Isolated Bone Marrow Research Articles

Effects of estrogen on osteoprogenitor cells and cytokines/bone-regulatory factors in postmenopausal women

Decreases in estrogen levels contribute not only to early postmenopausal bone loss but also to bone loss with aging. While estrogen is critical for the maintenance of bone formation, the mechanism(s) of this effect remain unclear. Thus, we assessed the effects of 4months of transdermal estradiol treatment (0.05mg/day) of postmenopausal women as compared to no treatment (n=16 per group) on the expression of genes in pre-specified pathways in freshly isolated bone marrow osteoprogenitor cells (hematopoietic lineage [lin]-/Stro1+). We also evaluated whether estrogen treatment modulated peripheral blood or bone marrow plasma levels of the Wnt antagonists, sclerostin and DKK1, as well as serotonin, OPG, RANKL, adiponectin, oxytocin, and inflammatory cytokines (TNFα, IL-1β, and IL-6), as each of these molecules have recently been shown to play an important role in regulating osteoblast function and/or being responsive to estrogen. We observed a significant decrease in the expression of several proliferation markers (cyclin B1, cyclin E1, E2F1) and increase in adhesion molecules (N-cadherin) in bone marrow lin-/Stro1+ cells from estrogen-treated compared to control women. None of the peripheral blood or bone marrow plasma marker levels differed between the two groups, with the exception of sclerostin levels, which were significantly lower in the estrogen-treated as compared to the control women in peripheral serum (by 32%, P=0.009) and in bone marrow plasma (by 34%, P=0.017). There were significant differences in bone marrow versus peripheral plasma levels of several factors: sclerostin and OPG levels were higher in bone marrow as compared to peripheral plasma, whereas serotonin and adiponectin levels were higher in peripheral as compared to bone marrow plasma. In summary, our data directly assessing possible regulation by estrogen of osteoprogenitor cells in humans indicate that, consistent with previous studies in mice, estrogen suppresses the proliferation of human bone marrow lin-/Stro1+ cells, which likely represent early osteoprogenitor cells. Further animal and human studies are needed to define the role of the changes we observed in mRNAs for adhesion molecules in these cells and in local sclerostin production in bone in mediating the effects of estrogen on bone metabolism in humans.

Use of In Vivo Gene Expression of Isolated Bone Marrow Mesenchymal Stromal Cells to Study the Pathophysiology of Osteoporosis in Patients With Severe Thalassemia

We compared osteoblast differentiation gene expressions in the isolated CD105 mesenchymal stromal cells from bone marrow in 10 patients with severe thalassemia and 12 normal donor controls. The fold expressions of osteoblast differentiation genes of CD105 cells from patients with thalassemia were lower than those of normal donors but increased after being cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Moreover, the fold expressions of these genes of CD105 cells from normal donors when cultured with 10% pooled serum of patients with thalassemia were lower than when cultured with 10% pooled serum of normal donors. We have also presented the evidence of reversible suppressed expression of these genes in CD105cells from patients with thalassemia when cultured in pooled serum of normal donors. Moreover, healthy donor CD105 cells exhibited lower expression of these genes when cultured in pooled serum of patients with thalassemia compared with pooled serum of normal donors indicating the existence of circulating factors in thalassemic serum impairing the differentiation of mesenchymal stromal cells to osteoblasts.

SerpinB1 functions in generating neutrophil extracellular traps (111.11)

Abstract SerpinB1 is a potent inhibitor of neutrophil serine proteases (NSPs), and mice deficient for serpinB1 showed reduced capacity to clear Pseudomonas aeruginosa lung infection, increase of proinflammatory cytokines and neutrophil death compared with wild type (WT) mice. Here we questioned whether this deficient anti-microbial protection might be caused by altered production of neutrophil extracellular traps (NETs). NETs, extruded chromatin associated with granule proteins including NSPs, capture and kill microbes, but excess NETs can cause tissue injury, e.g., impairment of lung function in patients with cystic fibrosis. On stimulation with phorbol myristate actetate (PMA) or platelet-activating factor (PAF), isolated bone marrow neutrophils of serpinB1-/- mice produced increased NETs compared with WT. Isolated lung neutrophils of LPS-treated serpinB1-/- mice also produced increased NETs compared with WT in response to PMA, and increased numbers of neutrophils with expanded nuclei. NET production was NADPH oxidase-dependent, and NADPH oxidase activity did not differ between the genotypes. Fluorescent microscopy showed that SerpinB1 translocated in PMA treated human neutrophils from the cytoplasm to the nucleus prior to nuclear membrane disintegration. SerpinB1 was also found as an extracellular component of NETs. These findings indicate that SerpinB1 contributes to regulation of NET formation and, by its presence in NETs, may protect against protease-induced tissue injury.

Indolent systemic mastocytosis without skin involvement vs. isolated bone marrow mastocytosis

We have read with great interest the paper by Zanotti et al. [1][1] about the description of “Isolated bone marrow mastocytosis” (BMM) as an underestimated subvariant of indolent systemic mastocytosis (ISM). This is a very interesting paper in which an in depth study was performed in a total of

Skeletal implications of isolated bone marrow mastocytosis (reply): Indolent systemic mastocytosis without skin involvement vs. isolated bone marrow mastocytosis (reply)

We are very grateful to Dr. Makras and Dr. Luis Escribano et al. for their comments about our letter Isolated bone marrow mastocytosis: an underestimated subvariant of indolent systemic mastocytosis”.[1][1] The request of Dr. Makras for more information on skeletal involvement, which was not

Skeletal implications of isolated bone marrow mastocytosis

In the article on the isolated bone marrow mastocytosis (IBMM) as an underestimated subvariant of indolent systemic mastocytosis (ISM), Zanotti et al.1 reported that 54.7% of patients with unexplained/recurrent anaphylaxis or severe allergic reactions were found to be suffering from IBMM. They conclude that the incidence of IBMM can be frequently underestimated, especially in the face of a lack of collaboration among medical specialities, while early recognition can potentially reduce life-threatening events and/or severe skeletal complications. Although this is an extremely interesting finding depicting the problem of unrecognized IBMM, the Authors do not provide an acceptable level of information regarding the skeletal problems of the patients. Since bone densitometry (BMD) was performed in all patients and X-rays in selected cases, it would be helpful for the reader to know the skeletal implications of these subjects. Osteoporosis or osteopenia is by far the most frequently observed pathological skeletal sign in systemic mastocytosis reported in more than one-third of all patients, while osteolysis and osteosclerosis can occur, even simultaneously, in the same patient. In addition, osteoporosis can be the only presentation in IBMM; specifically among men IBMM can be found in up to 9% of patients with idiopathic osteoporosis.2 Furthermore, fractures, mostly vertebral, have been reported in approximately 15% of all patients with systemic mastocytosis.3 Serum tryptase is currently considered the test of choice in the initial assessment of suspected systemic mastocytosis cases.4 However, its cost-effectiveness in the evaluation of secondary osteoporosis is yet to be defined due to the lack of data regarding the real incidence of the disease among osteoporotic patients. It would be very important to know whether the Authors can confirm or not a low BMD and/or pathological fractures in patients with IBMM, especially among young patients. In the former case, the importance of considering the diagnosis of IBMM in subjects with a low BMD and/or fractures and a history of recurrent allergic reactions would be highlighted.

Regenerative medicine for the treatment of musculoskeletal overuse injuries in competition horses.

Tissue repair in musculoskeletal injuries is often a slow and sometimes incomplete process. Regenerative medicine based on the use of growth factors (GFs) and cell therapy is aimed at improving the quality and speed of tendon and ligament healing. The aim of this study was to evaluate the potential for the administration of a combination of autologous platelet-rich plasma (PRP) and freshly isolated bone marrow mononucleated cells (BMMNCs) in 13 competition horses affected by overuse musculoskeletal injuries (suspensory ligament desmopathy and superficial flexor tendinopathy) and refractory to other therapies. After ultrasonographic localisation of the lesion, the autologous BMMNC suspension and PRP were injected directly into the core lesion. BMMNC and platelet count as well as growth factors in PRP were measured to determine factors influencing the clinical outcome. Horses showed a marked improvement in their degree of lameness and 84.6% were able to return to competition. Among all the factors studied, the platelet concentration predicted the healing time: significantly faster recovery (p = 0.049) was observed in cases of PRP with more than 750 × 10(3)/μl platelets. Competition horses are involved in highly demanding activities, thus being a similar model for the high mechanical overload typical of human athletes. The promising results obtained suggest that this combined biological approach may be useful even for the treatment of recalcitrant overuse musculoskeletal injuries in highly demanding patients if the appropriate dose of cells and GFs is applied.

Improvement of Cardiac Function by Intracoronary Freshly Isolated Bone Marrow Cells Transplantation in Patients With Acute Myocardial Infarction

We analyzed in the present study the influence of intracoronary autologous freshly isolated bone marrow cells transplantation (BMCs-Tx) on cardiac function in patients with acute myocardial infarction (AMI). The 32 patients with AMI were enrolled in this prospective nonrandomized study to either freshly isolated BMC-Tx or to a control group without cell therapy. Global left ventricular ejection fraction (LVEF) and the size of infarct area were determined by left ventriculography. We observed in patients with autologous freshly isolated BMCs-Tx at 6 months follow up a significant reduction of infarct size as compared to control group. Moreover, we found a significant increase of LVEF as well as infarct wall movement velocity at 6 months follow up in cell therapy group as compared to control group. In the control group there was no significant difference of LVEF, infarct size and infarct wall movement velocity between baseline and 6 months after AMI. These results demonstrate for the first time that intracoronary transplantation of autologous freshly isolated BMCs by use of a point of care system is safe, and may lead to improvement of cardiac function in patients with AMI.

Biologic characteristics of bone marrow–derived mesenchymal stem cells from a patient with thalassemia syndrome

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiating morphologically and functionally into several mesenchymal tissues. There have been contrasting data on whether MSCs are altered in various hematologic disorders. We isolated bone marrow (BM)-derived MSCs from a patient with thalassemia syndrome to compare phenotypic and functional characteristics to those from normal healthy donor. No differences were observed between MSCs from thalassemia syndrome (T-MSCs) and those from normal healthy donor in terms of morphology, phenotype, karyotype, multidifferentiation capacity. In mixed lymphocyte reaction, T-MSCs strongly inhibited the proliferation of allogeneic T cells in association with reduced proportion of CD3(+), CD4(+), and CD8(+) cells. Furthermore, the fraction of Treg cells was increased under the culture with T-MSCs, suggesting that T-MSCs exert normal immunomodulatory function. In addition, T-MSCs expressed hematopoietic cytokines and supported hematopoiesis, which was comparable to those from normal BM-derived MSCs. T-MSCs exhibited normal phenotype, karyotype as well as normal immunomodulatory function, and autologous MSCs from patients with thalassemia syndrome may be an attractive source of stem cell in terms of hematopoietic support as well as immunomodulatory activity.

Improved Mobilization of the CD34+and CD133+Bone Marrow-Derived Circulating Progenitor Cells by Freshly Isolated Intracoronary Bone Marrow Cell Transplantation in Patients with Ischemic Heart Disease

Cell therapy is a promising novel option for treatment of cardiovascular disease. Because the role of bone marrow-derived circulating progenitor cells (BM-CPCs) after cell therapy is less clear, we analyzed in this randomized, controlled study the influence of intracoronary autologous freshly isolated bone marrow cell transplantation (BMC-Tx) by using a point-of-care system on cardiac function and on the mobilization of BM-CPCs in patients with ischemic heart disease (IHD). Fifty-six patients with IHD were randomized to either receive freshly isolated BMC-Tx or a control group that did not receive cell therapy. Peripheral blood concentrations of CD34/45(+) and CD133/45(+) CPCs were measured by flow cytometry pre-, immediately post-, and at 3, 6, and 12 months postprocedure in both groups. Global ejection fraction and the size of infarct area were determined by left ventriculography. We observed in patients with IHD after intracoronary transplantation of autologous freshly isolated BMCs-Tx at 3 and 12 months follow-up a significant reduction of the size of infarct area and increase of global ejection fraction as well as infarct wall movement velocity. The mobilization of CD34/45(+) and CD133/45(+) BM-CPCs significantly increased at 3, 6, and 12 months after cell therapy when compared with baseline in patients with IHD, although no significant changes were observed between pre- and immediately postintracoronary cell therapy administration. In the control group without cell therapy, there was no significant difference of CD34/45(+) and CD133/45(+) BM-CPCs mobilization between pre- and at 3, 6, and 12 months postcoronary angiography. Intracoronary transplantation of autologous freshly isolated BMCs by using a point-of-care system in patients with IHD may enhance and prolong the mobilization of CD34/45(+) and CD133/45(+) BM-CPCs in peripheral blood and this might increase the regenerative potency in IHD.

Isolated bone marrow mastocytosis: an underestimated subvariant of indolent systemic mastocytosis

Systemic mastocytosis (SM) is a heterogeneous disorder characterized by the proliferation and accumulation of atypical mast cells (MC) in tissues, principally in the bone marrow (BM) and skin. The diagnosis of systemic mastocytosis requires the presence of multifocal dense mast cell infiltrates in

Improved Functional Activity of Bone Marrow Derived Circulating Progenitor Cells After Intra Coronary Freshly Isolated Bone Marrow Cells Transplantation in Patients with Ischemic Heart Disease

ObjectivesThere is growing evidence that intracoronary autologous bone marrow cells transplantation (BMCs-Tx) in patients with chronic myocardial infarction beneficially affects postinfarction remodelling. In this randomized controlled study we analyzed the influence of intracoronary autologous freshly isolated bone marrow cells transplantation by use of point of care system on cardiac function and on the functional activity of bone marrow derived circulating progenitor cells (BM-CPCs) in patients with ischemic heart disease (IHD).Methods56 patients with IHD were randomized to either received freshly isolated BMC-Tx or a control group that did not receive cell therapy. The functional activity of BM-CPCs in peripheral blood (PB) was measured by migration assay and colony forming unit assay pre- and 3, 6 as well as 12 months after procedure. Global ejection fraction (EF) and infarct size area were determined by left ventriculography.ResultsIntracoronary transplantation of autologous freshly isolated BMCs led to a significant reduction of infarct size and an increase of global EF as well as infarct wall movement velocity after 3 and 12 months follow-up compared to control group. The colony-forming capacity of BM-CPCs significantly increased 3, 6 and 12 months after cell therapy compared to pre BMCs-Tx and control group (CFU-E: p < 0.001, CFU-GM: p < 0.001). Likewise, we found significant increase of migratory response to stromal cell-derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) after cell therapy compared to pre BMCs-Tx (SDF-1: p < 0.001, VEGF: p < 0.001) and to control (SDF-1: p < 0.001, VEGF: p < 0.001). There was no significant difference of migratory- and colony forming capacity between pre- and 3, 6, 12 months after coronary angiography in control group without cell therapy.ConclusionsIntracoronary transplantation of autologous freshly isolated BMCs by use of point of care system may lead to improvement of BM-CPCs functional activity in peripheral blood, which might increase the regenerative potency in patients with IHD.

Ultraviolet Irradiation of Mice Reduces the Competency of Bone Marrow-Derived CD11c+ Cells via an Indomethacin-Inhibitable Pathway

Direct UV irradiation of dendritic cells and Langerhans cells reduces their Ag presenting ability. However, the effects of UV on CD11c(+) cells located distally to the point of irradiation are poorly understood. Three days after UV irradiation (8 kJ/m(2)) of BALB/c mice, bone marrow cells were isolated and cultured for 7 d with IL-4 and GM-CSF for the propagation of CD11c(+) cells. Bone marrow-derived CD11c(+) cells from UV-irradiated or nonirradiated mice were loaded with dinitrobenzene sulfonic acid and injected into the ear pinnas of naive BALB/c mice. After 7 d, the ears were painted with 2,4-dinitro-1-fluorobenzene and the ear swelling determined 24 h later. A reduced contact hypersensitivity response was found in mice injected with CD11c(+) cells from the UV-irradiated animals compared with those injected with cells from the nonirradiated animals. Further, a long-lasting suppression of the memory response to 2,4-dinitro-1-fluorobenzene was created. This suppressed response corresponded to increased IL-10 and PGE(2) secretion by freshly isolated bone marrow cells from UV-irradiated mice, and to increased myelopoiesis. The reduction in competence of bone marrow-derived CD11c(+) cells from UV-irradiated mice was not due to delayed maturation, as it was maintained upon LPS exposure prior to CD11c(+) cell purification. The UV-induced effect was reversed by the administration of indomethacin to mice prior to UV irradiation and could be reproduced by s.c. PGE(2). These results show that UV irradiation of mice can affect the function of bone marrow-derived CD11c(+) cells via a mechanism inhibitable by indomethacin; this pathway is likely to contribute to systemic UV-induced immunosuppression.

Differential Regulation of Expression of the Anti-Leukemogenic Orphan Nuclear Receptor Nur77 (nr4a1) In HSCs, CLPs, and CMPs

AbstractAbstract 4782The molecular basis for the balance between self-renewal and differentiation that regulates hematopoietic stem cell (HSC) development is poorly understood. Recent studies suggest that levels of expression of the orphan nuclear hormone receptor Nur77 (nr4a1), previously implicated in thymic selection, may play a pivotal role in HSC fate decisions. Previously published quantitative PCR data suggest that murine HSCs express relatively high levels of Nur77 mRNA. Genetic deletion of Nur77 and its close family member Nor-1 (nr4a3) in mice resulted in the development of acute myeloid leukemia (Mullican et al. Nat Med, 13:730;2007), consistent with Nur77's role as an anti-leukemogenic transcription factor, but also suggestive of a role in HSC homeostasis. To more closely investigate the role of Nur77 in normal hematopoiesis, we constructed a transgenic reporter mouse in which GFP is driven by the Nur77 promoter (eGFPNur77). Unexpectedly, we found that fewer than 3% of freshly isolated bone marrow HSCs (Lin-c-Kit+Sca-1+, IL-7R-) and common lymphoid progenitors (CLP = Lin-c-Kit+Sca-1+, IL-7R+), and no common myeloid progenitors (CMP) expressed Nur77 in our novel reporter system. Interestingly, culture of Lin- bone marrow cells with serum-containing medium resulted in a marked increase in Nur77 (GFP) expression among the majority of HSC's and CLP's, but not CMP's. Further studies indicate that a heat-insensitive component of serum, also present in plasma, is responsible for this induction. Taken together with published data, our studies suggest that induction and modulation of Nur77 may play a previously unappreciated role in self-renewal and lymphoid versus myeloid lineage commitment decisions of HSCs. We are currently working to understand the developmental difference in HSC/CLP/CMP responsiveness and to identify the serum component(s) responsible for upregulating Nur77. Disclosures:No relevant conflicts of interest to declare.

A Comparative Gene Expression Study of CD3+ T-Cells In Bone Marrow From Patients with Chronic ITP and Healthy Controls by DNA Microarray.

AbstractAbstract 1445 Background:The pathophysiology of ITP (immune thrombocytopenia) is more complex than initially believed. The importance of regulatory T-cells, platelet specific cytotoxic T-cells, suppression of megakaryocyte proliferation and maturation, functional thrombopoietin deficiency and inappropriately low bone marrow production of platelets has been unraveled over the last decade. T-cells are important in all forms of autoimmunity including ITP. However, very little is known about the T-cell activity in organs where platelets and their precursors are targeted by effectors of the immune system. To provide new insights into the pathophysiology of ITP, we performed a DNA microarray analysis on isolated bone marrow derived T-cells from chronic ITP patients and healthy controls. Patients and Method:Bone marrow was collected from 6 chronic ITP-patients (3 males and 3 females, mean age 39.5 years) and 6 healthy controls (3 males and3 females, mean age 33.8 years) through iliac crest bone marrow aspiration. T-cells were isolated by immunomagnetic cell sorting (Miltenyi Biotec, Surrey, UK). RNA was prepared using the Chomczynski method (Chomczynski and Sacchi, 1987), followed by RNeasy minielute clean-up (Qiagen, Hilden, Germany). RNA was amplified from 20 ng of starting total RNA with the Ovation RNA Amplification System V2 (NuGEN Technologies, Inc.), and cDNA was synthesized using the Encore Biotin Module kit (NuGEN Technologies, Inc.). After standard labeling, each sample was hybridized to an Affymetrix U133Plus 2.0 Human Genome array (Santa Clara, CA). Results:We identified 397 differently regulated genes in the bone marrow derived T-cells between ITP patients and controls by the DNA microarray analysis (P-value <0.05 and fold change ± 1.3). Some of the regulated genes have previously been implicated in ITP, such as integrin alpha 4 (ITGA4 or CD49D or VLA-4), Fas ligand (FASLG) and cytotoxic lymphocyte-associated 4 (CTLA-4). Furthermore, several apoptotic genes were lower expressed in ITP patients, such as Fas apoptotic inhibitory molecule 2 (FAIM2), BCL2-like 11 (BCL2L11), BCL2-like 13 (BCL2L13), peptidyl-tRNA hydrolase 2 (PTRH2) and the enzyme inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB).Integrin alpha 6, (ITGA6) and the adhesion molecule endothelial cell adhesion molecule (ESAM) were higher expressed in ITP patients compared to controls and are important for T-cell adhesion and migration or “homing” of lymphocytes to different lymphoid organs and inflammatory sites. Conclusion:We have identified a number of regulated genes in T-cells from bone marrow that differed between patients with ITP and healthy controls. The importance of several of the identified genes has already been implicated in ITP, proving the usefulness of this strategy. The identification of novel regulated genes may provide new insights into the physiology and pathophysiology of ITP and for autoimmunity in general. Disclosures:No relevant conflicts of interest to declare.

Umbilical Cord Blood-Derived Mesenchymal Cell Fate After Mouse Umbilical Cord Blood Transplantation

Umbilical Cord Blood-Derived Mesenchymal Cell Fate After Mouse Umbilical Cord Blood Transplantation

Effect of assemble flavone of Drynariae Rhizome on activity and calcium deposition of bone marrow stem cells of rabbit

To observe the effect of assemble flavone of Drynariae Rhizome on the activity and osteogenic differentiation of bone marrow stem cells of rabbit in vitro. Density gradient centrifugation was used to isolate bone marrow stem cells from SD rats. The MSCs were also undergone osteogenic induction. Cytochemical and immunocytochemical tainting were performed to verify their multipotential. High purity of bone marrow stem cells was successfully obtained. Both ALP activity and mineralization nodes tainting were positive in MSCs after osteogenic differentiation. The MSCs sustained their osteogenic differentiation potential in vitro by assemble flavone of rhizome drynaria.

Modulation of neutrophil apoptosis by murine pulmonary microvascular endothelial cell inducible nitric oxide synthase

Neutrophils contribute significantly to ALI (acute lung injury) through adhesion to pulmonary microvascular endothelial cells (PMEC), trans-PMEC migration and alveolar infiltration. Trans-PMEC migration delays expression of neutrophil apoptosis, which promotes intra-alveolar neutrophil survival and neutrophil mediated ALI. We assessed the role of neutrophil vs PMEC inducible nitric oxide (NO) synthase (iNOS) in modulating neutrophil apoptosis. Apoptosis of wild-type vs iNOS−/− neutrophils was quantified by microscopy and FACS annexin-V binding. In a murine model of ALI, neutrophils isolated by BAL(broncho-alveolar lavage) from iNOS−/− mice had increased expression of apoptosis after 24h culture ex vivo than wild-type neutrophils (15.2±3.3 vs 3.0±0.4%, mean±sd, p<0.01). Apoptosis rates of isolated bone marrow iNOS+/+ vs iNOS−/− neutrophils were similar under basal and LPS/IFN-γ stimulation, and following LPS/IFN-γ-stimulated trans-PMEC migration. Apoptosis of both iNOS+/+ and iNOS−/− neutrophils was inhibited by trans-PMEC migration only across iNOS+/+ PMEC (1.6±0.3 and 1.5±0.3%, respectively; p<0.05 for each vs non-migrated neutrophils) but not across iNOS−/− PMEC (4.3±1 and 3.1±0.6%, respectively). PMEC iNOS-dependent inhibition of neutrophil apoptosis was independent of changes in neutrophil caspase-3 activity. We conclude that PMEC iNOS, but not neutrophil iNOS, has an important inhibitory effect on neutrophil apoptosis during trans-PMEC neutrophil migration, which is independent of caspase-3 activity. Further studies will define the mechanism of PMEC iNOS-dependent inhibition of neutrophil apoptosis and assess the potential relevance of this phenomenon in human neutrophils and ALI.

Co-expression of the collagen receptors leukocyte-associated immunoglobulin-like receptor-1 and glycoprotein VI on a subset of megakaryoblasts

The collagen receptor glycoprotein VI generates activating signals through an immunoreceptor tyrosine-based activating motif on the co-associated Fc receptor gamma chain. Leukocyte-associated immunoglobulin-like receptor-1 also ligates collagen but generates inhibitory signals through immunoreceptor tyrosine-based inhibitory motifs. Thus far, the cellular expression of glycoprotein VI and leukocyte-associated immunoglobulin-like receptor-1 appears mutually exclusive. Using flow cytometry, we studied expression of collagen receptors on differentiating human megakaryocytes. CD34(+) cells were isolated from umbilical cord blood and matured to megakaryocytes in vitro. Freshly isolated bone marrow cells were used to study primary megakaryocytes. Upon cell sorting, cytospins were made to examine cytological characteristics of differentiation. Megakaryocyte maturation is accompanied by up-regulation of glycoprotein VI and down-regulation of leukocyte-associated immunoglobulin-like receptor-1. Interestingly, both in cultures from hematopoietic stem cells and primary cells obtained directly from bone marrow, we identified a subset of morphologically distinct megakaryocytes which co-express glycoprotein VI and leukocyte-associated immunoglobulin-like receptor-1. This is the first report of a primary cell that co-expresses these collagen receptors with opposite signaling properties. Since megakaryocytes mature in the collagen-rich environment of the bone marrow, these findings may point to a role for leukocyte-associated immunoglobulin-like receptor-1 in the control of megakaryocyte maturation/migration.

Compound heterozygosity for two new TERT mutations in a patient with aplastic anemia

Dyskeratosis congenita (DC) is a genetically heterogeneous syndrome characterized by reticular skin pigmentation, nail dystrophy, mucosal leukoplakia, short telomeres, and a predisposition to bone marrow failure and malignancy. Patients carrying mutations in TERT show a wide clinical spectrum of abnormalities, including classical DC, isolated bone marrow failure and lung fibrosis. Here, we report the clinical description and biological analysis of a patient with compound heterozygosity for two new missense mutations in TERT (V96L and V119L). Both mutations segregate with a short telomere phenotype, though only V96L segregates with clinical signs of DC.