a secreted factor is sufficient to inhibit osteoblasts in our model. The chemokine CCL-3 was recently reported to inhibit osteoblastic function in multiple myeloma. CCL3 protein was present at very high concentrations in both co-culture and conditioned media cultures (Osteoblasts: 14.08 ± 6.6 vs co-culture: 222.0 ± 14.2 p<0.0001; conditioned media: 130.2 ± 10.4 ng/ml p<0.0001 n=4 wells/group). CCL3 protein was also present at much higher levels in both the marrow plasma and blood serum of leukemic mice (Marrow: 4.53 ± 0.57 vs 122.0 ± 13.06 pg/ml N vs L p<0.0001, Blood: 0.103 ± 0.003 vs 26.12 ± 2.51 pg/ml N vs L p<0.0001 n=5 mice/group). CCL-3 mRNA which is expressed at very low levels in normal marrow was significantly elevated in malignant hematopoietic marrow cells compared to normal hematopoietic marrow cells from the same mice (0.47 ± 0.07 vs 3.70 ± 0.46 relative expression N vs L p<0.01 n=3 mice/group). To confirm that CCL3 signaling may play a role in human myeloid leukemias we analyzed CCL3 expression and protein levels in primary AML samples. Primitive CD34+CD38-CD123+ cells from 3 patients with AML expressed CCL3 mRNA at levels 54.7, 19.83, and 26.5 fold higher than 5 normal CD34+CD38- primary samples. CCL3 protein was also increased in marrow plasma from primary AML patients (10.76 ± 1.42 vs 18.97 ± 3.43 pg/ml N vs AML p<0.05 n=6–7 primary samples/group). Based on these results, we propose that therapeutic mitigation of leukemia-induced osteoblastic dysfunction may represent a novel approach to promote normal hematopoiesis in patients with myeloid neoplasms. Disclosures:No relevant conflicts of interest to declare.

Arachnoid ossificans containing metaplastic hematopoietic marrow resulting in diffuse thoracic intrathecal cysts and severe myelopathy

To present a rare case of multiple compressive thoracic intradural cysts with pathologic arachnoid ossification, review the literature and present the surgical options. Few reports have identified the existence of arachnoid calcifications and intrathecal cysts causing progressive myelopathy. The literature regarding each of these pathologies is limited to case reports. Their clinical significance is not well studied, although known to cause neurologic sequelae. An 81-year-old female clinically presents with rapidly progressive myelopathy. Pre-operative magnetic resonance imaging identified multiple compressive thoracic intrathecal cysts. Surgical exploration and decompression of these cysts identified calcified plaques within the arachnoid. Histopathologic examination revealed fibrocalcific tissue undergoing ossification with bone marrow elements. Due to progressive myelopathy, the thoracic cysts were decompressed and calcified plaques were excised, once identified intra-operatively. On last examination, the patient's neurologic status had not improved, but had stabilized. The rate of neurologic improvement from excision and decompression is variable, but it may still be warranted in the face of progressive neurologic deficits.

Informal caregiving in Hematopoietic Blood and Marrow Transplant patients

Purpose Hematopoietic cell transplant patients are among the most vulnerable and acutely ill cancer populations ( Bevans et al., 2008). The responsibility of caring for the daily physical and psychosocial needs of these patients after transplant is placed mostly on family caregivers ( Williams, 2007). The purpose of this descriptive correlational research study was to describe caregiving experiences of 56 caregivers of HCT patients 3–12 months following transplant. Methods & sample Patients and caregivers were recruited from two west coast regional transplant programs in the United States. Variables studied were: relationship quality, rewards of caregiving, predictability, role strain, patient function, caregiving activities, and caregiver quality of life (QOL). Key results Results indicated that all areas of role strain are significantly negatively correlated with caregiver’s QOL. Predictability was negatively associated with problem solving and emotional strain indicating that as the level of predictability of the situation decreases, caregiver strain and problem solving increase. Predictability was positively correlated to caregiver QOL indicating that as the situation is more predictable caregiver QOL increases. Emotional strain, problem-solving strain, and usual care strain were significantly positively related, indicating that emotional strain and problem-solving strain increased together. As usual care strain increased, so did problem-solving strain and emotional strain. Conclusions Suggestions for interventions include assessing and responding to caregiver issues such as emotional strain, problem-solving strain, usual care strain, unpredictability, and QOL. Examples of caregiver-focused interventions include providing timely appropriate information about these caregiver concerns including elements that make the caregiving situation predictable, and incorporating best practices for preventing and minimizing caregiver emotional strain.

Lenalidomide Enhances Clonogenic Activity, Proliferation and Erythroid Lineage Commitment of CD34+ Progenitor Cells While It Is Cytotoxic to CD34- Accessory Cells.

AbstractAbstract 1184Lenalidomide, a second generation immunomodulatory derivative of thalidomide, has demonstrated significant clinical benefit in myelodysplastic syndrome, multiple myeloma and B-cell malignancies. Its side effects include myelosuppression, particularly neutropenia, and impaired filgrastim induced stem cell mobilization in patients with myeloma. The precise mechanism of action of lenalidomide on hematopoietic and bone marrow microenvironment remains largely unknown. In this study, we investigated the effect of lenalidomide on survival, lineage commitment and differentiation of normal purified CD34+ hematopoietic stem cells.Mobilized peripheral blood progenitor cells (PBPCs) were obtained from a normal allogeneic stem cell donor with an informed consent, and were subjected to positive selection with the EasySep magnetic cell selection system to purify CD34+ cells (purity of 95%). Unsorted and CD34- accessory cells were used as controls.To assess the direct cytotoxic and proliferative effect of lenalidomide on purified CD34+ cells, lenalidomide (Celgene, NJ) was dissolved in DMSO and was diluted in culture medium to final concentration of 0.1, 1, 10, or 100 μM. Purified CD34+, CD34- and unsorted cells were plated in triplicates at a density of 2×104/mL in serum-free StemSpan® medium supplemented with growth factors and cytokines. 24 hours after plating the cells, lenalidomide or 0.1% DMSO as control was added. 72 hours following treatment, cells were counted with Beckman coulter counter and phenotypically analyzed using Accuri C6 flow-cytometer. The percentage of viable cells was determined using 7AAD staining. While no difference in viability was noted in the treated vs. untreated purified CD34+ cells, the viability of CD34- cells and unsorted cells was reduced to 30% and 40% respectively by lenalidomide. Contrary to our assumption, lenalidomide, in a dose dependant manner, induced proliferation of purified CD34+ cells. Maximal increase in proliferation was observed between 1mM and 10mM concentrations. The rate of growth plateaued beyond the 10mM concentration and 100mM concentration appeared to have no additional effect. At the peak of growth (10mM), the cell count nearly tripled. (Fig. 1) Lenalidomide appeared to be cytotoxic to CD34- and unsorted cells in a dose dependant manner. At 100mM concentration, the CD34- cell proliferation dropped by 4 fold and the unsorted cells by 7 fold.To examine the effect of lenalidomide on the clonogenic potential of purified CD34+ cells, 2,000 cells were seeded onto MethoCult® media (Stem cell technologies) supplemented with growth factors and cytokines. Cells were cultured in the presence of lenalidomide (0.1, 1, 10 and 100 μM) or 0.1 % DMSO as control. BFU-E, CFU-GM and CFU-mix were scored on day 14 with the use of an inverted microscope and standard morphological criteria. Significance of difference between groups was determined by student's t test for paired samples. Lenalidomide induced a significant increase in clonogenic activity of CD34+ cells (148 ± 27 colonies per 103 CD34+ cells, p = 0.01), most evident at the dose of 10 μM with enhanced formation of BFU-E (102 ± 23/ per 103 CD34+ cells, p = 0.01) but demonstrated no significant impact on CFU-GM (36 ± 14 per 103 CD34+ cells, p = 0.83) compared to control. (Fig. 2)This study demonstrates that lenalidomide has pronounced effects on both CD34+ and CD34- components of mobilized PBPCs. It enhances the proliferative and clonogenic potentials of CD34+ cells with lineage commitment towards erythroid differentiation. It also has cytotoxic activity towards CD34- cells. It is likely that neutropenia and impaired mobilization of stem cells in patients treated with lenalidomide is mediated via its impact on CD34- accessory cells in the bone marrow microenvironment. Further studies exploring the impact of lenalidomide on normal bone marrow stromal cells, T and NK cell mediated signaling are needed. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Correlation of measurable serum markers of inflammation with lung levels following bilateral femur fracture in a rat model

IntroductionEvaluation of the systemic inflammatory status following major orthopedic trauma has become an important adjunct in basing post-injury clinical decisions. In the present study, we examined the correlation of serum and lung inflammatory marker levels following bilateral femur fracture.Materials and methods45 Sprague Dawley rats underwent sham operation or bilateral femoral intramedullary pinning and mid-diaphyseal closed fracture via blunt guillotine. Animals were euthanized at specific time points after injury. Serum and lung tissue were collected, and 24 inflammatory markers were analyzed by immunoassay. Lung histology was evaluated by a blinded pathologist.ResultsBilateral femur fracture significantly increased serum markers of inflammation including interleukin (IL)-2, IL-6, IL-10, GM-CSF, KC/GRO, MCP-1, and WBC. Femur fracture significantly increased serum and lung levels of IL-1a and KC/GRO at 6 hours. Lung levels of IL-6 demonstrated a trend towards significance. Histologic changes in pulmonary tissue after fracture included pulmonary edema and bone elements including cellular hematopoietic cells, bone fragments and marrow emboli.Discussion and conclusionOur results indicate that bilateral femur fracture with fixation in rats results in increases in serum markers of inflammation. Among the inflammatory markers measured, rise in the serum KC/GRO (CINC-1), a homolog to human IL-8, correlated with elevated levels of lung KC/GRO. Ultimately, analysis of serum levels of KC/GRO (CINC-1), or human IL-8, may be a useful adjunct to guide clinical decisions regarding surgical timing.

Gelatinous Marrow Transformation and Hematopoietic Atrophy in a Miniature Horse Stallion

Gelatinous marrow transformation, or serous atrophy of bone marrow fat, has been noted in livestock, laboratory animals, and wildlife in association with an inadequate plane of nutrition, inanition, or intoxication. This is a report of gelatinous marrow transformation and hematopoietic marrow atrophy in a 5-year-old miniature horse stallion. The horse had oral malformations leading to poor food assimilation and emaciation. A bone marrow biopsy obtained to investigate persistent anemia and leukopenia showed hematopoietic atrophy and replacement of fat with a granular extracellular substance, which stained with alcian blue, consistent with acidic mucopolysaccharide content. Surgical correction of the dental abnormalities resulted in improved food assimilation, weight gain, and resolution of cytopenias. In humans, gelatinous bone marrow transformation and hematopoietic atrophy are commonly associated with malnutrition from anorexia nervosa and other causes. The cause of hematopoietic atrophy is unknown but may relate to a nonsupportive marrow microenvironment and inadequate hematopoietic substrate availability. Similar pathogenic mechanisms were suspected in this horse.

Treatment with a sclerostin antibody increases cancellous bone formation and bone mass regardless of marrow composition in adult female rats

The current report describes the skeletal effects of a sclerostin monoclonal antibody (Scl-AbIII) treatment at a yellow (fatty) marrow skeletal site in adult female rats. Ten-month-old female Sprague–Dawley rats were treated with vehicle or Scl-AbIII at 5 or 25 mg/kg, twice per week by s.c. injection for 4 weeks. Trabecular bone from a yellow (fatty) marrow site, the 5th caudal vertebral body (CVB), was processed undecalcified for quantitative bone histomorphometric analysis. Compared to vehicle controls, Scl-AbIII at both doses significantly increased bone formation parameters and trabecular bone volume and thickness and decreased bone resorption parameter in the trabecular bone of the CVB. As a reference, we also found that the Scl-AbIII at both doses significantly decreased bone resorption and increased bone formation and bone volume in a red (hematopoietic) marrow site, the 4th lumber vertebral body (LVB). It appears that the percentage of increase in trabecular bone volume induced by Scl-AbIII treatment was slightly larger in the LVB than in the CVB. In summary, these preclinical findings show that antibody-mediated sclerostin inhibition has significant bone anabolic effects at both red and yellow marrow skeletal sites.

Histological Differences in Iliac and Tibial Bone Graft

Cancellous bone graft is frequently used during orthopedic procedures. While the iliac crest has traditionally been the most common donor site, the proximal tibia is an alternative donor site, especially for foot and ankle procedures. This study tested the null hypothesis that the histologic composition of iliac and tibial bone grafts is similar. Specimens from the iliac crest (n = 10) and tibia (n = 10) in excess of that needed for patients undergoing foot or ankle fusion were examined histologically. Iliac samples were taken from the anterior iliac crest. Tibial samples were harvested from the region of Gerdy's tubercle. Specimens were graded based on the percent of bone surfaces that opposed active hematopoietic marrow, with Grade I at 0%, through Grade VI at 81% to 100%. Differences between iliac and tibial grafts were evaluated with Fisher's Exact Test. Iliac crest and tibial bone grafts both showed trabecular fragments with abundant osteocytes. All iliac grafts contained active hematopoietic marrow. In contrast, the medullary space of tibial grafts contained fat and little hematopoietic marrow. Nine iliac grafts were graded V or VI; whereas the ten tibial bone grafts were all graded I or II (p = 0.0001). The difference in the numbers of samples in each group ranked as grade VI was also statistically significant (p = 0.005). Iliac bone grafts contained active hematopoietic marrow, whereas quiescent medullary fat predominated in tibial grafts. These findings raise questions about the cellular contributions of different sources of bone graft to bone healing.

Whole-body MR imaging, bone diffusion imaging: how and why?

Whole-body MRI (W-B MRI) and diffusion-weighted imaging (DWI) are two novel techniques that greatly facilitate the evaluation of many disorders of childhood. In the musculoskeletal system, these techniques primarily aid in the evaluation of the marrow, although there is increasing interest in the study of soft-tissue abnormalities with W-B MRI and of cartilage with DWI.The normal pattern of marrow transformation affects both modalities throughout childhood. Haematopoietic marrow has a much higher signal intensity than fatty marrow on W-B MRI short tau inversion recovery (STIR) images (Darge et al. Eur J Radiol 68:289-298, 2008). Diffusion is greater in haematopoietic marrow than in fatty marrow and decreases in the skeleton with age (Jaramillo et al. Pediatr Radiol 34:S48, 2004). It is important therefore to remember that the entire skeleton is haematopoietic at birth and that there is a process of marrow transformation to fatty marrow. Marrow conversion proceeds from the fingers to the shoulders and from the toes to the hips. Within each bone, fatty marrow transformation begins in the epiphyses, and within the shaft of the long bones fatty marrow transformation begins at the diaphysis and proceeds towards the metaphyses.

Induction of bone formation by transforming growth factor‐β2 in the non‐human primate Papio ursinus and its modulation by skeletal muscle responding stem cells

Four adult non-human primates Papio ursinus were used to study induction of bone formation by recombinant human transforming growth factor-beta(2) (hTGF-beta(2)) together with muscle-derived stem cells. The hTGF-beta(2) was implanted in rectus abdominis muscles and in calvarial defects with and without addition of morcellized fragments of striated muscle, harvested from the rectus abdominis or temporalis muscles. Expression of osteogenic markers including osteogenic protein-1, bone morphogenetic protein-3 and type IV collagen mRNAs from generated specimens was examined by Northern blot analysis. Heterotopic intramuscular implantation of 5 and 25 microg hTGF-beta(2) combined with 100 mg of insoluble collagenous bone matrix yielded large corticalized mineralized ossicles by day 30 with remodelling and induction of haematopoietic marrow by day 90. Addition of morcellized rectus abdominis muscle to calvarial implants enhanced induction of bone formation significantly by day 90. In Papio ursinus, in marked contrast to rodents and lagomorphs, hTGF-beta(2) induced large corticalized and vascularized ossicles by day 30 after implantation into the rectus abdominis muscle. This striated muscle contains responding stem cells that enhance the bone induction cascade of hTGF-beta(2). Induction of bone formation by hTGF-beta(2) in the non-human primate Papio ursinus may occur as a result of expression of bone morphogenetic proteins on heterotopic implantation of hTGF-beta(2); the bone induction cascade initiated by mammalian TGF-beta proteins in Papio ursinus needs to be re-evaluated for novel molecular therapeutics for induction of bone formation in clinical contexts.

Chondrogenic pre-induction of human mesenchymal stem cells on β-TCP: Enhanced bone quality by endochondral heterotopic bone formation

New techniques to heal bone defects include the combination of bone substitute materials with mesenchymal stem cells (MSC). To find solutions not hampered by low material resorbability or high donor variability of human MSC, the potency of such composites is usually evaluated by heterotopic bone formation assays in immunocompromised animals. The aim of this study was to investigate whether resorbable phase-pure β-tricalcium-phosphate (β-TCP) could support heterotopic bone formation by MSC comparable to partially resorbable hydroxyapatite/tricalcium-phosphate (HA/TCP). Furthermore, in light of disappointing results with osteogenic in vitro priming of MSC, we tested whether chondrogenic pre-induction of constructs may allow for enhanced bone formation by triggering the endochondral pathway. β-TCP granules of three different sizes and HA/TCP were seeded with MSC and transplanted subcutaneously into immunocompromised mice either immediately or after a chondrogenic pre-induction for 6weeks. After 8weeks, explants were analysed by histology. β-TCP seeded with unprimed MSC revealed intramembranous bone formation without haematopoietic marrow with 3.8-fold more bone formed with granules smaller than 0.7mm than with 0.7–1.4mm particles (p⩽0.018). Chondrogenic pre-induction of β-TCP/MSC composites resulted in collagen type II and proteoglycan-rich cartilage-like tissue which, after transplantation, underwent endochondral ossification, yielding ectopic bone produced by human cells while haematopoietic marrow was derived from the mouse. Transdifferentiation of MSC-derived chondrocytes to osteoblasts or direct osteogenesis of cartilage-resident MSC is postulated to explain the human origin of new bone. In conclusion, β-TCP was significantly more osteo-permissive (p=0.004) than HA/TCP for human MSC, and chondrogenic priming of β-TCP/MSC represented a superior approach capable of supporting full bone formation, including marrow organization.

Dynamic Contrast Enhanced Magnetic Resonance Imaging of Diffuse Spinal Bone Marrow Infiltration in Patients with Hematological Malignancies

ObjectiveTo investigate the significance of the dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) parameters of diffuse spinal bone marrow infiltration in patients with hematological malignancies.Materials and MethodsDynamic gadolinium-enhanced MR imaging of the lumbar spine was performed in 26 patients with histologically proven diffuse bone marrow infiltration, including multiple myeloma (n = 6), acute lymphoblastic leukemia (n = 6), acute myeloid leukemia (n = 5), chronic myeloid leukemia (n = 7), and non-Hodgkin lymphoma (n = 2). Twenty subjects whose spinal MRI was normal, made up the control group. Peak enhancement percentage (Emax), enhancement slope (ES), and time to peak (TTP) were determined from a time-intensity curve (TIC) of lumbar vertebral bone marrow. A comparison between baseline and follow-up MR images and its histological correlation were evaluated in 10 patients. The infiltration grade of hematopoietic marrow with plasma cells was evaluated by a histological assessment of bone marrow.ResultsDifferences in Emax, ES, and TTP values between the control group and the patients with diffuse bone marrow infiltration were significant (t = -11.51, -9.81 and 3.91, respectively, p < 0.01). Emax, ES, and TTP values were significantly different between bone marrow infiltration groups Grade 1 and Grade 2 (Z = -2.72, -2.24 and -2.89 respectively, p < 0.05). Emax, ES and TTP values were not significantly different between bone marrow infiltration groups Grade 2 and Grade 3 (Z = -1.57, -1.82 and -1.58 respectively, p > 0.05). A positive correlation was found between Emax, ES values and the histological grade of bone marrow infiltration (r = 0.86 and 0.84 respectively, p < 0.01). A negative correlation was found between the TTP values and bone marrow infiltration histological grade (r = -0.54, p < 0.01). A decrease in the Emax and ES values was observed with increased TTP values after treatment in all of the 10 patients who responded to treatment (t = -7.92, -4.55, and 5.12, respectively, p < 0.01).ConclusionDCE-MRI of spine can be a useful tool in detecting diffuse marrow infiltration of hematological malignancies, while its parameters including Emax, ES, and TTP can reflect the malignancies' histological grade.

FDG Avid Patchy Bone Marrow Misinterpreted As Melanoma Metastases to Bone in a Case of Aplastic Anemia

A 65-year-old woman with resected melanoma underwent F-18 Fluorodeoxyglucose (FDG) PET/CT for the evaluation of a metastatic lesion. Focal increased accumulations were seen in the marrow space of several bones, and interpreted as metastases. MRI demonstrated localized abnormal signal in the corresponding sites. Since she had also suffered from aplastic anemia, additional In-111 chloride bone marrow scintigraphy was performed, and it revealed that the sites with increased accumulation of FDG were consistent with the patchy hematopoietic marrow. FDG PET/CT in a patient with aplastic anemia showing foci of increased bone accumulation occasionally indicates patchy functional hematopoiesis.

Non-p53 Dependent, Leukemia Initiating-Cell Selective, Therapy.

Abstract 2077Poster Board II-54Conventional drug therapy for AML is limited by toxic effects on normal hematopoietic stem cells (nHSC), and dependence on p53/apoptosis pathways that are impaired in malignancy. In hematopoiesis, key transcription factors (TF) determine cell-fate. Here, a difference in nHSC versus leukemia initiating-cell (LIC) TF expression is used to overcome the above limitations.The DNA methylating enzyme DNA methyl-transferase 1 (DNMT1) is also a component of multi-protein histone methyl-transferase complexes. Accordingly, shRNA mediated depletion of DNMT1 in hematopoietic cells hypomethylated DNA and decreased global H3K27 and H3K9 trimethylation (histone marks associated with transcription repression) by >70%. These epigenetic modifications were reproduced using a clinically relevant method: the cytosine analogue decitabine, added to normal human CD34+ hematopoietic precursor cells at 0.2–0.5uM 2–3X/week, depleted DNMT1, H3K27 and H3K9 trimethylation by >70% and significantly hypomethylated DNA (Illumina CpG Microarray). These decitabine levels did not cause measurable DNA damage (H2AX phosphorylation and Fast Micromethod) or apoptosis (Annexin staining and caspase 3 activity). Therefore, at low levels, decitabine can produce broad chromatin changes that increase TF access to target genes, without causing measurable DNA damage or apoptosis.The gene-expression/cell-fate consequences of opening chromatin with decitabine likely depend on the pre-existing TF expression pattern. HOXB4 (stem cell TF), CEBPa (lineage-specifying TF), and CEBPe (late differentiation TF) levels were measured by RQ-PCR in CD34+ cells from AML (n=3) versus normal bone marrow (n=3). AML CD34+ expressed >50-fold higher CEBPa, but HOXB4 and CEBPe levels comparable to normal CD34+ cells, a pattern confirmed in microarray gene expression analysis (CD34+ and myeloblasts, AML n=321, normal n=51 (GEO)). Repression of late differentiation TF likely involves chromatin-modification, regardless of underlying cause. Therefore, depleting DNMT1 to open chromatin in AML cells expressing high lineage-specifying TF could resume differentiation and terminate AML self-renewal, while nHSC, with high stem cell TF and little lineage-specifying TF, should continue to self-renew. nHSC and human MLL-AF9 AML cells were treated identically with decitabine for 7 days, then 300,000 each viable MLL-AF9 and nHSC were combined and transplanted into NSG mice (n = 8). Mice that received PBS treated cells died by week 5 (>90% human myeloblasts in bone marrow). Mice that received decitabine treated cells remained healthy until sacrifice for analysis at week 12 (log-rank p = 0.02, no detectable leukemia, >80% normal human hematopoietic cell marrow engraftment). Direct treatment of mice with established MLL-AF9 leukemia with very low dose decitabine 1mg/m2 3X/week extended survival by >20% (log-rank p = 0.04).Decitabine 0.5uM 2X/week induced morphologic differentiation, but not early apoptosis, in primary patient samples (n=15) and leukemia cell-lines (n=4). Cell-cycle exit by differentiation versus apoptosis may utilize different cyclin dependent kinase inhibitors (CDKN). The THP1 AML cell line contains a homozygous frame-shift mutation in TP53 (p.R174fs*3) and no detectable p53 RNA/protein. THP1 cells were treated with equimolar Ara-C or decitabine. Ara-C weakly upregulated CDKN1A (p21) but not CDKN2B (p15), and produced a transient decrease in cell-counts (D3-5) with recovery and growth similar to control by D7. Decitabine strongly upregulated p15, weakly upregulated p21, and produced gradual but complete and durable abrogation of cell growth by D7.A 66y patient with transfusion dependent RCMD with 5q-, 15q- and severe comorbidities was treated with metronomic (instead of cycled) very low dose SQ decitabine (0.2mg/kg [7.5mg/m2] 2X/week) to avoid cytotoxicity and sustain differentiation modification. Platelets increased by week 4, hematologic remission occurred by week 8 and cytogenetic remission by week 14 (without significant side-effects).Rationalizing dose and schedule of decitabine exploits a difference in nHSC and LIC TF expression to selectively terminate LIC self-renewal by a non-p53 dependent differentiation pathway. This approach, distinct from conventional apoptosis-based therapy, could have a very favorable safety profile, with efficacy in MDS/AML with complex cytogenetic abnormalities. Disclosures:Off Label Use: Decitabine, to treat myelodysplastic syndrome using a novel dose and schedule. Advani:Cephalon: Research Funding. Saunthararajah:HemaQuest: Consultancy.

Characterization of the Human Hematopoietic Niche During Ontogeny.

Abstract 3626Poster Board III-562Studies thus far have shown that there are at least two anatomically and physiologically distinct hematopoietic niches within the bone marrow (BM); the osteoblastic and the vascular. The former is thought to provide the appropriate support for quiescent hematopoietic stem cells (HSC), while the latter allows/induces expansion of the HSC pool. However, it is not entirely known which subpopulations of HSC interact with each niche, and what physiological role each niche type plays in the regulation of stem cell hierarchy and function. We hypothesized that the developmental process whereby the fetal marrow acquires the ability to support hematopoiesis can be used as a model for understanding both the interactions that occur between HSC and the cells comprising the marrow niches, and the role of these niche elements in the initiation/maintenance of hematopoiesis. To this end, human fetal bones between 10- 22 weeks of gestation (gw) were analyzed by flow cytometry and confocal microscopy to identify and characterize stromal/vascular/osteoblastic and hematopoietic elements. Since the development of the vascular bed has been reported to occur in humans between 9-10.5gw, we started the analysis of the human fetal bones at this time point (n=3). CD44 was the only marker widely expressed at this time point, suggesting that these bone rudiments were in an earlier (cartilaginous) stage of development. At 12gw (n=5), 47±4% of the cells isolated from the long bones were CD34+, but less than 1% of these cells were positive for CD45. Further characterization of the CD45−CD34+ population showed that 74±5.4% of these cells were CD106+; 65±7.2% were CD102+; 10±0.5% were CD31+; and 6.7±2.1 % KDR+. These data suggest that prior to the onset of hematopoiesis, a significant percentage of the cells in the BM are part of the vascular niche, and have an endothelial cell phenotype. Furthermore, the existence of a population of cells with a CD34+CD31+KDR+CD45− phenotype may indicate that, during this stage of development, a hemangioblast-like cell exists in the emergent BM. In addition, the existence of a large mesenchymal/stromal cell population was also found, with 21±2.9% of the cells expressing Stro-1+. CD44 was still widely expressed (55±7.8%) at this time point. Of particular note is the discovery of a cell population in which CD44+ cells co-expressed CD34 in the absence of CD45 (8±1.8%), and another in which CD34+ cells were co-expressing Stro-1 (9±1 %), suggesting the possibility that a common ancestor may exist for these cells. At 16gw (n=6) the overall percentage of CD34+ cells decreased to 15±3.1%, and the majority (72±7%) of the cells harvested from the long bones expressed CD45, showing the change to a predominantly hematopoietic marrow. Furthermore, 12±4.2% of the cells had a CD45+CD34+ phenotype consistent with that of HSC. At this time point, only 2.38±1% of the cells were CD34+CD106+; 3.5±1.5% were CD34+CD102+; and 1.34±0.8% were CD34+CD31+. In addition, the CD44+CD45- population decreased to only 20±1.2%. At 22gw (n=6), a similar flow cytometric profile was found except that the CD45+CD34+ population had decreased to 6±1.3%. Since phenotypic analysis showed a decrease in the percentage of BM stromal cell precursors from 12 to 22gw, we performed CFU-F assays and found that the frequency of CFU-F in 12gw BM was 0.34-0.45%, the highest found at any time point. At 16gw, the frequency of CFU-F was 0.23-0.25%, and at 22gw, it had decreased to 0.1-0.16%. Nevertheless, even this low level at 22gw was still higher than that of adult BM (0.001-0.02%). Thus, the results from CFU-F assays correlate well with the flow cytometry/confocal microscopy data showing that there is a decrease in mesenchymal/stromal cell precursor frequency/potential as the BM niches mature. To investigate the ability of the stromal layers of different gestational ages to interact with/retain HSC, adhesion assays (n=3) were performed with cord blood (CB) and adult BM-derived CD34+ cells. These studies showed that CB CD34+ cells adhered at similar levels to all of the fetal stromal layers, while adult BM-derived CD34+ cells only adhered efficiently to the 22gw stromal layers. Further studies addressing the cellular interactions that occur within each of the niches at the phenotypic, functional, and transcriptional levels are currently underway. Disclosures:No relevant conflicts of interest to declare.

Age dependent T2 changes of bone marrow in pediatric wrist MRI

Hyperintensity of the bone marrow on fluid-sensitive sequences can be seen on magnetic resonance imaging (MRI) during childhood, even in the absence of bone pathology. They can be related to hematopoietic marrow, normal and abnormal bone remodeling. We sought to investigate whether hyper intensity of the bone marrow on MRI of the wrist is age-dependent and to evaluate if this signal follows a consistent age-related pattern. Thirty-one wrist 1.5 T MR images of children (7-18 years) without suspected bone pathology were evaluated for foci of hyperintense bone marrow seen on fluid-sensitive coronal sequences using a scale of 1-3. Correlation of frequency, location and intensity of these foci with age was obtained. Results were analyzed for distribution in single bones and in the following regions: distal forearm, first/second carpal rows, and metacarpal bases. A total of 448 bones were evaluated. Eighty-eight out of 448 (21 out of 31 wrists) showed hyperintense bone marrow seen on fluid-sensitive sequences. The distribution was: radius in 19, ulna in 19, first metacarpal base in 11, scaphoid in 9, lunate in 6, pisiform in 6, and fifth metacarpal base in 1. The involvement of the first and second carpal rows and the metacarpal bases was almost similar (13, 12, and 12 respectively). In the distal forearm, the intensity was similar to or higher than that in the wrist (2.2 vs. 2.0). Frequency decreased with age (100% at 7-9 and 25% at 16-18 years). Foci of hyperintense bone marrow seen on fluid-sensitive sequences can be seen on MRI of the wrist during childhood even without apparent symptoms. It shows a consistent pattern with maturation: frequency and intensity decrease and there is distal-to-proximal resolution. This may be a normal finding that may represent normal bone remodeling or decreasing hematopoietic marrow and should not be confused with pathological bone marrow edema.

Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment

Osteoblasts and endothelium constitute functional niches that support hematopoietic stem cells (HSC) in mammalian bone marrow (BM) 1,2,3 . Adult BM also contains adipocytes, whose numbers correlate inversely with the hematopoietic activity of the marrow. Fatty infiltration of hematopoietic red marrow follows irradiation or chemotherapy and is a diagnostic feature in biopsies from patients with marrow aplasia 4. To explore whether adipocytes influence hematopoiesis or simply fill marrow space, we compared the hematopoietic activity of distinct regions of the mouse skeleton that differ in adiposity. By flow cytometry, colony forming activity, and competitive repopulation assay, HSCs and short-term progenitors are reduced in frequency in the adipocyte-rich vertebrae of the mouse tail relative to the adipocyte-free vertebrae of the thorax. In lipoatrophic A-ZIP/F1 “fatless” mice, which are genetically incapable of forming adipocytes8, and in mice treated with the PPARγ inhibitor Bisphenol-A-DiGlycidyl-Ether (BADGE), which inhibits adipogenesis9, post-irradiation marrow engraftment is accelerated relative to wild type or untreated mice. These data implicate adipocytes as predominantly negative regulators of the bone marrow microenvironment, and suggest that antagonizingmarrow adipogenesis may enhance hematopoietic recovery in clinical bone marrow transplantation.