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The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets ([Psmc1.sup.fl/fl] Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic by tamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets ([Psmc1.sup.fl/fl] PdgfCre-ER mice). These results indicate that proteasome function is critical for thrombopoiesis, and suggest inhibition of RhoA signaling as a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients., Introduction Thrombocytopenia (low platelet count) is observed in numerous diseases and can be life threatening due to bleeding complications. Bortezomib, a reversible chemotherapeutic inhibitor used to treat patients with relapsed [...]

Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the [...]

Myeloid differentiation factor 88 (MyD88), an adaptor critical for innate immune function, plays a role in neutrophil recruitment and myocardial injury after transient ischemia. However, how MyD88 signaling modulates neutrophil function and myocardial injury remains unclear. In an in vivo model of neutrophil migration and a chimeric model of MyD88 deletion, we demonstrated that Gr-1-positive ([Gr-1.sup.+]) neutrophil migration was significantly decreased by 68% in MyD88-deficient ([Myd88.sup.-/-]) mice and by 33% in knockout[right arrow]wild-type (KO [right arrow] WT; donor [right arrow] recipient) chimeric mice, which lacked MyD88 in bone marrow cells but maintained normal MyD88 expression in the heart. This marked attenuation in neutrophil migration was associated with decreased peritoneal neutrophil CXCR2 expression and lower peritoneal KC, a neutrophil chemoattractant, in [MyD88.sup.-/-] mice. Moreover, in vitro, KC induces significantly more downregulation of CXCR2 expression in [MyD88.sup.-/-] than WT neutrophils. In an in vivo model of myocardial ischemia-reperfusion (I/R) injury, KO [right arrow] WT chimeric mice had significantly smaller infarct sizes compared with the WT [right arrow] WT mice. While there was a marked increase in proinflammatory cytokine/chemokine expression in the myocardium following I/R, there was no significant difference between WT [right arrow] WT and KO[right arrow]WT mice. In contrast, [Gr-1.sup.+] neutrophil recruitment in the myocardium was markedly attenuated in [MyD88.sup.-/-] mice. Deletion of Toll-intefleukin-1 receptor (TIR)-domain-containing adaptor protein-inducing interferon-[beta]-mediated transcription factor (Trif), another innate immune adaptor, had no effect on the KC-mediated CXCR2 downregulation or on myocardial neutrophil recruitment after I/R. Taken together, these findings suggest that MyD88 signaling is essential for maintaining neutrophil migratory function and chemokine receptor expression. MyD88 signaling in bone marrow-derived circulating cells may play a specific and critical role in the development of myocardial I/R-induced injury. ischemia; reperfusion; MyD88; Toll-like receptor; inflammation doi: 10.1152/ajpcell.00155.2010.

5q- syndrome is a subtype of myelodysplastic syndrome characterized by severe anemia and variable neutropenia but normal or high platelet counts with dysplastic megakaryocytes. We examined expression of microRNAs (miRNAs) encoded on chromosome 5q as a possible cause of haploinsufficiency. We show that deletion of chromosome 5q correlates with loss of two miRNAs that are abundant in hematopoietic stem/progenitor cells (HSPCs), miR-145 and miR-146a, and we identify Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) and tumor necrosis factor receptor-associated factor-6 (TRAF6) as respective targets of these miRNAs. TIRAP is known to lie upstream of TRAF6 in innate immune signaling. Knockdown of miR-145 and miR-146a together or enforced expression of TRAF6 in mouse HSPCs resulted in thrombocytosis, mild neutropenia and megakaryocytic dysplasia. A subset of mice transplanted with TRAF6-expressing marrow progressed either to marrow failure or acute myeloid leukemia. Thus, inappropriate activation of innate immune signals in HSPCs phenocopies several clinical features of 5q- syndrome., Myelodysplastic syndrome (MDS), one of the most common hematopoietic malignancies, arises in primitive [CD34.sup.+] hematopoietic stem/ progenitor cells (1-4). MDS is characterized by ineffective hematopoiesis and dysplasia in one or [...]

It has been suggested in anesthetized animals that the occurrence of sequences of consecutive beats characterized by systolic arterial pressure (SAP) and RR or pulse interval (PI) changing in the opposite direction ([SAP.sup.+]/[RR.sup.-] and [SAP.sup.-]/[RR.sup.+], nonbaroreflex sequences) might represent the expression of neural cardiovascular regulatory mechanisms operating with feedforward characteristics. The aim of the present study was to study nonbaroreflex sequences in a more physiological experimental model, i.e., in conscious freely moving rats. We studied conscious rats before and after 1) complete autonomic blockade (n = 12), 2) sympathetic blockade (n = 10), 3) [alpha] (n = 7)- and [beta] (n = 8)-adrenergic blockade, and 4) parasympathetic blockade (n = 10). Nonbaroreflex sequences were defined as three or more beats in which SAP and PI of the following beat changed in the opposite direction. Complete autonomic blockade reduced the number of nonbaroreflex sequences (95.6 [+ or -] 9.0 vs. 45.2 [+ or -] 4.1, P < 0.001), as did sympathetic blockade (80.9 [+ or -] 12.6 vs. 30.9 [+ or -] 6.1, P < 0.001). The selective [alpha]-receptor blockade did not induce significant changes (80.9 [+ or -] 12.5 in baseline vs. 79.0 [+ or -] 14.7 after prazosin), whereas [beta]-receptor blockade significantly reduced nonbaroreflex sequence occurrence (80.9 [+ or -] 12.5 in baseline vs. 48.9 [+ or -] 15.3 after propranolol). Parasympathetic blockade produced a significant increase of nonbaroreflex sequences (95.1 [+ or -] 6.9 vs. 136.0 [+ or -] 12.4, P < 0.01). These results demonstrate the physiological role of the nonbaroreflex sequences as an expression of a feedforward type of short-term cardiovascular regulation able to interact dynamically with the feedback mechanisms of baroreflex origin in the neural control of the sinus node. feedforward mechanisms; negative feedback mechanisms; baroreceptots; nervous system autonomic; nervous system sympathetic

Phosphatidylinositol-4,5-bisphosphate ([PIP.sub.2]) is an abundant phospholipid that contributes to second messenger formation and has also been shown to contribute to the regulation of cytoskeletal dynamics in all eukaryotic cells. Although the [alpha], [beta], and [gamma] isoforms of phosphatidylinositol-4-phosphate-5-kinase I (PIP5KI) all synthesize PIP2, mammalian cells usually contain more than one PIP5KI isoform. This raises the question of whether different isoforms of PIP5KI fulfill different functions. Given the speculated role of [PIP.sub.2] in platelet and megakaryocyte actin dynamics, we analyzed murine megakaryocytes lacking individual PIP5KI isoforms. PIP5KI[gamma]-/- megakaryocytes exhibited plasma membrane blebbing accompanied by a decreased association of the membrane with the cytoskeleton. This membrane defect was rescued by adding back wild-type PIP5KI[gamma], but not by adding a catalytically inactive mutant or a splice variant lacking the talin-binding motif. Notably, both PIP5KI[beta]- and PIP5KI[gamma]-/- cells had impaired [PIP.sub.2] synthesis. However, PIP5KI[beta]-null cells lacked the membrane-cytoskeleton defect. Furthermore, overexpressing PIP5KI[beta] in PIP5KI[gamma]-/- cells failed to revert this defect. Megakaryocytes lacking the PIP5KI[gamma]-binding partner, talin1, mimicked the membrane-cytoskeleton defect phenotype seen in PIP5KI[gamma]-/- cells. These findings demonstrate a unique role for PIP5KI[gamma] in the anchoring of the cell membrane to the cytoskeleton in megakaryocytes, probably through a pathway involving talin. These observations further demonstrate that individual PIP5KI isoforms fulfill distinct functions within cells., Introduction In contrast to the head group of other phospholipids, the head group of phosphatidylinositol can be phosphorylated reversibly at the (3), (4), or (5) position to generate a family [...]

We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes [...]

We recently showed that a rare cell from murine bone marrow, which we termed multipotent adult progenitor cells (MAPCs), can be expanded for >120 population doublings. Mouse (m)MAPCs differentiate into mesenchymal lineage cells as well as endothelium and endoderm, and, when injected in the blastocyst, mMAPCs contribute to most if not all somatic cell lineages including the different cell types of the brain. Our results, reported herein, demonstrate that mMAPCs can also be induced to differentiate into cells having anatomical and electrophysiological characteristics similar to those of midbrain neurons. Differentiation to a neuronal phenotype was achieved by coculturing mMAPCs with astrocytes, suggesting that neuronal differentiation may require astrocyte-derived factors similar to what is required for the differentiation of embryonic stem cells and neural stem cells to neurons. Differentiation of mMAPCs to neuron-like cells follows similar developmental steps as described for embryonic stem cells and neural stem cells. MAPCs therefore may constitute a source of cells for treatment of central nervous system disorders.

We have tested the ability of bone marrow (BM) cells (BMCs) to form hepatocytes in liver injury models. We used three models: (i) carbon tetrachloride (C[Cl.sub.4]) treatment, (ii) albumin-urokinase transgenic mouse [TgN(Alb1Plau)], and (iii) hepatitis B transgenic mouse [TgN(Alb1HBV)]. As a nonselective liver injury model, irradiated C57BL/6 (B6) mice were transplanted with BMCs from GFP transgenic mouse [TgN(ActbEGFP)] or [beta]-galactosidase transgenic mouse [TgN(MtnLacZ)] followed by the administration of C[Cl.sub.4]. Irradiated TgN(Alb1HBV) and TgN(AlblPlau) were also transplanted with BMCs from TgN(ActbEGFP) or TgN(MtnLacZ). Approximately 1.5 x [10.sup.6] hepatocytes per liver were analyzed for GFP-positive cells, and the whole livers were inspected for [beta]-galactosidase expression. No GFP-positive hepatocytes and no gross blue staining of the livers with 5-bromo-4-chloro-3-indolyl [beta]-D-galactoside in any of the 18 recipient mice analyzed were detected. The livers from female animals with gender-mismatched BM transplantation were also tested with Y chromosome fluorescent in situ hybridization analysis to detect donor-derived cells. A total of five isolated hepatocytes were positive for Y chromosome in 4.1 x [10.sup.5] hepatocytes analyzed, Our results demonstrate that there is little or no contribution of BMCs to the replacement of injured livers in these models. We conclude that BM-derived cells cannot generally lead to a cure of liver damage.

We show here that cells within human adult bone marrow can contribute to cells in the adult human brain. Cerebellar tissues from female patients with hematologic malignancies, who had received chemotherapy, radiation, and a bone marrow transplant, were analyzed. Brain samples were obtained at autopsy from female patients who received male (sex-mismatched) or female (sex-matched, control) bone marrow transplants. Cerebella were evaluated in 10-[micro]m-thick, formaldehyde-fixed, paraffin-embedded sections that encompassed up to [approximately equal to] 50% of a human Purkinje nucleus. A total of 5,860 Purkinje cells from sex-mismatched females and 3,202 Purkinje cells from sex-matched females were screened for Y chromosomes by epifluorescence. Confocal laser scanning microscopy allowed definitive identification of the sex chromosomes within the morphologically distinct Purkinje cells. In the brains of females who received male bone marrow, four Purkinje neurons were found that contained an X and a Y chromosome and two other Purkinje neurons contained more than a diploid number of sex chromosomes. No Y chromosomes were detected in the brains of sex-matched controls. The total frequency of male bone marrow contribution to female Purkinje cells approximated 0.1%. This study demonstrates that although during human development Purkinje neurons are no longer generated after birth, cells within the bone marrow can contribute to these CNS neurons even in adulthood. The underlying mechanism may be caused either by generation de novo of Purkinje neurons from bone marrow-derived cells or by fusion of marrow-derived cells with existing recipient Purkinje neurons. stem cell | plasticity | cell fusion | cell fate change

Caspase-directed apoptosis usually fragments cells, releasing nonfunctional, prothrombogenic, membrane-bound apoptotic bodies marked for rapid engulfment by macrophages. Blood platelets are functional anucleate cells generated by specialized fragmentation of their progenitors, megakaryocytes (MKs), but committed to a constitutive caspase-independent death. Constitutive formation of the proplatelet-bearing MK was recently reported to be caspase-dependent, apparently involving mitochondrial release of cytochrome c, a known pro-apoptogenic factor. We extend those studies and report that activation of caspases in MKs, either constitutively or after Fas ligation, yields platelets that are functionally responsive and evade immediate phagocytic clearance, and retain mitochondrial transmembrane potential until constitutive platelet death ensues. Furthermore, the exclusion from the platelet progeny of caspase-9 present in the progenitor accounts for failure of mitochondrial release of cytochrome c to activate caspase-3 during platelet death. Thus, progenitor cell death by apoptosis can result in birth of multiple functional anucleate daughter cells.

Although it has been shown that unfractionated bone marrow, hematopoietic stem cells, common myeloid progenitors, and bipotent megakaryocyte/erythrocyte progenitors can give rise to megakaryocyte colonies in culture, monopotent megakaryocyte-committed progenitors (MKP) have never been prospectively isolated from the bone marrow of adult mice. Here, we use a monoclonal antibody to the megakaryocyte-associated surface protein, CD9, to purify MKPs from the c-[kit.sup.+][Sca-1.sup.-]IL7R[[alpha].sup.-][Thy1.1.sup.-][Lin.sup.-] fraction of adult C57BL/Ka-Thy1.1 bone marrow. The [CD9.sup.+] fraction contained a subset of [CD41.sup.+]Fc[gamma][R.sup.lo][CD34.sup.+][CD38.sup.+] cells that represent [approximately equal to] -0.01% of the total nucleated bone marrow cells. They give rise mainly to colony-forming unit-megakaryocytes and occasionally burst-forming unit-megakaryocytes, with a plating efficiency >60% at the single-cell level. In vivo, MKPs do not have spleen colony-forming activity nor do they contribute to long-term multilineage hematopoiesis; they give rise only to platelets for [approximately equal to] 3 weeks. Common myeloid progenitors and megakaryocyte/erythrocyte progenitors can differentiate into MKPs after 72 h in stromal cultures, indicating that MKPs are downstream of these two progenitors. These isolatable MKPs will be very useful for further studies of megakaryopoiesis as well as the elucidation of their gene expression patterns.

The angiogenic effect induced by autologous bone marrow cell implantation (BMCI) was examined in the ischemic hindlimbs of diabetic and nondiabetic rats. Diabetes mellitus was induced by the systemic administration of streptozotocin. We investigated the production of angiogenic factors and endothelial differentiation from bone marrow cells and the native recovery of blood flow in the ischemic hindlimbs. To observe the angiogenic effect induced by BMCI treatment, 6 x [10.sup.7] bone marrow cells were injected intramuscularly at six points into the ischemic limbs, and regional perfusion recovery was evaluated with colored microspheres 2 wk later. No difference was found between diabetic and nondiabetic rats in the release of angiogenic factors or endothelial differentiation from bone marrow cells in vitro. The levels of nitric oxide in plasma were significantly lower, and native perfusion recovery in the ischemic hindlimbs was significantly slower in the diabetic rats than in the nondiabetic rats. However, although perfusion recovery was achieved in the ischemic hindlimbs, there was no significant increase in systemic VEGF after BMCI treatment in either the diabetic or nondiabetic rats. Therefore, therapeutic angiogenesis induced by BMCI could be a safe and effective treatment for ischemic limb disease in diabetic patients. diabetes mellitus; angiogenic factor; endothelial differentiation; colored microsphere

Fibrinogen binding to integrin [alpha]llb[beta]3 mediates platelet aggregation and requires agonist-induced 'inside-out' signals that increase ([alpha]IIb[beta]3 affinity. Agonist regulation of alpha]IIb[beta]3 also takes place in megakaryocytes, the bone marrow cells from which platelets are derived. To facilitate mechanistic studies of inside-out signaling, we describe here the generation of megakaryocytes in quantity from murine embryonic stem (ES) cells. Coculture of ES cells for 8-12 days with OP9 stromal cells in the presence of thrombopoietin, IL-6, and IL-11 resulted in the development of large, polyploid megakaryocytes that produced proplatelets. These cells expressed [alpha]IIb[beta]3 and platelet glycoprotein Ib[alpha] but were devoid of hematopoietic stem cell, erythrocyte, and leukocyte markers. Mature megakaryocytes, but not megakaryocyte progenitors, specifically bound fibrinogen by way of ([alpha]llb[beta]3 in response to platelet agonists. Retrovirus-mediated expression of the reporter gene, green fluorescent protein, in ES cell-derived megakaryocytes did not affect viability or [alpha]IIb[beta]3 function. On the other hand, retroviral expression of CalDAG-GEFI, a Rap1 exchange factor identified by megakaryocyte gene profiling as a candidate integrin regulator, enhanced agonist-induced activation of Rap1b and fibrinogen binding to [alpha]IIb[beta]3 (P < 0.01). These results establish that ES cells are a ready source of mature megakaryocytes for integrin studies and other biological applications, and they implicate CalDAG-GEFI in inside-out signaling to [alpha]IIb[beta]3.

Human CD34 (hCD34) positive cells are used currently as a source for hematopoietic transplantation in humans. However, in steady-state murine hematopoiesis, hematopoietic stem cells (HSCs) with long-term reconstitution activity are found almost exclusively in the murine CD34 (mCD34)-negative to low fraction. To evaluate the possible differences in hCD34 and mCD34 gene expression in hematopoiesis, we made transgenic mouse trains with human genomic P1 artificial chromosome clones spanning the entire hCD34 genomic locus. In all transgenic mouse strains, a vast majority of phenotypic and functional HSC populations including mCD[34.sup.-/Io] express the hCD34 transgene. These data strongly support the notion that hCD[34.sup.+] human bone marrow cells contain long-term HSCs that can maintain hematopoiesis throughout life.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is critical for DNA repair via the nonhomologous end joining pathway. Previously, it was reported that bone marrow cells and spontaneously transformed fibroblasts from SCID (severe combined immunodeficiency) mice have defects in telomere maintenance. The genetically defective SCID mouse arose spontaneously from its parental strain CB17. One known genomic alteration in SCID mice is a truncation of the extreme carboxyl terminus of DNA-PKcs, but other as yet unidentified alterations may also exist. We have used a defined system, the DNA-PKcs knockout mouse, to investigate specifically the role DNA-PKcs specifically plays in telomere maintenance. We report that primary mouse embryonic fibroblasts (MEFs) and primary cultured kidney cells from 6-8 month-old DNA-PKcs-deficient mice accumulate a large number of telomere fusions, yet still retain wild-type telomere length. Thus, the phenotype of this defect separates the two-telomere related phenotypes, capping, and length maintenance. DNA-PKcs-deficient MEFs also exhibit elevated levels of chromosome fragments and breaks, which correlate with increased telomere fusions. Based on the high levels of telomere fusions observed in DNA-PKcs deficient cells, we conclude that DNA-PKcs plays an important capping role at the mammalian telomere.

Research shows that during bone marrow homing, the rare hematopoietic stem cells show an increase in the expression of CD34 and SCA-1. Data indicate that such adult bone marrow cells can differentiate into epithelial cells of various organs and tissues.

Thrombopoietin (TPO) acts as a lineage-specific late-acting factor to stimulate megakaryocyte and platelet formation. However, analysis of mice lacking either the cytokine or its receptor, c-Mpl, also revealed deficiencies in progenitor cells of multiple hematopoietic lineages, suggesting that TPO signaling may play an important role in the regulation of the hematopoietic stem cell compartment. To investigate this hypothesis, we determined preprogenitor and colony forming unit-spleen (CFU-S) numbers and analyzed the long-term hematopoietic repopulating capacity of bone marrow cells from [mpl.sup.-/-] mice. [mpl.sup.-/-] mice had 4- to 12-fold fewer preprogenitor cells than wild-type mice. In irradiated normal recipients, [mpl.sup.-/-] bone marrow generated 8- to 10-fold fewer spleen colonies than wild-type marrow at both 8 and 12 days after transplantation. This defect was intrinsic to the transplanted hematopoietic cells, as the microenvironment of [mpl.sup.-/-] recipients supported similar CFU-S growth to that observed in wild-type recipients. In definitive assays of stem cell function, bone marrow cells from [mpl.sup.-/-] mice failed to compete effectively with normal cells for long-term reconstitution of the hematopoietic organs of irradiated recipients, even when transplanted in 10-fold excess. Serial transplantation studies further suggested that stem cell self-renewal also may be compromised in [mpl.sup.-/-] mice. These data imply that TPO, signaling through c-Mpl, plays a vital physiological role in the regulation of hematopoietic stem cell production and function.

Marrow stromal cells from wild-type mice were infused into transgenic mice that had a phenotype of fragile bones resembling osteogenesis imperfecta because they expressed a human minigene for type I collagen. In mice that were irradiated with potentially lethal levels (700 cGy) or sublethal levels (350 cGy), DNA from the donor marrow stromal cells was detected consistently in marrow, bone, cartilage, and lung either 1 or 2.5 mo after the infusions. The DNA also was detected but less frequently in the spleen, brain, and skin. There was a small but statistically significant increase in both collagen content and mineral content of bone 1 mo after the infusion. Similar results were obtained with infusion of relatively large amounts of wild-type whole marrow cells into the transgenic mice. In experiments in which male marrow stromal cells were infused into a female osteogenesis imperfecta-transgenic mouse, fluorescense in situ hybridization assays for the Y chromosome indicated that, after 2.5 mo, donor male cells accounted for 4-19% of the fibroblasts or fibroblast-like cells obtained in primary cultures of the lung, calvaria, cartilage, long bone, tail, and skin. In a parallel experiment in which whole marrow cells from a male mouse were infused into a female immunodeficient rag-2 mouse, donor male cells accounted for 4-6% of the fibroblasts or fibroblast-like cells in primary cultures. The results support previous suggestions that marrow stromal cells or related cells in marrow serve as a source for continual renewal of cells in a number of nonhematopoietic tissues.

Marrow stromal cells can be isolated from other cells in marrow by their tendency to adhere to tissue culture plastic. The cells have many of the characteristics of stem cells for tissues that can roughly be defined as mesenchymal, because they can be differentiated in culture into osteoblasts, chondrocytes, adipocytes, and even myoblasts. Therefore, marrow stromal cells present an intriguing model for examining the differentiation of stem cells. Also, they have several characteristics that make them potentially useful for cell and gene therapy., Because circulating blood cells survive for only a few lays or months, hematopoietic stem cells (HSCs) in bone marrow must provide a continuous source of progenitors for red cells, platelets, [...]

The effects of NO on bone resorption and osteoclast formation were analyzed in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-stimulated mouse bone marrow cultures. Osteoclast formation of multinucleated cells was attenuated by the administration of sodium nitroprusside (SNS) which increased NO release. Furthermore, the inhibitory effects of NO on 1,25-(OH)2D3-stimulated bone marrow cells during the bone remodelling cycle involved the stimulation of soluble guanylate cyclase.

The granulocyte colony-stimulating factor (G-CSF) contributes to the maturation and proliferation of precursor cells to functional granulocytes. To understand this mechanism, kinetic analyses of receptor-mediated endocytosis (RME) of a G-CSF derivative, nartograstim, in rat bone marrow cells were performed. Similar rate constants were obtained for the RME processes. However, the association constant for initial binding was fivefold greater than steady-state results. The difference was due to the effect of ligand binding on the process of internalization.

Murine bone marrow cells were placed in co-culture with ST-2 stromal cells to examine how macrophage colony-stimulating factor (CSF-1) promotes the growth and differentiation of osteoclasts from precursor cells derived from bone marrow. The results show that CSF-1 was most effective in promoting differentiation into osteoclasts up to day six of co-culture, and its effect was significantly diminished beyond day nine of co-culture. These results indicate that CSF-1 promotes differentiation into osteoclasts before precursor cells are committed to any specific differentiation path.

Analysis of cytotoxic lymphocytes in adult bone marrow and newborn cord blood reveals that the BY55 monoclonal antibody (BY55 mAb) differentiates between the cytotoxic lymphocytes of different cell subsets in the cord blood and bone narrow, that are sources of hematological stem cells for allogenic transplantation. Cytotoxic T lymphocyte activity is absent in cord blood but the CD3- BY55+ cell subset exhibits the entire natural killer activity.

The long-term disposition of circulating neutrophils and the site of disappearance from circulation remain unclear. We investigated neutrophil localization in mice using [sup.111]In-labeled murine peripheral blood neutrophils, mature bone marrow neutrophils, and peritoneal exudate neutrophils to track in vivo localization of these different cell populations. Infused peripheral neutrophils were found to localize equally between liver and marrow sites by 4 h (31.2 [+ or -] 1.9 vs. 31.9 [+ or -] 1.8%), whereas exudate neutrophils predominantly localized to liver (42.0 [+ or -] 1.1%) and marrow-derived neutrophils to the marrow (65.9 [+ or -] 6.6%) where they were found to localize predominantly in the hematopoietic cords. Stimulation of marrow neutrophils before infusion caused a shift in localization from marrow to liver, and subsequent induction of an inflammatory site after infusion and marrow sequestration led to remobilization of infused marrow neutrophils but not of peripheral neutrophils. These results indicate that the marrow participates in removing neutrophils from circulation, with evidence supporting both storage and perhaps disposal functions. Furthermore, models for circulating neutrophil homeostasis should consider that the site of retention is governed by the maturation and activation states of the cell. bone marrow; liver; lipopolysaccharide; inflammation

Attempts to repair myocardial infarcts by transplanting cardiomyocytes or skeletal myoblasts have failed to reconstitute healthy myocardium and coronary vessels integrated structurally and functionally with the remaining viable portion of the ventricular wall. The recently discovered growth and transdifferentiation potential of primitive bone marrow cells (BMC) prompted us, in an earlier study, to inject in the border zone of acute infarcts [Lin.sup.-] c-[kit.sup.POS] BMC from syngeneic animals. These BMC differentiated into myocytes and vascular structures, ameliorating the function of the infarcted heart. Two critical determinants seem to be required for the transdifferentiation of primitive BMC: tissue damage and a high level of pluripotent cells. On this basis, we hypothesized here that BMC, mobilized by stem cell factor and granulocyte-colony stimulating factor, would home to the infarcted region, replicate, differentiate, and ultimately promote myocardial repair. We report that, in the presence of an acute myocardial infarct, cytokine-mediated translocation of BMC resulted in a significant degree of tissue regeneration 27 days later. Cytokine-induced cardiac repair decreased mortality by 68%, infarct size by 40%, cavitary dilation by 26%, and diastolic stress by 70%. Ejection fraction progressively increased and hemodynamics significantly improved as a consequence of the formation of 15 x [10.sup.6] new myocytes with arterioles and capillaries connected with the circulation of the unaffected ventricle. In conclusion, mobilization of primitive BMC by cytokines might offer a noninvasive therapeutic strategy for the regeneration of the myocardium lost as a result of ischemic heart disease and, perhaps, other forms of cardiac pathology.

Increasing membrane-bound MCSF does not enhance OPGL-driven osteoclastogenesis from marrow cells. Am J Physiol Endocrinol Metab 280: E103-E111, 2001.--Macrophage colony-stimulating factor (MCSF) and osteoprotegerin ligand (OPGL), both produced by osteoblasts/ stromal cells, are essential factors for osteoclastogenesis. Whether local MCSF levels regulate the amount of osteoclast formation is unclear. Two culture systems, ST-2 and Chinese hamster ovary-membrane-bound MCSF (CHO-mMCSF)-Tet-OFF cells, were used to study the role of mMCSF in osteoclast formation. Cells from bone marrow (BMM) or spleen were cultured with soluble OPGL on glutaraldehyde-fixed cell layers; osteoclasts formed after 7 days. Osteoclast number was proportional to the amount of soluble OPGL added. In contrast, varying mMCSF levels in the ST-2 or CHO-mMCSF-Tet-OFF cell layers, respectively by variable plating or by addition of doxycycline, did not affect BMM osteoclastogenesis: 20-450 U of mMCSF per well generated similar osteoclast numbers. In contrast, spleen cells were resistant to mMCSF: osteoclastogenesis required [is greater than or equal to] 250 U per well and further increased as mMCSF rose higher. Our results demonstrate that osteoclast formation in the local bone environment is dominated by OPGL. Increasing mMCSF above basal levels does not further enhance osteoclast formation from BMMs, indicating that mMCSF does not play a dominant regulatory role in the bone marrow. tetracycline regulation; osteoclast differentiation factor; TRANCE; colony stimulating factor-1; bone marrow

A variable skeleto-hematopoietic phenotype was observed in collagen X null mice which mirrored the defects in transgenic (Tg) mice with dominant interference collagen X mutations (Jacenko, O., P. LuValle, and B.R. Olsen. 1993. Nature. 365:56-61). Specifically, perinatal lethality was seen in ~10.8% of null mutants at week three after birth, and in another subset by 12 wk. In perinatal lethal mutants, growth plates were compressed, trabecular bone reduced, and hematopoietic aplasia and erythrocyte-filled vascular sinusoids were apparent in marrows. Lymphatic organs, reduced to ~80% that of controls, displayed altered architecture and lymphocyte content. In thymuses, a paucity of cortical [CD3.sup.+]/[CD4.sup.+]/[CD8.sup.+] lymphocytes was consistent with the marrow's inability to replenish maturing T cells. In spleens, an unaltered T cell distribution was coupled with diffuse staining for Ig[D.sup.+]/[B220.sup.+] B cells, whose reduction was prominent in poorly organized lymphatic nodules. Disorderly arrays of splenic macrophages surrounding periarteriolar lymphatic sheaths and a red pulp depletion further complemented the Tg perinatal lethal phenotype. Moreover, subtle growth plate compressions and hematopoietic changes were seen in all null mice. Data from Tg and null mice implicate the disruption of collagen X function in the observed skeleto-hematopoietic defects, and suggest that hypertrophic cartilage and endochondral skeletogenesis may contribute to the marrow microenvironment prerequisite for blood cell differentiation. Key words: endochondral ossification * hypertrophic cartilage * skeletogenesis * marrow * lymphopenia

Hematopoiesis is the development of erythrocytes and leukocytes within bone marrow compartments. There is a dense array of nerve fibers innervating the bone marrow that supply these compartments with neuropeptides. [...]