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Ex vivo expanded peripheral blood progenitor cells (PBPC) have been shown to provide rapid neutrophil engraftment, and in some patients, to eliminate neutropenia after transplantation to support high-dose chemotherapy. However, the effect of expansion culture on stem cell content and potential loss of stem cells caused by induction of differentiation remains a concern. We have transplanted 21 patients with breast cancer with expanded autologous PBPC, with 11 patients receiving expanded PBPC as their sole hematopoietic cell source. In these studies, the CD34+ cells were selected and cultured for 10 days in defined media containing 100 ng/mL each of recombinant human stem cell factor (rhSCF), recombinant human granulocyte colony stimulating factor (rhG-CSF), and recombinant human megakaryocyte growth and developmental factor (rhMGDF) in 1-liter Teflon bags at 20,000 to 50,000 cells/mL. After culture the cells were washed and reinfused after high-dose chemotherapy followed by daily administration of rhG-CSF....

Background aims Hematopoietic stem cell transplantation of mobilized peripheral blood progenitor cell (PBPC) products results in rapid platelet engraftment, whereas the use of cord blood (CB) shows significant delays. The difference in the quality and number of megakaryocyte (MK) progenitors that may be responsible for the delay in platelet engraftment has not been fully defined. The objective of this study was to quantify the cells of the MK lineage in PBPC and CB products to determine whether potential differences exist. Methods We examined PBPC or CB for differences in surface markers and subpopulations as well as polyploidization status within the MK lineage. Colony-forming assays were used to determine whether differences exist in the clonogenic MK progenitor cell. Finally, we transplanted PBPC and CB mononuclear cells into NOD/SCID/IL2Rγ−/− (NSG) mice to study platelet engraftment rates. Results Equivalent MK populations and polyploidization was observed in PBPCs and CB. MK progenitors were present only in CD34+ cells and had little difference in colony growth between PBPC and CB. Additionally, MK subpopulations were similar in either product with a slightly more progenitor-enriched phenotype in CB. Finally, when PBPC or CB was transplanted at similar doses, equivalent platelet engraftment rates were observed. Conclusions PBPC and CB contain similar frequencies of MK populations, and, when transplanted in comparable doses, CB is as effective as PBPCs in producing platelet engraftment in vivo. Understanding the differences in MK populations between PBPC and CB could help generate protocols to improve platelet engraftment after CB transplantation.

Many cyber attacks can be attributed to poorly configured software, where administrators are often unaware of insecure settings due to the configuration complexity or the novelty of an attack. A resilient configuration management approach would address this problem by updating configuration settings based on current threats while continuing to render useful services. This responsive and adaptive behavior can be obtained using an evolutionary algorithm, where security measures of current configurations are employed to evolve new configurations. Periodically, these configurations are applied across a collection of computers, changing the systems' attack surfaces and reducing their exposure to vulnerabilities. The effectiveness of this evolutionary strategy for defending RedHat Linux Apache web-servers is analyzed experimentally through a study of configuration fitness, population diversity, and resiliency observations. Configuration fitness reflects the level of system confidentiality, integrity and availability; whereas, population diversity gauges the heterogeneous nature of the configuration sets. The computers' security depends upon the discovery of a diverse set of highly fit parameter configurations. Resilience reflects the evolutionary algorithm's adaptability to new security threats. Experimental results indicate the approach is able to determine and maintain secure parameter settings when confronted with a variety of simulated attacks over time.

Longitudinal studies can provide individual histories of educational attainment and are becoming widely used in educational research. Two national longitudinal studies, the National Child Development Study (NCDS) and the British Cohort Study of 1970 (BCS70), are used here to investigate changing trends in the educational attainment of children in the UK over time. Multilevel modelling is used to examine variation between different social groups in attainment in mathematics and reading and to examine educational progress during secondary education; the results of these analyses are compared for the two different cohorts. In both cohorts, the main source of variation in achievement is due to differences in social background; differences between regions and local education authorities are found to be negligible. Changes in the mathematics and reading attainment of the different social groups between the cohorts reflect recognized trends in educational attainment and highlight some trends not previously reported.

Information is rapidly emerging regarding the important role of the arterial vasa vasorum in a variety of systemic vascular diseases. In addition, increasing evidence suggests that progenitor cells of bone marrow (BM) origin may contribute to postnatal neovascularization and/or vascular wall thickening that is characteristic in some forms of systemic vascular disease. Little is known regarding postnatal vasa formation and the role of BM-derived progenitor cells in the setting of pulmonary hypertension (PH). We sought to determine the effects of chronic hypoxia on the density of vasa vasorum in the pulmonary artery and to evaluate if BM-derived progenitor cells contribute to the increased vessel wall mass in a bovine model of hypoxia-induced PH. Quantitative morphometric analyses of lung tissue from normoxic and hypoxic calves revealed that hypoxia results in a dramatic expansion of the pulmonary artery adventitial vasa vasorum. Flow cytometric analysis demonstrated that cells expressing the transmembrane tyrosine kinase receptor for stem cell factor, c-kit, are mobilized from the BM in the circulation in response to hypoxia. Immunohistochemistry revealed an increase in the expression of c- kit+cells together with vascular endothelial growth factor, fibronectin, and thrombin in the hypoxia-induced remodeled pulmonary artery vessel wall. Circulating mononuclear cells isolated from neonatal calves exposed to hypoxia were found to differentiate into endothelial and smooth muscle cell phenotypes depending on culture conditions. From these observations, we suggest that the vasa vasorum and circulating progenitor cells could be involved in vessel wall thickening in the setting of hypoxia-induced PH.

To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic stem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded cells, we expanded mononuclear cells (MNCs) and CD34+/c-kit+ cells from mouse bone marrow and transplanted the expanded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34+/c-kit+ cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34+/c-kit+ cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells, CD34+ cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34+/c-kit+ cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of expanded cells by the co-culture with MSCs may result in more rapid engraftment of neutrophils following infusion to transplant recipients.

Umbilical cord blood (CB) from unrelated donors is increasingly used to restore hematopoiesis after myeloablative therapy. CB transplants are associated with higher rates of delayed and failed engraftment than are bone marrow transplants, particularly for adult patients. We studied the ex vivo expansion of CB in an attempt to improve time to engraftment and reduce the graft failure rate in the recipients. In this feasibility study, 37 patients (25 adults, 12 children) with hematologic malignancies (n = 34) or breast cancer (n = 3) received high-dose therapy followed by unrelated allogeneic CB transplantation. A fraction of each patient's CB allograft was CD34-selected and cultured ex vivo for 10 days prior to transplantation in defined media with stem cell factor, granulocyte colony-stimulating factor, and megakaryocyte growth and differentiation factor. The remainder of the CB graft was infused without further manipulation. Two sequential cohorts of patients were accrued to the study. The first cohort had 40% and the second cohort had 60% of their CB graft expanded. Patients received a median of 0.99 x 10(7) total nucleated cells (expanded plus unexpanded) per kilogram. The median time to engraftment of neutrophils was 28 days (range, 15-49 days) and of platelets was 106 days (range, 38-345 days). All evaluable patients who were followed for 28 days or longer achieved engraftment of neutrophils. Grade III/IV acute GVHD was documented in 40% and extensive chronic GVHD in 63% of patients. At a median follow-up of 30 months, 13 (35%) of 37 of patients survived. This study demonstrates that the CD34 selection and ex vivo expansion of CB prior to transplantation of CB is feasible. Additional accrual will be required to assess the clinical efficacy of expanded CB progenitors. Biol Blood Marrow Transplant 2002;8(7):368-76.

Adoptive therapy with regulatory T cells (Tregs) to prevent graft-versus-host disease (GVHD) would benefit from a strategy to improve homing to the sites of inflammation. We hypothesized that adding fucose to human Tregs, forming the Sialyl Lewis X moiety on P-selectin glycoprotein ligand-1, would improve their trafficking pattern. The selectin pathway recruiter, α-1,3-fucosyltransferase-VI enzyme, significantly increased Treg surface fucosylation (66% vs 8%). In a xenogenic GVHD mouse model, fucosylated Tregs showed prolonged periods of in vivo persistence. When given at a lower dose compared with the untreated Tregs, the murine recipients of fucosylated Tregs maintained weight, had ameliorated clinical GVHD, and improved survival (70% vs 30%; P < .0001). These preclinical data indicate that fucosylated human Tregs is an effective strategy for prevention of GVHD and, as such, warrants consideration for future clinical trials.

Understanding the mechanisms regulating islet growth and survival is critical for developing novel approaches to increasing or sustaining β cell mass in both type 1 and type 2 diabetes patients. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that is important for the regulation of cell growth and adhesion. Increased SPARC can be detected in the serum of type 2 diabetes patients. The aim of this study was to investigate the role of SPARC in the regulation of β cell growth and survival. We show using immunohistochemistry that SPARC is expressed by stromal cells within islets and can be detected in primary mouse islets by western blot. SPARC is secreted at high levels by pancreatic stellate cells and is regulated by metabolic parameters in these cells, but SPARC expression was not detectable in β cells. In islets, SPARC expression is highest in young mice, and is also elevated in the islets of non-obese diabetic (NOD) mice compared to controls. Purified SPARC inhibits growth factor-induced signalling in both INS-1 β cells and primary mouse islets, and inhibits IGF-1-induced proliferation of INS-1 β cells. Similarly, exogenous SPARC prevents IGF-1-induced survival of primary mouse islet cells. This study identifies the stromal-derived matricellular protein SPARC as a novel regulator of islet survival and β cell growth.

OBJECTIVES:: This study examined the relationship between mental health symptoms and the use of tobacco and caffeinated beverages during and just before a counseling session in a population of adult polysubstance abusers. METHODS:: The participants were all polysubstance users in substance abuse treatment. The participants completed the Beck Depression Inventory II and the State-Trait Anxiety Inventory immediately after a treatment episode. They also reported whether or not they had drunk a caffeinated beverage or smoked a cigarette just before or during that treatment episode. RESULTS:: Coffee drinkers scored significantly higher (mean = 20.3) on the Beck Depression Inventory II than did noncoffee drinkers (mean = 9.2). The differences between these groups on the State-Trait Anxiety Inventory were nonsignificant. There was no relationship between other caffeinated beverages or tobacco use and depression or anxiety. CONCLUSIONS:: Caffeine use is associated with depression in adult polysubstance abusers. Implications for using coffee drinking as a predictor of depression in substance abuse treatment settings are discussed. Language: en

Regenerative medicine offers the potential for treatment and possibly cures debilitating diseases including heart disease, diabetes, Parkinson’s disease, and liver failure. Approaches using stem cells from various sources are in preclinical and clinical testing. The goal of these studies is to deliver cellular products capable of replacing damaged tissue and/or cells. However, the balance between cellular proliferation and differentiation is a carefully controlled process involving a range of growth factors and cytokines produced in large part by tissue stromal cells. These stromal cells make up the tissue microenvironment and appear to be essential for normal homeostasis. We hypothesize that tissue damage in many instances involves damage to the microenvironment resulting in a lack of signals through growth factor networks necessary to maintain survival and proliferation of tissue-specific stem cells and progenitor cells. Therefore, optimal repair of disease tissue must account for the damage to the stromal environment and will require reconstitution of the microenvironment to support the survival, proliferation, and differentiation of the tissue-specific stem cells or progenitor cells. Further, stromal cells from different tissues have distinct gene profiles and so a homologous source of stromal cells would minimize potential differences that could result in unwanted toxicities or biological effects.

A subset of regulatory B cells (Bregs) in mice negatively regulate T-cell immune responses through the secretion of regulatory cytokines such as IL-10 and direct cell-cell contact and have been linked to experimental models of autoimmunity, inflammation, and cancer. However, the regulatory function of Bregs in human disease is much less clear. Here we demonstrate that B cells with immunoregulatory properties are enriched within both the CD19(+)IgM(+)CD27(+) memory and CD19(+)CD24(hi)CD38(hi) transitional B-cell subsets in healthy human donors. Both subsets suppressed the proliferation and interferon-γ production of CD3/CD28-stimulated autologous CD4(+) T cells in a dose-dependent manner, and both relied on IL-10 secretion as well as cell-cell contact, likely mediated through CD80 and CD86, to support their full suppressive function. Moreover, after allogeneic stem cell transplantation, Bregs from patients with chronic graft-versus-host disease (cGVHD) were less frequent and less likely to produce IL-10 than were Bregs from healthy donors and patients without cGVHD. These findings suggest that Bregs may be involved in the pathogenesis of cGVHD and support future investigation of regulatory B cell-based therapy in the treatment of this disease.

With improving expertise and supportive care, cord blood (CB) transplants are now associated with outcomes comparable to unrelated and sibling donor transplants.1 There is a need to increase the size and diversity of international CB inventories and to retain cryopreserved CB units for as long as safely possible. There are in vitro data supporting >90% recovery of hematopoietic progenitor cells from frozen CB cells stored for up to 12 years,2 and data showing that progenitor cell recoveries from short-term freeze samples ranging from 2 to 8 weeks are comparable to those cryopreserved for 10–15 years.3,4 Broxmeyer et al.5 reported adequate recovery (80–100%) of granulocyte–macrophage and multi-potential hematopoietic progenitors from functional CB units cryopreserved for up to 23.5 years as well as recovery of viable progenitor cells from CBs frozen for 15 years, which were able to generate CD45+ human cell engraftment when infused into sublethally irradiated NOD-SCID mice and were comparable to that reported with fresh CB.6 Despite this in vitro evidence, transplant physicians still remain concerned about the possible loss of integrity of frozen CB units associated with longer durations of storage.7 In one report, the incidence of bag breaks over a 6.5-year period was 3.5%, where 75% of the breaches occurred in units that had been cryopreserved for >2 years.7 Therefore, it is important to determine whether prolonged time of cryopreservation and storage may adversely affect clinical transplant outcomes, and with the new US Food and Drug Administration licensing regulations for CB units this question has become even more relevant: ‘is there an expiration date for CB unit in storage?’.

Human papillomaviruses (HPVs) are frequently detected in a variety of lesions in the oral mucosa and upper respiratory tract. The pathogenesis in these areas is not as clearly elucidated as in other anatomical regions, but most experts agree that HPVs are responsible for the commonly observed benign lesions, such as squamous papillomas, verruca vulgaris and recurrent respiratory papillomatosis. Transformation of these benign lesions is well described, but it is not clear what role the virus plays, if any, in carcinogenesis. HPV types 6 and 11 are most frequently detected in oral cavity and respiratory tract lesions, though several other types have also been reported. Despite an opaque understanding of these lesions' pathogeneses, it is essential for the clinician to recognize these diseases, to provide appropriate treatment and to promote patient awareness of potential oral transmission. In this paper, we review the major HPV-associated diseases of the oral mucosa and upper respiratory tract, focusing specifically on clinical features, histopathological characteristics and disease management.

Rationale: Transendocardial stem cell injection (TESI) with mesenchymal stem cells improves remodeling in chronic ischemic cardiomyopathy, but the effect of the injection site remains unknown. Objective: To address whether TESI exerts its effects at the site of injection only or also in remote areas, we hypothesized that segmental myocardial scar and segmental ejection fraction improve to a greater extent in injected than in noninjected segments. Methods and Results: Biplane ventriculographic and endocardial tracings were recorded. TESI was guided to 10 sites in infarct-border zones. Sites were mapped according to the 17-myocardial segment model. As a result, 510 segments were analyzed in 30 patients before and 13 months after TESI. Segmental early enhancement defect (a measure of scar size) was reduced by TESI in both injected (−43.7±4.4%; n=95; P P P P =0.003) but not in noninjected scar segments (21.3±2.6–23.5±3.2%; P =0.20; between-group comparison P P =0.003), versus P =0.33, between-group comparison P Conclusions: These findings illustrate a dichotomy in regional responses to TESI. Although scar size reduction was evident in all scar segments, scar size reduction and ventricular functional responses preferentially occurred at the sites of TESI versus non-TESI sites. Furthermore, improvement was greatest when segmental left ventricular dysfunction was severe.