High-throughput surface marker screen on primary human breast tissues reveals further cellular heterogeneity.

BACKGROUND: Normal human breast tissues are a heterogeneous mix of epithelial and stromal subtypes in different cell states. Delineating the spectrum of cellular heterogeneity will provide new insights into normal cellular properties within the breast tissue that might become dysregulated in the initial stages of cancer. Investigation of surface marker expression provides a valuable approach to resolve complex cell populations. However, the majority of cell surface maker expression of primary breast cells have not been investigated. METHODS: To determine the differences in expression of a range of uninvestigated cell surface markers between the normal breast cell subpopulations, primary human breast cells were analysed using high-throughput flow cytometry for the expression of 242 cell surface proteins in conjunction with EpCAM/CD49f staining. RESULTS: We identified 35 surface marker proteins expressed on normal breast epithelial and/or stromal subpopulations that were previously unreported. We also show multiple markers were equally expressed in all cell populations (e.g. CD9, CD59, CD164) while other surface markers were confirmed to be enriched in different cell lineages: CD24, CD227 and CD340 in the luminal compartment, CD10 and CD90 in the basal population, and CD34 and CD140b on stromal cells. CONCLUSIONS: Our dataset of CD marker expression in the normal breast provides better definition for breast cellular heterogeneity

Chondrosarcoma of the Pelvis: Oncologic and Functional Outcome

Purpose. Chondrosarcoma (CS) most commonly involves the pelvis. The factors that influence local and systemic control of pelvic CS and the functional outcome should be evaluated

Index sorting resolves heterogeneous murine hematopoietic stem cell populations.

Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation, often performed using multiparameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolution of the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single-cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AAD(dim) fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information.This work was supported by grants from Leukaemia and Lymphoma Research, the Medical Research Council, the National Institute for Health Research Cambridge Biomedical Research Centre, and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute. DGK is the recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship and a European Hematology Association non-clinical advanced research fellowship. The authors declare that they have no conflict of interest.This is the author accepted manuscript. The final version will be available from Elsevier at http://dx.doi.org/10.1016/j.exphem.2015.05.006

Mucosal prior to systemic application of recombinant adenovirus boosting is more immunogenic than systemic application twice but confers similar protection against SIV-challenge in DNA vaccine-primed macaques

AbstractWe investigated the immunogenicity and efficacy of a bimodal prime/boost vaccine regimen given by various routes in the Simian immunodeficiency virus (SIV) rhesus monkey model for AIDS. Twelve animals were immunized with SIV DNA-vectors followed by the application of a recombinant adenovirus (rAd5) expressing the same genes either intramuscularly (i.m.) or by oropharyngeal spray. The second rAd5-application was given i.m. All vaccinees plus six controls were challenged orally with SIVmac239 12 weeks post-final immunization.Both immunization strategies induced strong SIV Gag-specific IFN-γ and T-cell proliferation responses and mediated a conservation of CD4+ memory T-cells and a reduction of viral load during peak viremia following infection. Interestingly, the mucosal group was superior to the systemic group regarding breadth and strength of SIV-specific T-cell responses and exhibited lower vector specific immune responses. Therefore, our data warrant the inclusion of mucosal vector application in a vaccination regimen which makes it less invasive and easier to apply

Teaching child and adolescent psychiatry to undergraduate medical students - A survey in German-speaking countries

Objective To conduct a survey about teaching child and adolescent psychiatry to undergraduate medical students in German-speaking countries. Methods A questionnaire was sent to the 33 academic departments of child and adolescent psychiatry in Germany, Austria, and the German-speaking part of Switzerland. Results All departments responded. For teaching knowledge, the methods most commonly reported were lectures and case presentations. The most important skills to be taught were thought to be how to assess psychopathology in children and how to assess families. For elective courses, the departments reported using a wide range of teaching methods, many with active involvement of the students. An average of 34 hours per semester is currently allocated by the departments for teaching child and adolescent psychiatry to medical students. Required courses are often taught in cooperation with adult psychiatry and pediatrics. Achievement of educational objectives is usually assessed with written exams or multiple-choice tests. Only a minority of the departments test the achievement of skills. Conclusions Two ways of improving education in child and adolescent psychiatry are the introduction of elective courses for students interested in the field and participation of child and adolescent psychiatrists in required courses and in longitudinal courses so as to reach all students. Cooperation within and across medical schools can enable departments of child and adolescent psychiatry, despite limited resources, to become more visible and this specialty to become more attractive to medical students. Compared to the findings in earlier surveys, this survey indicates a trend towards increased involvement of academic departments of child and adolescent psychiatry in training medical students

Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations.

Heterogeneity within the self-renewal durability of adult hematopoietic stem cells (HSCs) challenges our understanding of the molecular framework underlying HSC function. Gene expression studies have been hampered by the presence of multiple HSC subtypes and contaminating non-HSCs in bulk HSC populations. To gain deeper insight into the gene expression program of murine HSCs, we combined single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays. Through bioinformatic integration of these datasets, we designed an unbiased sorting strategy that separates non-HSCs away from HSCs, and single-cell transplantation experiments using the enriched population were combined with RNA-seq data to identify key molecules that associate with long-term durable self-renewal, producing a single-cell molecular dataset that is linked to functional stem cell activity. Finally, we demonstrated the broader applicability of this approach for linking key molecules with defined cellular functions in another stem cell system.Work in the author’s laboratory is supported by grants from the Leukaemia and Lymphoma Research, the Medical Research Council, Cancer Research UK, Biotechnology and Biological Sciences Research Council, Leukemia Lymphoma Society, and the National Institute for Health Research Cambridge Biomedical Research Centre and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute. D.G.K. is the recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship. F.B. and F.J.T. are funded by the European Research Council (starting grant “LatentCauses”). For funding for the open access charge, the core support grant was provided by the Wellcome Trust-MRC Cambridge Stem Cell Institute. We acknowledge the support of the University of Cambridge, Cancer Research UK Institute (core grant C14303/A17197), and Hutchison Whampoa Limited.This is the final published version. It first appeared at http://www.cell.com/cell-stem-cell/abstract/S1934-5909%2815%2900162-9

Mammary stem cells have myoepithelial cell properties.

Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt actin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using two independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage-tracing approach we follow the progeny of myoepithelial cells that express α-smooth muscle actin and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy.This work was funded by Cancer Research UK, Breast Cancer Campaign, the University of Cambridge, Hutchison Whampoa Limited, La Ligue Nationale Contre le Cancer (Equipe Labelisée 2013) and a grant from Agence Nationale de la Recherche ANR- 08-BLAN-0078-01 to M.A.G.This is the author accepted manuscript. The final version is available from Nature at http://www.nature.com/ncb/journal/vaop/ncurrent/full/ncb3025.html

Strong mucosal immune responses in SIV infected macaques contribute to viral control and preserved CD4+ T-cell levels in blood and mucosal tissues

<p>Abstract</p> <p>Background</p> <p>Since there is still no protective HIV vaccine available, better insights into immune mechanism of persons effectively controlling HIV replication in the absence of any therapy should contribute to improve further vaccine designs. However, little is known about the mucosal immune response of this small unique group of patients. Using the SIV-macaque-model for AIDS, we had the rare opportunity to analyze 14 SIV-infected rhesus macaques durably controlling viral replication (controllers). We investigated the virological and immunological profile of blood and three different mucosal tissues and compared their data to those of uninfected and animals progressing to AIDS-like disease (progressors).</p> <p>Results</p> <p>Lymphocytes from blood, bronchoalveolar lavage (BAL), and duodenal and colonic biopsies were phenotypically characterized by polychromatic flow cytometry. In controllers, we observed higher levels of CD4+, CD4+CCR5+ and Gag-specific CD8+ T-cells as well as lower immune activation in blood and all mucosal sites compared to progressors. However, we could also demonstrate that immunological changes are distinct between these three mucosal sites.</p> <p>Intracellular cytokine staining demonstrated a significantly higher systemic and mucosal CD8+ Gag-specific cellular immune response in controllers than in progressors. Most remarkable was the polyfunctional cytokine profile of CD8+ lymphocytes in BAL of controllers, which significantly dominated over their blood response. The overall suppression of viral replication in the controllers was confirmed by almost no detectable viral RNA in blood and all mucosal tissues investigated.</p> <p>Conclusion</p> <p>A strong and complex virus-specific CD8+ T-cell response in blood and especially in mucosal tissue of SIV-infected macaques was associated with low immune activation and an efficient suppression of viral replication. This likely afforded a repopulation of CD4+ T-cells in different mucosal compartments to almost normal levels. We conclude, that a robust SIV-specific mucosal immune response seems to be essential for establishing and maintaining the controller status and consequently for long-term survival.</p

Effects of prostratin on Cyclin T1/P-TEFb function and the gene expression profile in primary resting CD4(+ )T cells

BACKGROUND: The latent reservoir of human immunodeficiency virus type 1 (HIV-1) in resting CD4(+ )T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART). Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4(+ )T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular kinase composed of Cyclin T1 and Cyclin-dependent kinase-9 (CDK9) that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its kinase activity. RESULTS: Prostratin up-regulated Cyclin T1 protein expression, modestly induced CDK9 protein expression, and did not affect Cyclin T2a protein expression. Although the kinase activity of CDK9 in vitro was up-regulated by prostratin, we observed a large increase in the association of 7SK snRNA and the HEXIM1 protein with CDK9. Using HIV-1 reporter viruses with and without a functional Tat protein, we found that prostratin stimulation of HIV-1 gene expression appears to require a functional Tat protein. Microarray analyses were performed and several genes related to HIV biology, including APOBEC3B, DEFA1, and S100 calcium-binding protein genes, were found to be regulated by prostratin. CONCLUSION: Prostratin induces Cyclin T1 expression and P-TEFb function and this is likely to be involved in prostratin reactivation of latent HIV-1 proviruses. The large increase in association of 7SK and HEXIM1 with P-TEFb following prostratin treatment may reflect a requirement in CD4(+ )T cells for a precise balance between active and catalytically inactive P-TEFb. Additionally, genes regulated by prostratin were identified that have the potential to regulate HIV-1 replication both positively and negatively