Mononuclear Fraction Research Articles

Abstract 5377: Low donor thymic output predicts increased incidence of extensive chronic GVHD and worse overall survival after TCD-matched sibling SCT

Abstract Low CD4+ T cell counts early post-SCT are associated with a greater risk of developing chronic GVHD (cGVHD). However, the factors affecting post graft CD4+ T cell recovery are not known. Furthermore, the CD4 T cell subset correlating best with protection from cGvHD is not defined. We hypothesized that the donor immune repertoire determines CD4+ reconstitution after SCT. We studied 220 donor-recipient pairs undergoing a myeloablative matched-sibling T-cell depleted SCT for a variety of hematological malignancies including AML, ALL, CML, and MDS. Median donor and patient age was 36 years (range, 9 - 70). Median CD34+ cell dose was 5.56 x106/kg (range, 2.3-14.5), with a fixed T cell dose per protocol of 1-5 x104/kg. Ninety-four patients (43%) developed chronic GvHD (32 limited, 64 extensive). Prior to SCT a cryopreserved mononuclear fraction (PBMC) was obtained by leukopheresis on all donors. PBMC from 139 donors were stained with fluorescently conjugated antibodies and acquired on a custom-built LSR Fortessa (BD) flow cytometer. Regulatory T cells (Tregs) and Treg subsets were identified within the CD4+ T cell population using a combination of FOXP3 and Helios. The combination of FOXP3 with Helios has recently been shown to identify natural, thymus-derived Tregs (nTregs), whereas CD4+FOXP3+ T cells lacking Helios represent a pool of induced Tregs (Thornton et al., 2010, J Immunol. 184:3433-3441). Combined expression of CD31 and CD45RA in the CD4+ and nTreg populations was used to identify recent thymic emigrants (RTE). Absolute lymphocyte counts in 220 donors were 2.11/µl (range 0.89 - 4.12). A low absolute lymphocyte count was significantly correlated with a higher cumulative probability for time to incidence of extensive cGVHD (HR 2.38, p = 0.0008). This relationship between lymphocytes and cGVHD was retained in CD4+ T cell subsets: CD3+CD4+ (HR 2.48, p = 0.005); CD3+CD4+CD25+ < 30 cells/μL (HR 2.1, p = 0.02); nTregs < 8 cells/μL (HR 1.97, p = 0.043). RTE of total CD4 did not correlate with RTE nTregs, indicating that nTreg production varies among donors with similar levels of thymic activity. Furthermore recipients of donors with low RTE nTregs relative to overall thymic output had a significantly higher likelihood of developing extensive chronic GvHD (HR 2.78, p=0.0014). In multivariate analysis including patient age, acute GvHD (grade II - IV), graft direction (female donor to male recipient vs. other combinations), disease and status at transplant, and administration of post SCT donor lymphocyte infusion, only thymic output of nTregs (HR = 0.8, p = 0.037) and graft direction (HR = 2.1, p = 0.023) remained significant risk factors for extensive cGvHD. These data show that the thymic output of nTreg varies inherently among donors. Donors with low thymic output of nTreg may recreate a less tolerant immune system in the recipient leading to cGVHD of greater severity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5377. doi:1538-7445.AM2012-5377

Abstract 3116: The Effect of Stroke on Bone Marrow: Studies from a Cell-Therapy Clinical Trial

Objective: The application of autologous bone marrow cells has emerged as an investigational cell-based therapy for ischemic stroke. Previous animal studies have reported that stroke affects leukocytes in the bone marrow. In addition, older age and presence of comorbidities raise concerns about the variability in yield of bone marrow stem cells. These issues may potentially impact autologous applications of bone marrow cell therapies in stroke patients. We examined whether acute ischemic stroke (AIS) affects the bone marrow in patients by assessing various cell subpopulations within the mononuclear fraction of bone marrow harvested from healthy donors and study patients in our clinical trial testing mononuclear cells (MNCs) in patients with AIS. Methods: This study examined the bone marrow composition of 22 consecutive patients with AIS enrolled into our clinical trial testing the safety of autologous bone marrow MNCs administered intravenously within 24 to 72 hours after symptom onset. After bone marrow harvest, MNCs are isolated, separated, and characterized at a GMP facility. The bone marrow from 15 healthy donors was also processed at the same GMP facility. Descriptive analysis comprised calculation of means for absolute cell counts and determination of proportions for subtypes of different cell subpopulations. Samples of healthy bone marrow donors were compared with that of AIS patients. Results: AIS patients had a median age of 61 (IQR 50-73), had CAD (5%), Afib (14%), diabetes (32%), hypertension (50%), hyperlipidemia (32%), or were actively smoking (27%). Onset time to harvest was 48 ±11 hours after stroke onset. Figure 1 shows no significant differences among the proportions of cell populations including lineage negative, CD34+ cells (a marker of hematopoietic stem cells - HSCs). There was no significant difference in the variance of the cell subpopulations between healthy donors and stroke patients, except for NK cells which were significantly higher among the stroke patients (p = 0.0074). Age, history of DM, or the location of the infarct depending on vascular territory did not affect the proportion of the different cell populations. Conclusions: AIS does not cause significant changes in the proportions of different cell types in the MNCs of bone marrow including the HSCs. We found no evidence that autologous MNCs are different, in terms of cell composition, from study patients who are older and have vascular comorbidities compared with healthy donors.

Células mononucleares da medula óssea no tratamento da ruptura total dos tendões flexores e ligamento suspensor do boleto em equino

This paper describes the case of a horse treated in a Teaching Veterinary Hospital. The sutures of the flexor apparatus associated with cell therapy, the stem cell, provided a rapid response of repair compared to other cases treated in routine of the Hospital. The association of conventional treatments with the mononuclear fraction of bone marrow significantly contributed to the reduction of the hyperextension of the metatarsophalangeal joint. Ultrasound images showed rapid fill cell and decrease in inflammation.

MOLECULAR Karyotyping In CLL - COST Neutral Alternative to FISH SCREENING for Detection of Clinically Relevant Genome Imbalances,

Abstract 3561Specific genome aberrations are recognised prognostic factors in CLL. FISH-based genome risk classifications used in clinical decision making for over a decade. However molecular karyotyping is gaining acceptance as an alternative, albeit cost and skill demanding, that not only overcomes the limitations of FISH but provides comprehensive whole genome scanning. Indeed, several recent studies (Rinaldi et al., BJH 2011, Parker et al., Leukemia 2011, Quilette et al, Blood 2011) using different array platforms have revealed novel clinically relevant cryptic genome aberrations. Here we compare FISH and molecular karyotyping data of 40 diagnostic and follow up samples from 22 CLL patients. The whole genome screening (WGS) was performed with 8×60K oligonucleotide arrays (Agilent) using DNA from mononuclear fraction of p.blood/bone marrow samples as well as from fixed chromosome preparations and commercial reference DNA. Following manufacturerÕs protocols and bio-informatics routines (Z score and ADM1/2 algorithms) WGS was done at a cost comparable to the expense of routine FISH screening for 13q14, p53, ATM loss and trisomy 12. Genome array screening identified clonal imbalances recognised by FISH if present above 13%. In addition, WGS provided novel information in 73% of the samples, the results of which are summarised as follows (see figure below): (i) The 13q genome loss found in 10/13 presentation samples was stratified by size and location resulting in a positive assessment for 40% of the cases positive by FISH for del 13q14 as class II/type 2 deletions. These are known to be associated with high risk for disease progression; (ii) A common deleted region of 206.2Kb at @ 50556688–50699677 (hg19) was found to encompass the DLEU2 gene and is seen in 5/13 presentation samples with 13q loss; (iii) In 3 out of 13 cases FISH-positive for 13q loss, disease progression was accompanied by a proximal extension of the deletion to include the RB1 gene region thus acquiring the status of class II/type 2 deletion (high risk) (iv) Genome loss involving IGH sequences at 14q32.32, consistent with B cell type VDJ gene rearrangements was detected in all samples, while deletions within IG kappa and IG lambda were found in 5/25 patients; (v) Cryptic deletion of the ATM gene region was detected in 2 samples undetected by FISH; (vi) Total genome imbalances (TGA) in presentation samples varied between 3 and 17 with higher levels found in samples with p53 loss and class II, type 2 13q loss; (vii) Persistent disease was found to be associated with increase of TGA, some of which appeared to be non-random, such as loss of the whole or part of chromosome 19 and deletions of 4q21 region. [Display omitted] In conclusion, the application of 8×60K genome arrays provides established and growing genome-wide clinically relevant information as a cost neutral assay. A strategy by which array interrogation is carried out at presentation, followed by therapy response monitoring using FISH on a selected marker in CLL is a plausible alternative to routine practice. In cases where FISH has provided evidence for persistent disease, array screening will assess the genome damage, especially if carried on CD19(+)cell isolates, to assist treatment decisions. Disclosures:No relevant conflicts of interest to declare.

Low Thymic Natural Regulatory T Cell Output in the Donor Is Associated with An Increased Incidence of Extensive Chronic GvHD After T Cell Depleted Matched Sibling Stem Cell Transplantation (SCT)

Abstract 328Low CD4+ T cell counts early post-SCT are associated with a greater risk of developing chronic GVHD (cGVHD). However, the factors affecting post graft CD4+ T cell recovery are not known. Furthermore, the CD4 T cell subset correlating best with protection from cGvHD is not defined. We hypothesized that the donor immune repertoire determines CD4+ reconstitution after SCT. We studied 220 donor-recipient pairs undergoing a myeloablative matched-sibling T-cell depleted SCT for a variety of hematological malignancies including AML, ALL, CML, and MDS. Median donor and patient age was 36 years (range, 9 – 70). Median CD34+ cell dose was 5.56 ×106/kg (range, 2.3–14.5), with a fixed T cell dose per protocol of 1–5 ×104/kg. Ninety-four patients (43%) developed chronic GvHD (32 limited, 64 extensive). Prior to SCT a cryopreserved mononuclear fraction (PBMC) was obtained by leukopheresis on all donors. PBMC from 139 donors were stained with fluorescently conjugated antibodies against CD3, CD4, CD8, CD27, CD45RA, CD31, CD25, CD14, CD19, FOXP3, and Helios and acquired on a custom-built LSR Fortessa (BD) flow cytometer. The following T cell subsets were examined: naive, CD27+CD45RA+; central memory, CD27+CD45RA-; effector memory, CD27-CD45RA-; and effector T cells, CD27-CD45RA+. Regulatory T cells (Tregs) and Treg subsets were identified within the CD4+ T cell population using a combination of FOXP3 and Helios The combination of FOXP3 with Helios has recently been shown to identify natural, thymus-derived Tregs (nTregs), whereas CD4+FOXP3+ T cells lacking Helios represent a pool of induced Tregs (Thornton et al., 2010, J Immunol. 184:3433–3441). Combined expression of CD31 and CD45RA in the CD4+ and nTreg populations was used to identify recent thymic emigrants (RTE). Data were analyzed by FlowJo (Treestar, Ashland, OH, version 8.6.6), and both univariate and multivariate analysis analyses using the Cox regression models with right censored time-to-event variables were performed on the readouts (SPSS 15.0, S-Plus 8 and Prism 4 Software). All p-values were computed based on the log-rank test statistics. Absolute lymphocyte counts in 220 donors were 2.11/μl (range 0.89 – 4.12). A low absolute lymphocyte count was significantly correlated with a higher cumulative probability for time to incidence of extensive cGVHD (HR 2.38, p = 0.0008). This relationship between lymphocytes and cGVHD was retained in CD4+ T cell subsets: CD3+CD4+ (HR 2.48, p = 0.005); CD3+CD4+CD25+ < 30 cells/μL (HR 2.1, p = 0.02); nTregs < 8 cells/μL (HR 1.97, p = 0.043). RTE of total CD4 did not correlate with RTE nTregs, indicating that nTreg production varies among donors with similar levels of thymic activity. Furthermore recipients of donors with low RTE nTregs relative to overall thymic output had a significantly higher likelihood of developing extensive chronic GvHD (HR 2.78, p=0.0014). In multivariate analysis including patient age, acute GvHD (grade II – IV), graft direction (female donor to male recipient vs. other combinations), disease and status at transplant, and administration of post SCT donor lymphocyte infusion, only thymic output of nTregs (HR = 0.8, p = 0.037) and graft direction (HR = 2.1, p = 0.023) remained significant risk factors for extensive cGvHD. These data show that the thymic output of nTreg varies inherently among donors. Donors with low thymic output of nTreg may recreate a less tolerant immune system in the recipient leading to cGVHD of greater severity.(ZAM and JJM contributed equally to this work). [Display omitted] Disclosures:No relevant conflicts of interest to declare.

59 Endothelial colony forming cells (ECFC) are senescent and dysfunctional in COPD due to reduced sirtuin-1 levels

IntroductionCardiovascular disease (CVD) is a major cause of death in chronic obstructive pulmonary disease (COPD). Numerous studies describe clinical evidence of endothelial dysfunction in COPD but the molecular pathways which...

Circulating CD133+CD34+ progenitor cells inversely correlate with soluble ICAM-1 in early ischemic stroke patients

Background and PurposeBoth endothelial progenitor cells (EPC) and markers of neuroinflammation are candidate biomarkers for stroke severity and outcome prediction. A relationship between EPC and neuroinflammatory markers in early stroke is not fully elucidated. The objectives were to investigate correlations between EPC and neuroinflammation markers (adhesion molecules ICAM-1, VCAM-1, E-selectin, tumor necrosis factor (TNF)-α, interleukin (IL)-6, endothelin (ET)-1, markers of tissue injury (matrix metalloproteinases (MMP)-9 and tissue inhibitor of matrix metalloproteinases (TIMP)-1) in early stroke patients.MethodsWe prospectively recruited symptomatic patients with ischemic cerebrovascular disease. We assessed stroke severity by using of acute (diffusion-weighted imaging (DWI) and final lesion volumes (fluid attenuated inversion recovery (FLAIR). We measured serum soluble ICAM-1, VCAM-1, E-selectin, MMP-9, TIMP-1 and plasma TNF-α, IL-6, ET-1 by ELISA, and quantified EPC in mononuclear fraction of peripheral blood on days 1 and 3 in 17 patients (mean(SD) age 62(14), with admission National Institutes of Health Stroke Scale (NIHSS) 10(8)) selected from 175 patients with imaging confirmed ischemic stroke. Non-parametric statistics, univariate and multivariate analysis were used.ResultsOnly ICAM-1 inversely correlated with EPC subset CD133+CD34+ on day 1 (Spearman r = -0.6, p < 0.01) and on day 3 (r = -0.967, p < 0.001). This correlation remained significant after adjustment for age and NIHSS (beta -0.992, p < 0.004), for glucose and systolic blood pressure (beta -0.86, p < 0.005), and for white blood cells and hematocrit (beta -1.057, p < 0.0001) on day 3. MMP-9 (r = 0.509, p < 0.04) and MMP-9/TIMP-1 (r = 0.59, p < 0.013) on day 1 correlated with acute lesion volume. Both IL-6 (r = 0.624, p < 0.01) and MMP-9/TIMP-1 (r = 0.56, p < 0.02) correlated with admission NIHSS.ConclusionOur study showed that high ICAM-1 is associated with low CD133+CD34+subset of EPC. Biomarkers of neuroinflammation may predict tissue injury and stroke severity in early ischemia.

Venous vessel pathology — chronic cerebrospinal venous insufficiency in multiple sclerosis

Background: Now it is not known which cell type is best for inducing therapeutic angiogenesis in end-stage critical limb ischemia (CLI). We hypothesized that mononuclear cells mobilized with granulocyte colony stimulating factor (G-CSF) into peripheral blood are as effective as highly selective and purified CD133+ and CD34+ endothelia progenitor cells (EPCs). This would have significant clinical implications with regard to study designs and costs of stem cell treatment regimens. Methods: Patients with critical ischemia of lower extremity donated peripheral blood stem cells after 5 days of stimulation with G-CSF. Five patients in each group were treated with CD 34, CD 133 or mononuclear cells after G-CSF stimulation. We analyzed expression of cells surface markers CD34, VEGFR-2, CD133, CD31 and CD144. Results: There were no differences in pain, ABI, ulcer healing or amputation rate between the three groups. Overall limb salvage rate was 12/15. Only in 4/15 patients, after a mean follow-up of 11 months, did control MRA examinations show stem-cell-induced neoangiogenesis: in two patients after CD34 cell injections, in one case after CD 133 treatment and in one case after treatment with mononuclear cells. In the remaining cases, MRA analysis did not show any traces of neoangiogenesis. Injection of purified CD133+ or CD34+ EPCs did not yield better results than unselected mononuclear cells after G-CSF stimulation. Paracrine factors produced by the mononuclear fraction of mobilized peripheral blood may contribute more to the amelioration of critical limb ischemia than one particular cell type. Conclusion: Stem cell therapy will play an increasingly important role in the near future. Most patients can be treated with unselected cells, which further reduce costs of cell-based therapy.

Streamlining the generation of an osteogenic graft by 3D culture of unprocessed bone marrow on ceramic scaffolds

Mesenchymal stromal cells are present in very low numbers in the bone marrow, necessitating their selective expansion on tissue culture plastic prior to their use in tissue-engineering applications. MSC expansion is laborious, time consuming, unphysiological and not economical, thus calling for automated bioreactor-based strategies. We and others have shown that osteogenic grafts can be cultured in bioreactors by seeding either 2D-expanded cells or by direct seeding of the mononuclear fraction of bone marrow. To further streamline this protocol, we assessed in this study the possibility of seeding the cells onto porous calcium phosphate ceramics directly from unprocessed bone marrow. Using predetermined volumes of bone marrow from multiple human donors with different nucleated cell counts, we were able to grow a confluent cell sheath on the scaffold surface in 3 weeks. Cells of stromal, endothelial and haematopoietic origin were detected, in contrast to grafts grown from 2D expanded cells, where only stromal cells could be seen. Upon implantation in nude mice, similar quantities of bone tissue were generated as compared to that obtained by using the conventional number of culture expanded cells from the same donor. We conclude that human osteogenic grafts can be efficiently prepared by direct seeding of cells from unprocessed bone marrow.

Stimulation of angiogenesis in rat ischemic limbby intramuscular implantation of mononuclear fraction cells from autologous bone marrow

We studied angiogenic effects of two variants of indirect revascularization (transplantation of mononuclear fraction cells from autologous bone marrow alone or in combination with laser tunneling) after modeled ischemia of the lower limbs in rats. Doppler sonography and immunofluorescence studies showed that intramuscular implantation of isolated fraction of autologous bone marrow mononuclear cells in combination with laser tunneling of the muscles is most effective and can be recommended as a method of angiogenesis stimulation in non-reconstructable distal vascular pathology of the extremities.

Protein microarray analysis of aberrant signaling pathways in Acute Myeloid Leukemia to predict the patients responsiveness to PI3K/Akt/mTOR inhibitors

Mapping of deregulated kinases and protein signalling networks within tumors can provide a means to stratify patients with shared biological characteristics to the most optimal treatment, and identify drug targets. In particular, the PI3K/AKT/mTOR signaling pathways are frequently activated in blast cells from patients with acute myelogenous leukemia (AML), a neoplastic disorder characterized by the accumulation of genetically altered myelogenous cells displaying deregulated intracellular signalling pathways and aggressive clinical behavior with poor prognosis. Using Reverse Phase Protein Microarrays (RPMA), we have analyzed the phosphorylated epitopes of signal pathway proteins of 81 peripheral blood and bone marrow specimens with newly diagnosed AML. Patients are diagnosed according to blast content, FAB classification and cytogenetic analysis. Samples are enriched for leukemic cells by performing Ficoll separation to yield a mononuclear fraction with >60% blast cells. The objective of the study was to predict the sensitivity of each patient to PI3K/Akt/mTor inhibitors, to avoid unnecessary and toxic ineffective treatment of non-responsive patients. To this goal, fresh blast cells were grown for 16 h untreated or treated with phase I or phase II mTor or Akt inhibitors either alone or in combination. Remarkably, by unsupervised hierarchical clustering a strong phosphorylation/activity of most of the sampled members of the PI3K/Akt/mTOR pathway was observed in 70% of samples from AML patients. This confirms that this pathway might indeed represent a pharmacological target in many patients. Moreover, treatment with the above inhibitors had no effect on the phosphorylation of other selected targets, demonstrating the specificity of the above results (more than one different inhibitor was used to avoid off-target effects). More importantly, by the use of the above drugs, we have been able to discriminate within the “high pAkt” population a PI3K/Akt/mTOR inhibitor-responsive group of patients and a PI3K/Akt/mTOR inhibitor non-responsive group. In addition, our data indicate that the Akt pathway is hyper-activated in M4, M5 patients, compared to M0, M2 patients, and that a strong activation of most upstream and downstream Akt effectors correlates with an over-expression of the c-kit receptor (CD117). We believe these data are important because they, have the potential to define a profile for the personalized administration of targeted drugs.

Genetically modified human umbilical cord blood cells expressing vascular endothelial growth factor and fibroblast growth factor 2 differentiate into glial cells after transplantation into amyotrophic lateral sclerosis transgenic mice

Current therapy of a number of neuropsychiatric maladies has only symptomatic modality. Effective treatment of these neuro-degenerative diseases, including amyotrophic lateral sclerosis (ALS), may benefit from combined gene/stem-cell approaches. In this report, mononuclear fraction of human umbilical cord blood cells (hUCBCs) were transfected by electroporation with dual plasmid constructs, simultaneously expressing vascular endothelial growth factor 165 (VEGF(165)) and human fibroblast growth factor 2 (FGF(2)) (pBud-VEGF-FGF(2)). These genetically modified hUCBCs were injected retro-orbitally into presymptomatic ALS transgenic animal models ((G)93(A) mice). Lumbar spinal cords of rodents were processed for immunofluoresent staining with antibodies against human nuclear antigen (HNA), oligodendrocyte-specific protein, S100, iba1, neuronal β(3)-tubulin and CD34. Co-localization of HNA and S100 was found in the spinal cord of mice after transplantation of genetically modified hUCBCs over-expressing VEGF-FGF(2). Double staining in control animals treated with unmodified hUCBCs, however, revealed HNA+ cells expressing iba1 and CD34. Neuron-specific β(3)-tubulin or oligodendrocyte-specific protein were not expressed in hUCBCs in either control or experimental mice. These results demonstrate that genetically naïve hUCBCs may differentiate into endothelial (CD34+) and microglial (iba1+) cells; however when over-expressing VEGF-FGF(2), hUCBCs transform into astrocytes (S100+). Autocrine regulation of VEGF and FGF(2) on hUCBCs, signal molecules from dying motor neurons in spinal cord, as well as self-differentiating potential may provide a unique microenvironment for the transformation of hUCBCs into astrocytes that eventually serve as a source of growth factors to enhance the survive potential of surrounding cells in the diseased regions.

Age-dependent changes in osteoclast formation in a new strain of P2X7 receptor knockout mice

Event Abstract Back to Event Age-dependent changes in osteoclast formation in a new strain of P2X7 receptor knockout mice A Agrawal1*, S Syberg2, NR Jørgensen2 and A Gartland1 1 the University of Sheffield, Department of Human Metabolism, United Kingdom 2 University Hospital of Copenhagen Glostrup, Research Center for Ageing and Osteoporosis and Dept of Clinical Chemistry, Denmark The P2X7 receptor (P2X7R) is an ion-gated channel belonging to the family of purinergic receptors that get activated by extracellular nucleotides. The role of the P2X7R in regulating osteoclastogenesis has been described using knockout mice as well as primary human osteoclast cultures. These studies have contradictory findings as blocking the receptor in vitro prevented fusion of human monocytes to form multinucleated osteoclasts, whilst two different models of knockout mice retained the ability to form multinucleated osteoclasts in vivo and in vitro. The mice used in these studies have a natural mutation in the gene coding for P2X7R leading to reduced receptor function. Therefore, we generated mice with no known mutation in the P2X7R to better determine its role in the process of osteoclastogenesis. Spleens from age-matched WT and KO BALB/c mice were removed and the mononuclear fraction isolated. Equal numbers of cells were seeded onto glass coverslips or dentine discs and cultured in the presence of M-CSF and RANKL. Cells on glass coverslips were cultured for 7 days and TRAP stained to assess the number of multinucleated osteoclasts. Dentine discs were stained after 9 days to assess resorption. KO mice had a significantly higher number of multinucleated, TRAP positive cells and increased resorption compared to WT controls at 3 months of age. Mature, 6 month old, KO mice had a higher number of multinucleated TRAP positive cells, however, the resorption was the same in KO and WT cultures. These data suggest that receptor deletion has a role in enhancing osteoclast formation and subsequently function in young and developing mice. In older mice whilst there was an increase in osteoclast formation and activity in both WT and KO mice compared to 3 months of age, the higher number of osteoclasts observed in KO mice did not lead to a significantly higher amount of resorption compared to WT. We therefore propose the P2X7-/- BALB/c mice as a new model for studying the role of P2X7 receptor and that P2X7R in these mice enhances the differentiation and survival of osteoclasts but reduces their resorptive activity with increasing age. Keywords: Bones, Bone Research Conference: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society, Cambridge, United Kingdom, 27 Jun - 29 Jun, 2011. Presentation Type: Poster Topic: Abstracts Citation: Agrawal A, Syberg S, Jørgensen N and Gartland A (2011). Age-dependent changes in osteoclast formation in a new strain of P2X7 receptor knockout mice. Front. Endocrinol. Conference Abstract: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society. doi: 10.3389/conf.fendo.2011.02.00001 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2011; Published Online: 30 Sep 2011. * Correspondence: Prof. A Agrawal, the University of Sheffield, Department of Human Metabolism, Sheffield, S10 2RX, United Kingdom, a.gartland@sheffield.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers A Agrawal S Syberg NR Jørgensen A Gartland Google A Agrawal S Syberg NR Jørgensen A Gartland Google Scholar A Agrawal S Syberg NR Jørgensen A Gartland PubMed A Agrawal S Syberg NR Jørgensen A Gartland Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Effect of Dynorphin A (1-17) on Proliferation and IL-2, IL-4, IFN-γ Production by Peripheral Blood Mononuclears

Dynorphin A (1-17) in concentrations of 10(-8)-10(-9) M inhibits phytohemagglutinin (2.5 μg/ml)-induced proliferative response of mononuclear fraction lymphocytes. In mitogen-stimulated cultures, 10(-8) M dynorphin A (1-17) stimulates the production of IL-4, inhibits the production of IL-2, and does not modify the production of IFN-γ. Nonselective κ-receptor antagonist naloxone and selective antagonist binaltorphimine hydrochloride abolish the inhibitory effects of both dynorphin A concentrations on the lymphocyte proliferative response. On the other hand, evaluation of the effect of κ-receptor blockade on the production of IL-2 and IL-4 showed that this effect depends on peptide concentration and antagonist type. Hence, the results attest to an important role of κ-receptors in modulation of functional activity of immune cells.

LR11 Is a Novel Surface Marker for Normal Leukocytes and Leukemia Cells

AbstractAbstract 4834 Introduction:LR11 (also called SorLA or SORL1) is a type I membrane protein, from which a large extracellular part, sLR11, is released by proteolytic shedding. LR11 plays a key role in the migration of undifferentiated vascular smooth muscle cells, and circulating sLR11 is a biomarker of carotid intima-media thickness. In accordance with sLR11 levels correlating with the fraction of immature vascular cells, human CD34+CD38- immature hematopoietic precursors display high levels of LR11 mRNA. We investigated the expression of LR11 in normal leukocytes, leukemia cell lines and acute leukemia cells. Methods:A2-2-3 anti LR11 monoclonal antibody was used for immunoblotting. Biotinylated or FITC-conjugated anti LR11 monoclonal antibodies, M3 and R15 were used for flow cytometric analysis and immunohistochemistry. Normal mononuclear cells were obtained from healthy volunteer donors. Leukemia cells were obtained from patients' bone marrow or peripheral blood. LR11 protein levels and sLR11 in the culture supernatant of human leukemic cell lines were examined by Western blotting and ELISA using specific monoclonal antibody against LR11. The expression of LR11 mRNA of the cells was examined by Real-Time PCR. Flow cytometric analysis of cell surface LR11 was performed with desktop cell sorter JSAN (Bay Bioscience). Results:Most human leukemia cell lines expressed high level of LR11 mRNA and protein. sLR11 was also detected in the culture supernatant. The levels of LR11 mRNA, the amount of cellular LR11 protein, and the amount of released sLR11 protein were significantly correlated with each other. Flow cytometric analysis of peripheral leukocytes using the anti-LR11 mAb M3, showed expression of LR11 in most CD14+ monocytes. LR11 was not significantly expressed on most T cells (CD4+, CD8+), B cells (CD19+), or granulocytes. However, the leukemia cell lines HL-60 (acute promyelocytic), CCRF-SB (lymphoblastic), and U937 (monocytic), but not K562 (chronic myelogenous) expressed LR11. Since LR11 is expressed by leukemia cells of different origins, we explored the expression of LR11 on the surface of patients' leukemia cells. We have examined 7 AML cases (M0, M1, M2, M3, M4, M5 and M6) and 3 ALL cases. Although expression level of LR11 differs among these cases, LR11 was detected in every case except one ALL case. The most dramatic M3-stained population was the clonally expanded CD19+ mononuclear fraction in MLL-AF4 positive early precursor B acute lymphoblastic leukemia (ALL). In addition, more than 50% blastic cells were positive for LR11 in a Philadelphia chromosome positive ALL patient. Over 50% of CD34+ mononuclear cells in AML (M0) were LR11-positive, whereas LR11-positive blasts predominated in the CD38- fraction. The majority of mononuclear cells in AML (M4) with high CD11b-expression were also LR11-positive. Thus, LR11 is specifically expressed on the surface of leukemic blasts in both ALL and AML. Furthermore, immunohistochemistry of bone marrow clot sections of AML and ALL revealed that cytoplasm of leukemia cells are specifically reacted against the anti-LR11 antibody. Thus, LR11 is expressed both in the cytoplasm and on the cell surface of acute leukemia cells. Conclusion:LR11 is specifically and highly expressed on cell surface of acute leukemia cells in addition to normal leukocytes. Together with our finding that sLR11 is a novel marker for acute leukemia, the identification of novel surface antigen sheds light on leukocyte biology and leukemia cell development. Disclosures:No relevant conflicts of interest to declare.

Stem Cell Research in Cell Transplantation: Sources, Geopolitical Influence, and Transplantation

If the rapidly progressing field of stem cell research reaches its full potential, successful treatments and enhanced understanding of many diseases are the likely results. However, the full potential of stem cell science will only be reached if all possible avenues can be explored and on a worldwide scale. Until 2009, the US had a highly restrictive policy on obtaining cells from human embryos and fetal tissue, a policy that pushed research toward the use of adult-derived cells. Currently, US policy is still in flux, and retrospective analysis does show the US lagging behind the rest of the world in the proportional increase in embryonic/fetal stem cell research. The majority of US studies being on either a limited number of cell lines, or on cells derived elsewhere (or funded by other sources than Federal) rather than on freshly isolated embryonic or fetal material. Neural, mesenchymal, and the mixed stem cell mononuclear fraction are the most commonly investigated types, which can generally be classified as adult-derived stem cells, although roughly half of the neural stem cells are fetal derived. Other types, such as embryonic and fat-derived stem cells, are increasing in their prominence, suggesting that new types of stem cells are still being pursued. Sixty percent of the reported stem cell studies involved transplantation, of which over three quarters were allogeneic transplants. A high proportion of the cardiovascular systems articles were on allogeneic transplants in a number of different species, including several autologous studies. A number of pharmaceutical grade stem cell products have also recently been tested and reported on. Stem cell research shows considerable promise for the treatment of a number of disorders, some of which have entered clinical trials; over the next few years it will be interesting to see how these treatments progress in the clinic.

Decreased collagen types I and IV, laminin, CK-19 and α-SMA expression after bone marrow cell transplantation in rats with liver fibrosis

Bone marrow cells have frequently been tested in animal models of liver fibrosis to assess their role in hepatic regeneration. The mononuclear fraction of bone marrow cells is of particular interest, as many studies show that these cells may be beneficial to treat hepatic fibrosis. In this study, we used the bile duct ligation model to induce hepatic fibrosis in an irreversible manner, and rats were treated with bone marrow mononuclear (BMMN) cells after fibrosis was established. Analysis of collagen types I and IV, laminin and α-SMA showed a decreased expression of these proteins in fibrotic livers after 7 days of BMMN cell injection. Moreover, cytokeratin-19 analysis showed a reduction in bile ducts in the BMMN cell-treated group. These results were accompanied by ameliorated levels of hepatic enzymes GPT (Glutamic-pyruvic transaminase), GOT (glutamic-oxaloacetic transaminase) and alkaline phosphatase (AP). Therefore, we showed that BMMN cells decrease hepatic fibrosis by significantly reducing myofibroblast numbers and through reduction of the collagen and laminin-rich extracellular matrix of fibrotic septa and hepatic sinusoids.

Selected Abstracts from the “Best of Europe” Session of the 2nd Congress of the European Association of Societies of Aesthetic Plastic Surgery (EASAPS)

Selected Abstracts from the ‘‘Best of Europe’’ Session of the 2nd Congress of the European Association of Societies of Aesthetic Plastic Surgery (EASAPS) Best of Europe in Aesthetic Plastic Surgery 2009 (Editorial Coordinator: Cristino Suarez Lopez de Vergara). Strahil Efremov • Chavdar Botev • Ivan Tonev • Milcho Mincheff • James Henderson • Marketa Duskova • Dirk F. Richter • Andrea Grisotti • Emeri Jean-Francois • Toma Mugea • Jose Carlos Parreira • Ercan Cihandide • E. E. Aydin • A. Uzunismail • Juan Monreal • Alain Bzowski • Hassan Tazzi • Cristino Suarez Lopez de Vergara Springer Science+Business Media, LLC and International Society of Aesthetic Plastic Surgery 2010 7. Use of Autologous Stem Cells for Stimulation of Neoangiogenesis in Plastic Surgery Strahil Efremov, Chavdar Botev, Ivan Tonev, Milcho Mincheff (Medical Center ‘‘Liliya’’, Sofia, Bulgaria) Recent years have witnessed an increase in the therapeutic use of stem cells in both animal models and humans. Embryonic stem cells have shown great promise in regenerative medicine due to their capability to differentiate into mature stem cells of all lineages. Their use, however, has been hampered by the possibility of them forming tumors and for religious reasons. Adult stem cells are easier to obtain and several different ‘‘stem’’ cells such as tissue-specific, endothelial precursor, or ‘‘embryonic’’ -like cells have been described. These can be obtained from different sources such as bone marrow, peripheral blood, fat tissue, and cord blood. We have found that the mononuclear fraction from both adult bone marrow and fat tissue contains stem cells (endothelial precursors) whose application leads to neoangiogenesis and better nutrition of the affected tissue. In this presentation we will discuss two cases. The first is a young female who suffered a trauma after an explosion that totally destroyed her right ear and the basin of the right a. temporalis. The lack of adequate blood supply resulted in two unsuccessful otoplasties using implants. The application of autologous stem cells at the surgery site a month prior to and during otoplasty, however, led to neoangiogenesis at the affected area and a successful third otoplasty. The second case involves a female with a breast defect that was resolved successfully by the application of a mixture of autologous fat tissue and bone marrow-derived stem cells. 9. The Cosmetic Surgery Postcode Lottery James Henderson (Addenbrooke’s Hospital, Cambridge, UK) Introduction: There is wide variation of availability for many treatments, leading to a ‘‘postcode lottery’’ for health-care provision in England. The aim of this work was to evaluate the extent of the inequality of funding for the plastic surgery procedures which are viewed by PCTs as cosmetic. Materials and methods: All PCTs in the UK were asked for their funding policies for cosmetic procedures, including breast reduction and augmentation, removal of implants, mastopexy, abdominoplasty, face-lift, blepharoplasty, rhinoplasty, pinnaplasty, body lifting, surgery for gynecomastia, and tattoo removal. Results: Details of policies were received from 122 PCTs (82% response rate). Guidelines varied widely; some refuse all procedures while others allow a range. Different and sometimes contradictory rules governing symptoms, body S. Efremov C. Botev I. Tonev M. Mincheff J. Henderson M. Duskova D. F. Richter A. Grisotti E. Jean-Francois T. Mugea J. C. Parreira E. Cihandide E. E. Aydin A. Uzunismail J. Monreal A. Bzowski H. Tazzi C. S. Lopez de Vergara (&) EASAPS—Cardiff 2009, Wales, UK e-mail: cristinosuarez@gmail.com 123 Aesth Plast Surg (2010) 34:551–554 DOI 10.1007/s00266-010-9560-4

Optimization of immunomagnetic selection of myeloma cells from bone marrow using magnetic activated cell sorting

Plasma cells (PCs) enrichment from bone marrow samples of multiple myeloma (MM) patients is frequently performed by immunomagnetic separation (magnetic activated cell sorting, MACS) using anti-CD138 MicroBeads. The aim of our work was to find optimal strategy for immunomagnetic separation of PCs and determine optimal algorithm of separation techniques for samples with various percentage of neoplastic cells. From 2007 to 2008, selection of PCs using separation programs Possels and Posseld(2) was carried out on 234 bone marrow samples obtained from 208 MM patients. In 2008, an optimal algorithm for separation programs was introduced based on the analysis of the previous experiments. The Possels program is applicable for samples with >10% PCs in the mononuclear fraction, while the Posseld(2) program is used for samples with 5-10% PCs in the mononuclear fraction. Median purity of 92.6% for the positive fraction of cells (range 14.5-99.6%) and median recovery of 60.4% (range 25.7-99.5%) were obtained when the Possels program was applied (n = 45). A total of 80% (36/45) of processed samples had purity of >70%. Median purity for the positive fraction of 83.7% (range 14.3-99.7%) and median recovery of 14.3% (range 3.6-50.0%) were achieved using the Posseld(2) program (n = 99). A total of 68% (67/99) of processed samples reached >70% purity. This separation strategy enabled us to obtain sufficient amounts of highly purified PCs required for subsequent research purposes. The MACS method has been unsuccessful if the percentage of PCs in the initial sample was <5%. These samples were processed by fluorescence activated cell sorting (FACS).

The Effect of Human Umbilical Cord Blood Cells on Survival and Cytokine Production by Post-Ischemic Astrocytes in Vitro

Cerebral ischemia induces death of all neural cell types within the region affected by the loss of blood flow. We have shown that administering human umbilical cord blood cells after a middle cerebral artery occlusion in rats significantly reduces infarct size, presumably by rescuing cells within the penumbra. In this study we examined whether the cord blood cells enhanced astrocyte survival in an in vitro model of hypoxia with reduced glucose availability. Primary astrocyte cultures were incubated for 2 h in no oxygen (95% N, 5% CO(2)) and low glucose (1% compared to 4.5%) media. Cord blood mononuclear cells were added to half the cultures at the beginning of hypoxia. Astrocyte viability was determined using fluorescein diacetate/propidium iodide (FDA/PI) labeling and cytokine production by the astrocytes measured using ELISA. In some studies, T cells, B cells or monocytes/macrophages isolated from the cord blood mononuclear fraction with magnetic antibody cell sorting (MACS) were used instead to determine which cellular component of the cord blood mononuclear fraction was responsible for the observed effects. Co-culturing mononuclear cord blood cells with astrocytes during hypoxia stimulated production of IL-6 and IL-10 during hypoxia. The cord blood T cells decreased survival of the astrocytes after hypoxia but had no effect on the examined cytokines. Our data demonstrate that the tested cord blood fractions do not enhance astrocyte survival when delivered individually, suggesting there is either another cellular component that is neuroprotective or an interaction of all the cells is essential for protection.