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Sample records for epithelial subpopulations identifies

  1. Transcriptome analysis of mammary epithelial subpopulations identifies novel determinants of lineage commitment and cell fate

    PubMed Central

    Kendrick, Howard; Regan, Joseph L; Magnay, Fiona-Ann; Grigoriadis, Anita; Mitsopoulos, Costas; Zvelebil, Marketa; Smalley, Matthew J

    2008-01-01

    Background Understanding the molecular control of cell lineages and fate determination in complex tissues is key to not only understanding the developmental biology and cellular homeostasis of such tissues but also for our understanding and interpretation of the molecular pathology of diseases such as cancer. The prerequisite for such an understanding is detailed knowledge of the cell types that make up such tissues, including their comprehensive molecular characterisation. In the mammary epithelium, the bulk of the tissue is composed of three cell lineages, namely the basal/myoepithelial, luminal epithelial estrogen receptor positive and luminal epithelial estrogen receptor negative cells. However, a detailed molecular characterisation of the transcriptomic differences between these three populations has not been carried out. Results A whole transcriptome analysis of basal/myoepithelial cells, luminal estrogen receptor negative cells and luminal estrogen receptor positive cells isolated from the virgin mouse mammary epithelium identified 861, 326 and 488 genes as highly differentially expressed in the three cell types, respectively. Network analysis of the transcriptomic data identified a subpopulation of luminal estrogen receptor negative cells with a novel potential role as non-professional immune cells. Analysis of the data for potential paracrine interacting factors showed that the basal/myoepithelial cells, remarkably, expressed over twice as many ligands and cell surface receptors as the other two populations combined. A number of transcriptional regulators were also identified that were differentially expressed between the cell lineages. One of these, Sox6, was specifically expressed in luminal estrogen receptor negative cells and functional assays confirmed that it maintained mammary epithelial cells in a differentiated luminal cell lineage. Conclusion The mouse mammary epithelium is composed of three main cell types with distinct gene expression patterns

  2. Comparative Secretome Analysis of Epithelial and Mesenchymal Subpopulations of Head and Neck Squamous Cell Carcinoma Identifies S100A4 as a Potential Therapeutic Target*

    PubMed Central

    Rasanen, Kati; Sriswasdi, Sira; Valiga, Alexander; Tang, Hsin-Yao; Zhang, Gao; Perego, Michela; Somasundaram, Rajasekharan; Li, Ling; Speicher, Kaye; Klein-Szanto, Andres J.; Basu, Devraj; Rustgi, Anil K.; Speicher, David W.; Herlyn, Meenhard

    2013-01-01

    Epithelial-mesenchymal transition (EMT) is a key contributor in tumor progression and metastasis. EMT produces cellular heterogeneity within head and neck squamous cell carcinomas (HNSCC) by creating a phenotypically distinct mesenchymal subpopulation that is resistant to conventional therapies. In this study, we systematically characterized differences in the secretomes of E-cadherin high epithelial-like and E-cadherin low mesenchymal-like subpopulations using unbiased and targeted proteomics. A total 1765 proteins showed significant changes with 177 elevated in the epithelial subpopulation and 173 elevated in the mesenchymal cells. Key nodes in affected networks included NFκB, Akt, and ERK, and most implicated cellular components involved various aspects of the extracellular matrix. In particular, large changes were observed in multiple collagens with most affected collagens at much higher abundance levels in the mesenchymal subpopulation. These cells also exhibited a secretome profile resembling that of cancer-associated fibroblastic cells (CAF). S100A4, a commonly used marker for cancer-associated fibroblastic cells, was elevated more than 20-fold in the mesenchymal cells and this increase was further verified at the transcriptome level. S100A4 is a known mediator of EMT, leading to metastasis and EMT has been proposed as a potential source of cancer-associated fibroblastic cells in solid tumors. S100A4 knockdown by small interfering RNA led to decreased expression, secretion and activity of matrix metalloproteinase 2, as verified by quantitative PCR, multiple reaction monitoring and zymography analyses, and reduced invasion in collagen-embedded spheroids. Further confirmation in three-dimensional organotypic reconstructs showed less invasion and advanced differentiation in the S100A4 RNA interference samples. Orthotopic metastasis model, developed to validate the findings in vivo, demonstrated a decrease in spontaneous metastasis and augmented differentiation

  3. Genetic and epigenetic changes in mammary epithelial cells identify a subpopulation of cells involved in early carcinogenesis.

    PubMed

    Berman, H; Zhang, J; Crawford, Y G; Gauthier, M L; Fordyce, C A; McDermott, K M; Sigaroudinia, M; Kozakiewicz, K; Tlsty, T D

    2005-01-01

    Morphologically normal foci of epithelial cells exhibiting p16 inactivation have been found in several tissues and may be precursors to cancer. Our previous work demonstrates that cells lacking p16(INK4A) activity exhibit phenotypes associated with malignancy (Romanov et al. 2001). The acquisition of genomic instability occurs through the activation of telomeric and centrosomal dysfunction. Additionally, the activation of stress pathways such as COX-2 provides these cells with the mutagenic potential to survive adverse environments as well as the ability to migrate, evade apoptosis and immune surveillance, and summon sustaining vasculature. Examination of archived tissue from women with DCIS (ductal carcinoma in situ) reveals epithelial cells that overexpress markers of premalignant stress activation pathways and mirror the distinctive expression patterns of these markers observed in vitro. These epithelial cells are found within the premalignant lesion as well as in the field of morphologically normal tissue that surrounds the lesion. Here, we show that p16(INK4A)-silenced vHMEC cells exhibit a gene expression profile which is distinct, reproducible, and extends beyond the changes mediated by p16(INK4A) inactivation. The present work suggests that cells lacking p16(INK4A) activity exhibit critical activities which allow cells to evade differentiation processes that would be expected to terminate proliferation. All of these properties are critical to malignancy. These events may be useful biomarkers to detect the earliest events in breast cancer. PMID:16869768

  4. Flow Cytometry Analysis of Thymic Epithelial Cells and Their Subpopulations.

    PubMed

    Ohigashi, Izumi; Takahama, Yousuke

    2016-01-01

    The parenchyma of the thymus is compartmentalized into the cortex and the medulla, which are constructed by cortical thymic epithelial cells (cortical TECs, cTECs) and medullary thymic epithelial cells (mTECs), respectively. cTECs and mTECs essentially and differentially regulate the development and repertoire selection of T cells. Consequently, the biology of T cell development and selection includes the study of TECs in addition to the study of developing T cells and other hematopoietic cells including dendritic cells. In this chapter, we describe the methods for flow cytometric analysis and sorting of TECs and their subpopulations, including cTECs and mTECs. PMID:26294398

  5. Combinations of differentiation markers distinguish subpopulations of alveolar epithelial cells in adult lung.

    PubMed

    Liebler, Janice M; Marconett, Crystal N; Juul, Nicholas; Wang, Hongjun; Liu, Yixin; Flodby, Per; Laird-Offringa, Ite A; Minoo, Parviz; Zhou, Beiyun

    2016-01-15

    Distal lung epithelium is maintained by proliferation of alveolar type II (AT2) cells and, for some daughter AT2 cells, transdifferentiation into alveolar type I (AT1) cells. We investigated if subpopulations of alveolar epithelial cells (AEC) exist that represent various stages in transdifferentiation from AT2 to AT1 cell phenotypes in normal adult lung and if they can be identified using combinations of cell-specific markers. Immunofluorescence microscopy showed that, in distal rat and mouse lungs, ∼ 20-30% of NKX2.1(+) (or thyroid transcription factor 1(+)) cells did not colocalize with pro-surfactant protein C (pro-SP-C), a highly specific AT2 cell marker. In distal rat lung, NKX2.1(+) cells coexpressed either pro-SP-C or the AT1 cell marker homeodomain only protein x (HOPX). Not all HOPX(+) cells colocalize with the AT1 cell marker aquaporin 5 (AQP5), and some AQP5(+) cells were NKX2.1(+). HOPX was expressed earlier than AQP5 during transdifferentiation in rat AEC primary culture, with robust expression of both by day 7. We speculate that NKX2.1 and pro-SP-C colocalize in AT2 cells, NKX2.1 and HOPX or AQP5 colocalize in intermediate or transitional cells, and HOPX and AQP5 are expressed without NKX2.1 in AT1 cells. These findings suggest marked heterogeneity among cells previously identified as exclusively AT1 or AT2 cells, implying the presence of subpopulations of intermediate or transitional AEC in normal adult lung. PMID:26545903

  6. FSP1+ fibroblast subpopulation is essential for the maintenance and regeneration of medullary thymic epithelial cells

    PubMed Central

    Sun, Lina; Sun, Chenming; Liang, Zhanfeng; Li, Hongran; Chen, Lin; Luo, Haiying; Zhang, Hongmei; Ding, Pengbo; Sun, Xiaoning; Qin, Zhihai; Zhao, Yong

    2015-01-01

    Thymic epithelial cells (TECs) form a 3-dimentional network supporting thymocyte development and maturation. Besides epithelium and thymocytes, heterogeneous fibroblasts are essential components in maintaining thymic microenvironments. However, thymic fibroblast characteristics, development and function remain to be determined. We herein found that thymic non-hematopoietic CD45-FSP1+ cells represent a unique Fibroblast specific protein 1 (FSP1)—fibroblast-derived cell subset. Deletion of these cells in FSP1-TK transgenic mice caused thymus atrophy due to the loss of TECs, especially mature medullary TECs (MHCIIhigh, CD80+ and Aire+). In a cyclophosphamide-induced thymus injury and regeneration model, lack of non-hematopoietic CD45-FSP1+ fibroblast subpopulation significantly delayed thymus regeneration. In fact, thymic FSP1+ fibroblasts released more IL-6, FGF7 and FSP1 in the culture medium than their FSP1- counterparts. Further experiments showed that the FSP1 protein could directly enhance the proliferation and maturation of TECs in the in vitro culture systems. FSP1 knockout mice had significantly smaller thymus size and less TECs than their control. Collectively, our studies reveal that thymic CD45-FSP1+ cells are a subpopulation of fibroblasts, which is crucial for the maintenance and regeneration of TECs especially medullary TECs through providing IL-6, FGF7 and FSP1. PMID:26445893

  7. Mathematical modelling of transcriptional heterogeneity identifies novel markers and subpopulations in complex tissues

    PubMed Central

    Wang, Niya; Hoffman, Eric P.; Chen, Lulu; Chen, Li; Zhang, Zhen; Liu, Chunyu; Yu, Guoqiang; Herrington, David M.; Clarke, Robert; Wang, Yue

    2016-01-01

    Tissue heterogeneity is both a major confounding factor and an underexploited information source. While a handful of reports have demonstrated the potential of supervised computational methods to deconvolute tissue heterogeneity, these approaches require a priori information on the marker genes or composition of known subpopulations. To address the critical problem of the absence of validated marker genes for many (including novel) subpopulations, we describe convex analysis of mixtures (CAM), a fully unsupervised in silico method, for identifying subpopulation marker genes directly from the original mixed gene expressions in scatter space that can improve molecular analyses in many biological contexts. Validated with predesigned mixtures, CAM on the gene expression data from peripheral leukocytes, brain tissue, and yeast cell cycle, revealed novel marker genes that were otherwise undetectable using existing methods. Importantly, CAM requires no a priori information on the number, identity, or composition of the subpopulations present in mixed samples, and does not require the presence of pure subpopulations in sample space. This advantage is significant in that CAM can achieve all of its goals using only a small number of heterogeneous samples, and is more powerful to distinguish between phenotypically similar subpopulations. PMID:26739359

  8. Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers

    PubMed Central

    Smalley, Matthew J; Iravani, Marjan; Leao, Maria; Grigoriadis, Anita; Kendrick, Howard; Dexter, Tim; Fenwick, Kerry; Regan, Joseph L; Britt, Kara; McDonald, Sarah; Lord, Christopher J; MacKay, Alan; Ashworth, Alan

    2007-01-01

    Introduction To understand which signalling pathways become deregulated in breast cancer, it is necessary to identify functionally significant gene expression patterns in the stem, progenitor, transit amplifying and differentiated cells of the mammary epithelium. We have previously used the markers 33A10, CD24 and Sca-1 to identify mouse mammary epithelial cell subpopulations. We now investigate the relationship between cells expressing these markers and use gene expression microarray analysis to identify genes differentially expressed in the cell populations. Methods Freshly isolated primary mouse mammary epithelial cells were separated on the basis of staining with the 33A10 antibody and an α-Sca-1 antibody. The populations identified were profiled using gene expression microarray analysis. Gene expression patterns were confirmed on normal mouse and human mammary epithelial subpopulations and were examined in a panel of breast cancer samples and cell lines. Results Analysis of the separated populations demonstrated that Sca-1- 33A10High stained cells were estrogen receptor α (Esr1)- luminal epithelial cells, whereas Sca-1+ 33A10Low/- stained cells were a mix of nonepithelial cells and Esr1+ epithelial cells. Analysis of the gene expression data identified the gene Rgs2 (regulator of G-protein signalling 2) as being highly expressed in the Sca-1- 33A10Low/- population, which included myoepithelial/basal cells. RGS2 has previously been described as a regulator of angiotensin II receptor signalling. Gene expression analysis by quantitative real-time RT-PCR of cells separated on the basis of CD24 and Sca-1 expression confirmed that Rgs2 was more highly expressed in mouse myoepithelial/basal mammary cells than luminal cells. This expression pattern was conserved in normal human breast cells. Functional analysis demonstrated RGS2 to be a modulator of oxytocin receptor signalling. The potential significance of RGS2 expression in breast cancer was demonstrated by semi

  9. Identifying vulnerable subpopulations for climate change health effects in the United States.

    PubMed

    Balbus, John M; Malina, Catherine

    2009-01-01

    Climate change can be expected to have differential effects on different subpopulations. Biological sensitivity, socioeconomic factors, and geography may each contribute to heightened risk for climate-sensitive health outcomes, which include heat stress, air pollution health effects, extreme weather event health effects, water-, food-, and vector-borne illnesses. Particularly vulnerable subpopulations include children, pregnant women, older adults, impoverished populations, people with chronic conditions and mobility and cognitive constraints, outdoor workers, and those in coastal and low-lying riverine zones. For public health planning, it is critical to identify populations that may experience synergistic effects of multiple risk factors for health problems, both related to climate change and to other temporal trends, with specific geographic factors that convey climate-related risks. PMID:19136871

  10. Two distinct subpopulations of neurons in the thalamic intergeniculate leaflet identified by subthreshold currents.

    PubMed

    Chrobok, Lukasz; Palus, Katarzyna; Lewandowski, Marian Henryk

    2016-08-01

    The intergeniculate leaflet (IGL) is a flat retinorecipient thalamic structure implicated in orchestrating circadian rhythm, historically considered to be a subdivision of the neighboring ventrolateral geniculate nucleus (VLG). IGL consists of two main neuronal subpopulations: enkephalinergic and neuropeptide Y (NPY)-synthesizing cells. These cell types have different functions, connectivity and firing pattern in vivo, which suggest that they have different membrane currents to support their functional differences. We therefore performed patch-clamp experiments combined with immunohistochemical staining to clarify possible differences in the subthreshold currents of IGL neurons. Our results suggest that IGL neurons can be divided into two subpopulations based on two ionic currents. A T-type calcium current (IT) was identified in neurons that do not synthesise NPY, whereas all NPY-positive neurons were found to express a marked A-type potassium current (IA). Due to the fact that the clear electrophysiological discriminants between IGL and VLG are lacking, we decided to compare the amplitudes of the identified currents between those two structures. Our data suggest that VLG neurons can be characterized by a high amplitude IT and a low IA. Finally, we compared both currents with WAG/Rij rats, a well-established model of absence epilepsy, with co-occurring retinal pathologies, sleep-onset disturbances, and seizures exhibiting circadian rhythmicity. Data presented in this study uncovered pathologies in the IT exhibiting neurons of the IGL and VLG. In conclusion, the data presented here suggest that different subthreshold current expression supports the functional differences of thalamic nuclei. Those differences are promising for possible pharmacological manipulations of specified cell types in pathophysiologies including absence epilepsy. PMID:27208616

  11. Circulating tumor cells and epithelial, mesenchymal and stemness markers: characterization of cell subpopulations

    PubMed Central

    Fici, Pietro; Gallerani, Giulia; Fabbri, Francesco; Zoli, Wainer; Rigaud, Michel

    2014-01-01

    Until now detection and numeration of circulating tumor cells (CTCs) were essentially used as a prognostic factor in cancer progression. To extend the role of these kinds of analysis, it seems necessary to improve analytical methods related to isolation and characterization of CTCs. Discrepancies between published results corroborates this requirement. In this review we suggest a combination of markers able to reach the goal. Moreover to improve the clinical utility of CTC analysis, particularly in the therapeutic follow up of the disease, epithelial mesenchymal transition (EMT) level of a global CTC population should be studied. PMID:25489583

  12. Glycosaminoglycan synthesis by subpopulations of epithelial cells from a mammary adenocarcinoma

    SciTech Connect

    Angello, J.C.; Danielson, K.G.; Anderson, L.W.; Hosick, H.L.

    1982-06-01

    Glycosaminoglycan synthesis by two subpopulations of a mouse mammary tumor cell line was compared. The two sublines express distinctly different growth characteristics in vitro and in vivo which indicate differences in growth regulation. Newly made glycosaminoglycans were recovered from the culture media, the cell surfaces, and residual cellular material. The cell population which grows more aggressively in vivo (+SA subline, a subline that grows in soft agarose) incorporated about 8 times more (/sup 14/C)glucosamine per cell into total glycosaminoglycans than did the slower-growing population (-SA subline, which does not grow in soft agarose). Appropriate control experiments indicated that the apparent difference in rates of synthesis was not due to discrepancies in glucosamine uptake. The main residual cellular molecule labeled was heparan sulfate, but the predominant molecule at the cell surface and in the culture fluid was hyaluronic acid. Overall, +SA cells synthesized more hyaluronic acid and -SA cells synthesized more heparan sulfate; in both cell populations, these two molecules accounted for about 90% of total glycosaminoglycans produced.

  13. CD169 identifies an anti-tumour macrophage subpopulation in human hepatocellular carcinoma.

    PubMed

    Zhang, Yi; Li, Jin-Qing; Jiang, Ze-Zhou; Li, Lian; Wu, Yan; Zheng, Limin

    2016-06-01

    Macrophages are a major component of most solid tumours and can exert both anti- and pro-tumourigenic functions. Although the immunosuppressive/pro-tumour roles of macrophages have been widely examined, significantly less is known about macrophage subpopulations that have potential anti-tumour properties in humans. In the present study, a population of CD169(+) macrophages with relatively high expression levels of HLA-DR and CD86 was identified in human hepatocellular carcinoma tissues. The frequency of CD169-expressing macrophages within cancer nests was significantly lower than that found in paired non-tumour areas. In vitro experiments revealed that in the presence of anti-CD3 stimulation, CD169(+) macrophages could significantly enhance the proliferation, cytotoxicity, and cytokine production capacity of CD8(+) T cells in a CD169 molecule-dependent manner. Autocrine TGF-β produced by tumour-stimulated macrophages was involved in the down-regulation of CD169 expression on these cells. Moreover, the accumulation of CD169(+) macrophages in tumour tissues was negatively associated with disease progression and predicted favourable survival in hepatocellular carcinoma patients, which was in contrast to the trend observed for total CD68(+) macrophages. Therefore, CD169 might act as a co-stimulatory molecule for cytotoxic T-cell activation, and could define a population of tumour-infiltrating macrophages with potential anti-tumour properties in human hepatocellular carcinoma tissues. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:27174787

  14. Use of stable isotopes to identify dietary differences across subpopulations and sex for a free-ranging generalist herbivore.

    PubMed

    Walter, W David

    2014-01-01

    Carbon and nitrogen isotopes in tissues can be used to understand plants consumed by various taxa, but can they provide additional information about consumers? Values of δ(13)C and δ(15)N were assessed from tissue of free-ranging elk (Cervus elaphus) occupying disparate habitats of mixed prairie-oak savannah that contained C3 agricultural crops in a C4-dominated landscape and in key plants consumed by elk. Muscle and hoof samples were collected from female and male elk in two subpopulations (forested land and grassland) from private land and one subpopulation from the Wichita Mountains Wildlife Refuge (refuge) in 2001-2006. Previous research identified differences between mean muscle δ(13)C and δ(15)N and mean hoof δ(13)C and δ(15)N indicating that isotopes differed between tissues of varying metabolic activity. Mean δ(13)C in hoof of elk on forested land and grassland were lower than hoof δ(13)C from elk in the refuge indicating greater long-term consumption of C3 plants by elk on forested land and grassland subpopulations. The δ(15)N in hoof was greater for elk outside the refuge than that for elk in the refuge. Interaction of sex and subpopulation only occurred for hoof δ(15)N suggesting that factors such as tissue type, sex, and habitat need to be considered to understand free-ranging ecology of generalist herbivores using stable isotopes. Availability of C3 agricultural crops high in percent nitrogen on a nearly annual basis in a C4-dominated landscape was likely driving differences in tissue δ(13)C and δ(15)N among subpopulations of free-ranging elk. An increase in tissue δ(15)N resulted from an increase in the consumption of higher δ(15)N in forage for sexes and subpopulations of a free-ranging ungulate in North America but δ(15)N should be further evaluated as an index of nutrition for subpopulations of generalist herbivores. PMID:24450704

  15. An activation antigen on a subpopulation of B lymphocytes identified by the monoclonal antibody CMRF-17.

    PubMed

    Peach, S F; Davidson, S E; McKenzie, J L; Nimmo, J C; Hart, D N

    1989-07-01

    The identification of membrane molecules expressed on subpopulations of B lymphocytes is of potential significance because these molecules may be candidates for regulating the activation, proliferation and differentiation of B cells. A new monoclonal antibody, CMRF-17, which reacts with a subpopulation of tonsil B lymphocytes has been produced. The antibody did not react with T lymphocytes in tonsil or peripheral blood nor most peripheral blood B lymphocytes but did label erythrocytes and some platelets. In tonsil, the germinal centre cells, cells in the interfollicular region and endothelial cells were positive, but mantle zone B cells were negative. Double labelling experiments showed that CMRF-17 reacted with activated tonsillar lymphocytes. The antigen recognized by CMRF-17 was heat stable, resistant to treatment with proteolytic enzymes and neuraminidase and was shown to be a carbohydrate determinant on one or more glycolipids. These characteristics of the antigen recognized by CMRF-17 and its pattern of reactivity distinguish this antibody from other monoclonal antibodies recognizing B-cell activation markers. It was notable that of the B-lymphoid malignancies tested to date, including those of probable follicular origin, few stained with CMRF-17. PMID:2474491

  16. Annexin A8 identifies a subpopulation of transiently quiescent c-kit positive luminal progenitor cells of the ductal mammary epithelium.

    PubMed

    Iglesias, Juan Manuel; Cairney, Claire J; Ferrier, Roderick K; McDonald, Laura; Soady, Kelly; Kendrick, Howard; Pringle, Marie-Anne; Morgan, Reginald O; Martin, Finian; Smalley, Matthew J; Blyth, Karen; Stein, Torsten

    2015-01-01

    We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8's association with this breast cancer subgroup we established ANXA8's cellular distribution in the mammary gland and ANXA8's effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (-ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G0/G1 arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8's association with their specific cells of origin. PMID:25803307

  17. Analysis of EpCAM positive cells isolated from sentinel lymph nodes of breast cancer patients identifies subpopulations of cells with distinct transcription profiles

    PubMed Central

    2011-01-01

    Introduction The presence of tumor cells in the axillary lymph nodes is the most important prognostic factor in early stage breast cancer. However, the optimal method for sentinel lymph node (SLN) examination is still sought and currently many different protocols are employed. To examine two approaches for tumor cell detection we performed, in sequence, immunomagnetic enrichment and RT-PCR analysis on SLN samples from early stage breast cancer patients. This allowed us to compare findings based on the expression of cell surface proteins with those based on detection of intracellular transcripts. Methods Enrichment of EpCAM and Mucin 1 expressing cells from fresh SLN samples was achieved using magnetic beads coated with the appropriate antibodies. All resulting cell fractions were analyzed by RT-PCR using four chosen breast epithelial markers (hMAM, AGR2, SBEM, TFF1). Gene expression was further analyzed using RT-PCR arrays and markers for epithelial to mesenchymal transition (EMT). Results Both EpCAM and Mucin 1 enriched for the epithelial-marker expressing cells. However, EpCAM-IMS identified epithelial cells in 71 SLNs, whereas only 35 samples were positive with RT-PCR targeting breast epithelial transcripts. Further analysis of EpCAM positive but RT-PCR negative cell fractions showed that they had increased expression of MMPs, repressors of E-cadherin, SPARC and vimentin, all transcripts associated with the process of epithelial to mesenchymal transition. Conclusions The EpCAM IMS-assay detected tumor cells with epithelial and mesenchymal-like characteristics, thus proving to be a more robust marker than pure epithelial derived biomarkers. This finding has clinical implications, as most methods for SLN analysis today rely on the detection of epithelial transcripts or proteins. PMID:21816090

  18. High mitochondrial mass identifies a sub-population of stem-like cancer cells that are chemo-resistant

    PubMed Central

    Farnie, Gillian; Sotgia, Federica; Lisanti, Michael P.

    2015-01-01

    Chemo-resistance is a clinical barrier to more effective anti-cancer therapy. In this context, cancer stem-like cells (CSCs) are thought to be chemo-resistant, resulting in tumor recurrence and distant metastasis. Our hypothesis is that chemo-resistance in CSCs is driven, in part, by enhanced mitochondrial function. Here, we used breast cell lines and metastatic breast cancer patient samples to begin to dissect the role of mitochondrial metabolism in conferring the CSC phenotype. More specifically, we employed fluorescent staining with MitoTracker (MT) to metabolically fractionate these cell lines into mito-high and mito-low sub-populations, by flow-cytometry. Interestingly, cells with high mitochondrial mass (mito-high) were specifically enriched in a number of known CSC markers, such as aldehyde dehydrogenase (ALDH) activity, and they were ESA+/CD24-/low and formed mammospheres with higher efficiency. Large cell size is another independent characteristic of the stem cell phenotype; here, we observed a >2-fold increase in mitochondrial mass in large cells (>12-μm), relative to the smaller cell population (4–8-μm). Moreover, the mito-high cell population showed a 2.4-fold enrichment in tumor-initiating cell activity, based on limiting dilution assays in murine xenografts. Importantly, primary human breast CSCs isolated from patients with metastatic breast cancer or a patient derived xenograft (PDX) also showed the co-enrichment of ALDH activity and mitochondrial mass. Most significantly, our investigations demonstrated that mito-high cells were resistant to paclitaxel, resulting in little or no DNA damage, as measured using the comet assay. In summary, increased mitochondrial mass in a sub-population of breast cancer cells confers a stem-like phenotype and chemo-resistance. As such, our current findings have important clinical implications for over-coming drug resistance, by therapeutically targeting the mito-high CSC population. PMID:26421710

  19. Identifying the cellular mechanisms of symbiont-induced epithelial morphogenesis in the squid-Vibrio association.

    PubMed

    Koropatnick, Tanya; Goodson, Michael S; Heath-Heckman, Elizabeth A C; McFall-Ngai, Margaret

    2014-02-01

    The symbiotic association between the Hawaiian bobtail squid Euprymna scolopes and the luminous marine bacterium Vibrio fischeri provides a unique opportunity to study epithelial morphogenesis. Shortly after hatching, the squid host harvests bacteria from the seawater using currents created by two elaborate fields of ciliated epithelia on the surface of the juvenile light organ. After light organ colonization, the symbiont population signals the gradual loss of the ciliated epithelia through apoptosis of the cells, which culminates in the complete regression of these tissues. Whereas aspects of this process have been studied at the morphological, biochemical, and molecular levels, no in-depth analysis of the cellular events has been reported. Here we describe the cellular structure of the epithelial field and present evidence that the symbiosis-induced regression occurs in two steps. Using confocal microscopic analyses, we observed an initial epithelial remodeling, which serves to disable the function of the harvesting apparatus, followed by a protracted regression involving actin rearrangements and epithelial cell extrusion. We identified a metal-dependent gelatinolytic activity in the symbiont-induced morphogenic epithelial fields, suggesting the involvement of Zn-dependent matrix metalloproteinase(s) (MMP) in light organ morphogenesis. These data show that the bacterial symbionts not only induce apoptosis of the field, but also change the form, function, and biochemistry of the cells as part of the morphogenic program. PMID:24648207

  20. ABCG2 Transporter Identifies a Population of Clonogenic Human Limbal Epithelial Cells

    PubMed Central

    de Paiva, Cintia S.; Chen, Zhuo; Corrales, Rosa M.; Pflugfelder, Stephen C.; Li, De-Quan

    2010-01-01

    ABCG2, a member of the ATP binding cassette (ABC) transporters, has been identified as a molecular determinant for bone marrow stem cells and proposed as a universal marker for stem cells. This study investigates ABCG2 expression and its potential as a marker that identifies human limbal epithelial stem cells. ABCG2 expression was evaluated by immunofluorescent and immunohistochemical staining, laser scanning confocal microscopy, flow cytometry, and semiquantitative reverse transcription–polymerase chain reaction. Cells selected from primary limbal epithelial cultures by flow cytometry with ABCG2 monoclonal antibody (mAb) or Hoechst 33342 dye staining were evaluated for their gene expression and colony-forming efficiency (CFE). ABCG2 protein was mainly located in the basal cells of limbal epithelia but not in the limbal suprabasal and corneal epithelia. ABCG2 staining was also observed in primary limbal epithelial cultures. Limbal epithelia express higher levels of ABCG2 and ΔNp63 mRNAs than corneal epithelia. Labeling with ABCG2 mAb yielded 2.5%–3.0% positive cells by flow cytometry. The ABCG2-positive cells exhibited greater CFE on a 3T3 fibroblast feeder layer than ABCG2-negative cells. A side population (SP) was detected by the Hoechst 33342 exclusion assay. SP cells displayed stronger expression of ABCG2 and ΔNp63 mRNA and greater CFE than the non-SP cells. In conclusion, these findings demonstrate that ABCG2 transporter was exclusively expressed by limbal basal cells and that the ABCG2-positive and SP cells possess enriched stem cell properties, suggesting for the first time that ABCG2 could serve as a marker to identify the putative limbal epithelial stem cells. PMID:15625123

  1. The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells.

    PubMed

    Ferrell, Patrick I; Xi, Jiafei; Ma, Chao; Adlakha, Mitali; Kaufman, Dan S

    2015-04-01

    Derivation of hematopoietic stem cells (HSCs) from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom(+) hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of the tdTom(+) population. Notably, RUNX1c/tdTom(+) cells represent only a limited subpopulation of the CD34(+) CD45(+) and CD34(+) CD43(+) cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom(+) cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom(+) population to CD34(+) CD45(+) umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs. PMID:25546363

  2. Identifying Inhibitors of Epithelial-Mesenchymal Transition by Connectivity-Map Based Systems Approach

    PubMed Central

    Reka, Ajaya Kumar; Kuick, Rork; Kurapati, Himabindu; Standiford, Theodore J.; Omenn, Gilbert S.; Keshamouni, Venkateshwar G.

    2011-01-01

    Background Acquisition of mesenchymal phenotype by epithelial cells by means of epithelial mesenchymal transition (EMT) is considered as an early event in the multi-step process of tumor metastasis. Therefore, inhibition of EMT might be a rational strategy to prevent metastasis. Methods Utilizing the global gene expression profile from a cell culture model of TGF-β-induced EMT, we identified potential EMT inhibitors. We used a publicly available database (www.broad.mit.edu/cmap) comprising gene expression profiles obtained from multiple different cell lines in response to various drugs to derive negative correlations to EMT gene expression profile using Connectivity Map (C-Map), a pattern matching tool. Results Experimental validation of the identified compounds showed rapamycin as a novel inhibitor of TGF-β signaling along with 17-AAG, a known modulator of TGF-β pathway. Both of these compounds completely blocked EMT and the associated migratory and invasive phenotype. The other identified compound, LY294002, demonstrated a selective inhibition of mesenchymal markers, cell migration and invasion, without affecting the loss of E-cadherin expression or Smad phosphorylation. Conclusions Collectively, our data reveals that rapamycin is a novel modulator of TGF-β signaling, and along with 17-AAG and LY294002, could be used as therapeutic agent for inhibiting EMT. Also, this analysis demonstrates the potential of a systems approach in identifying novel modulators of a complex biological process. PMID:21964532

  3. A systematic approach identifies FOXA1 as a key factor in the loss of epithelial traits during the epithelial-to-mesenchymal transition in lung cancer

    PubMed Central

    2013-01-01

    Background The epithelial-to-mesenchymal transition is an important mechanism in cancer metastasis. Although transcription factors including SNAIL, SLUG, and TWIST1 regulate the epithelial-to-mesenchymal transition, other unknown transcription factors could also be involved. Identification of the full complement of transcription factors is essential for a more complete understanding of gene regulation in this process. Chromatin immunoprecipitation-sequencing (ChIP-Seq) technologies have been used to detect genome-wide binding of transcription factors; here, we developed a systematic approach to integrate existing ChIP-Seq and transcriptome data. We scanned multiple transcription factors to investigate their functional impact on the epithelial-to-mesenchymal transition in the human A549 lung adenocarcinoma cell line. Results Among the transcription factors tested, impact scores identified the forkhead box protein A1 (FOXA1) as the most significant transcription factor in the epithelial-to-mesenchymal transition. FOXA1 physically associates with the promoters of its predicted target genes. Several critical epithelial-to-mesenchymal transition effectors involved in cellular adhesion and cellular communication were identified in the regulatory network of FOXA1, including FOXA2, FGA, FGB, FGG, and FGL1. The implication of FOXA1 in the epithelial-to-mesenchymal transition via its regulatory network indicates that FOXA1 may play an important role in the initiation of lung cancer metastasis. Conclusions We identified FOXA1 as a potentially important transcription factor and negative regulator in the initial stages of lung cancer metastasis. FOXA1 may modulate the epithelial-to-mesenchymal transition via its transcriptional regulatory network. Further, this study demonstrates how ChIP-Seq and expression data could be integrated to delineate the impact of transcription factors on a specific biological process. PMID:24093963

  4. An epithelial marker promoter induction screen identifies histone deacetylase inhibitors to restore epithelial differentiation and abolishes anchorage independence growth in cancers.

    PubMed

    Tang, H M; Kuay, K T; Koh, P F; Asad, M; Tan, T Z; Chung, V Y; Lee, S C; Thiery, J P; Huang, Ry-J

    2016-01-01

    Epithelial-mesenchymal transition (EMT), a crucial mechanism in development, mediates aggressiveness during carcinoma progression and therapeutic refractoriness. The reversibility of EMT makes it an attractive strategy in designing novel therapeutic approaches. Therefore, drug discovery pipelines for EMT reversal are in need to discover emerging classes of compounds. Here, we outline a pre-clinical drug screening platform for EMT reversal that consists of three phases of drug discovery and validation. From the Phase 1 epithelial marker promoter induction (EpI) screen on a library consisting of compounds being approved by Food and Drug Administration (FDA), Vorinostat (SAHA), a histone deacetylase inhibitor (HDACi), is identified to exert EMT reversal effects by restoring the expression of an epithelial marker, E-cadherin. An expanded screen on 41 HDACi further identifies 28 compounds, such as class I-specific HDACi Mocetinosat, Entinostat and CI994, to restore E-cadherin and ErbB3 expressions in ovarian, pancreatic and bladder carcinoma cells. Mocetinostat is the most potent HDACi to restore epithelial differentiation with the lowest concentration required for 50% induction of epithelial promoter activity (EpIC-50).The HDACi exerts paradoxical effects on EMT transcriptional factors such as SNAI and ZEB family and the effects are context-dependent in epithelial- and mesenchymal-like cells. In vitro functional studies further show that HDACi induced significant increase in anoikis and decrease in spheroid formation in ovarian and bladder carcinoma cells with mesenchymal features. This study demonstrates a robust drug screening pipeline for the discovery of compounds capable of restoring epithelial differentiation that lead to significant functional lethality. PMID:27551531

  5. α-Amanitin Restrains Cancer Relapse from Drug-Tolerant Cell Subpopulations via TAF15

    PubMed Central

    Kume, Kohei; Ikeda, Miyuki; Miura, Sawako; Ito, Kohei; Sato, Kei A.; Ohmori, Yukimi; Endo, Fumitaka; Katagiri, Hirokatsu; Ishida, Kaoru; Ito, Chie; Iwaya, Takeshi; Nishizuka, Satoshi S.

    2016-01-01

    Cancer relapse occurs with substantial frequency even after treatment with curative intent. Here we studied drug-tolerant colonies (DTCs), which are subpopulations of cancer cells that survive in the presence of drugs. Proteomic characterization of DTCs identified stemness- and epithelial-dominant subpopulations, but functional screening suggested that DTC formation was regulated at the transcriptional level independent from protein expression patterns. We consistently found that α-amanitin, an RNA polymerase II (RNAPII) inhibitor, effectively inhibited DTCs by suppressing TAF15 expression, which binds to RNA to modulate transcription and RNA processing. Sequential administration of α-amanitin and cisplatin extended overall survival in a cancer-relapse mouse model, namely peritonitis carcinomatosa. Therefore, post-treatment cancer relapse may occur through non-distinct subpopulations and may be effectively prevented by α-amanitin to disrupt transcriptional machinery, including TAF15. PMID:27181033

  6. α-Amanitin Restrains Cancer Relapse from Drug-Tolerant Cell Subpopulations via TAF15.

    PubMed

    Kume, Kohei; Ikeda, Miyuki; Miura, Sawako; Ito, Kohei; Sato, Kei A; Ohmori, Yukimi; Endo, Fumitaka; Katagiri, Hirokatsu; Ishida, Kaoru; Ito, Chie; Iwaya, Takeshi; Nishizuka, Satoshi S

    2016-01-01

    Cancer relapse occurs with substantial frequency even after treatment with curative intent. Here we studied drug-tolerant colonies (DTCs), which are subpopulations of cancer cells that survive in the presence of drugs. Proteomic characterization of DTCs identified stemness- and epithelial-dominant subpopulations, but functional screening suggested that DTC formation was regulated at the transcriptional level independent from protein expression patterns. We consistently found that α-amanitin, an RNA polymerase II (RNAPII) inhibitor, effectively inhibited DTCs by suppressing TAF15 expression, which binds to RNA to modulate transcription and RNA processing. Sequential administration of α-amanitin and cisplatin extended overall survival in a cancer-relapse mouse model, namely peritonitis carcinomatosa. Therefore, post-treatment cancer relapse may occur through non-distinct subpopulations and may be effectively prevented by α-amanitin to disrupt transcriptional machinery, including TAF15. PMID:27181033

  7. Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma.

    PubMed

    Kodama, Takahiro; Newberg, Justin Y; Kodama, Michiko; Rangel, Roberto; Yoshihara, Kosuke; Tien, Jean C; Parsons, Pamela H; Wu, Hao; Finegold, Milton J; Copeland, Neal G; Jenkins, Nancy A

    2016-06-14

    Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets. PMID:27247392

  8. An epithelial marker promoter induction screen identifies histone deacetylase inhibitors to restore epithelial differentiation and abolishes anchorage independence growth in cancers

    PubMed Central

    Tang, H M; Kuay, K T; Koh, P F; Asad, M; Tan, T Z; Chung, V Y; Lee, S C; Thiery, J P; Huang, RY-J

    2016-01-01

    Epithelial–mesenchymal transition (EMT), a crucial mechanism in development, mediates aggressiveness during carcinoma progression and therapeutic refractoriness. The reversibility of EMT makes it an attractive strategy in designing novel therapeutic approaches. Therefore, drug discovery pipelines for EMT reversal are in need to discover emerging classes of compounds. Here, we outline a pre-clinical drug screening platform for EMT reversal that consists of three phases of drug discovery and validation. From the Phase 1 epithelial marker promoter induction (EpI) screen on a library consisting of compounds being approved by Food and Drug Administration (FDA), Vorinostat (SAHA), a histone deacetylase inhibitor (HDACi), is identified to exert EMT reversal effects by restoring the expression of an epithelial marker, E-cadherin. An expanded screen on 41 HDACi further identifies 28 compounds, such as class I-specific HDACi Mocetinosat, Entinostat and CI994, to restore E-cadherin and ErbB3 expressions in ovarian, pancreatic and bladder carcinoma cells. Mocetinostat is the most potent HDACi to restore epithelial differentiation with the lowest concentration required for 50% induction of epithelial promoter activity (EpIC-50).The HDACi exerts paradoxical effects on EMT transcriptional factors such as SNAI and ZEB family and the effects are context-dependent in epithelial- and mesenchymal-like cells. In vitro functional studies further show that HDACi induced significant increase in anoikis and decrease in spheroid formation in ovarian and bladder carcinoma cells with mesenchymal features. This study demonstrates a robust drug screening pipeline for the discovery of compounds capable of restoring epithelial differentiation that lead to significant functional lethality. PMID:27551531

  9. Integrative genomic analysis identifies epigenetic marks that mediate genetic risk for epithelial ovarian cancer

    PubMed Central

    2014-01-01

    Background Both genetic and epigenetic factors influence the development and progression of epithelial ovarian cancer (EOC). However, there is an incomplete understanding of the interrelationship between these factors and the extent to which they interact to impact disease risk. In the present study, we aimed to gain insight into this relationship by identifying DNA methylation marks that are candidate mediators of ovarian cancer genetic risk. Methods We used 214 cases and 214 age-matched controls from the Mayo Clinic Ovarian Cancer Study. Pretreatment, blood-derived DNA was profiled for genome-wide methylation (Illumina Infinium HumanMethylation27 BeadArray) and single nucleotide polymorphisms (SNPs, Illumina Infinium HD Human610-Quad BeadArray). The Causal Inference Test (CIT) was implemented to distinguish CpG sites that mediate genetic risk, from those that are consequential or independently acted on by genotype. Results Controlling for the estimated distribution of immune cells and other key covariates, our initial epigenome-wide association analysis revealed 1,993 significantly differentially methylated CpGs that between cases and controls (FDR, q < 0.05). The relationship between methylation and case-control status for these 1,993 CpGs was found to be highly consistent with the results of previously published, independent study that consisted of peripheral blood DNA methylation signatures in 131 pretreatment cases and 274 controls. Implementation of the CIT test revealed 17 CpG/SNP pairs, comprising 13 unique CpGs and 17 unique SNPs, which represent potential methylation-mediated relationships between genotype and EOC risk. Of these 13 CpGs, several are associated with immune related genes and genes that have been previously shown to exhibit altered expression in the context of cancer. Conclusions These findings provide additional insight into EOC etiology and may serve as novel biomarkers for EOC susceptibility. PMID:24479488

  10. Monocyte Subpopulations in Angiogenesis

    PubMed Central

    Dalton, Heather J.; Armaiz-Pena, Guillermo; Gonzalez-Villasana, Vianey; Lopez-Berestein, Gabriel; Bar-Eli, Menashe; Sood, Anil K.

    2014-01-01

    Growing understanding of the role of the tumor microenvironment in angiogenesis has brought monocyte-derived cells into focus. Monocyte subpopulations are an increasingly attractive therapeutic target in many pathologic states, including cancer. Before monocyte-directed therapies can be fully harnessed for clinical use, understanding of monocyte-driven angiogenesis in tissue development and homeostasis, as well as malignancy, is required. Here, we provide an overview of the mechanisms by which monocytic subpopulations contribute to angiogenesis in tissue and tumor development, highlight gaps in our existing knowledge, and discuss opportunities to exploit these cells for clinical benefit. PMID:24556724

  11. Use of Plasma Metabolomics to Identify Diagnostic Biomarkers for Early Stage Epithelial Ovarian Cancer

    PubMed Central

    Fan, Lijun; Yin, Mingzhu; Ke, Chaofu; Ge, Tingting; Zhang, Guangming; Zhang, Wang; Zhou, Xiaohua; Lou, Ge; Li, Kang

    2016-01-01

    The early detection of ovarian carcinoma is difficult due to the absence of recognizable physical symptoms and a lack of sensitive screening methods. The currently available biomarkers (such as CA125 and HE4) are insufficiently reliable to distinguish early stage (I/II) epithelial ovarian cancer (EOC) patients from normal individuals because they possess a relatively poor sensitivity and specificity. To evaluate the application of metabolomics to biomarker discovery in the early stages of epithelial ovarian cancer (EOC), plasma samples from 21 early stage EOC patients and 31 healthy controls were analyzed with ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/Q-Tof/MS) in conjunction with multivariate statistical analysis. Eighteen metabolites, including lysophospholipids, 2-piperidone and MG (18:2), were found to be disturbed in early stage EOC with satisfactory diagnostic accuracy (AUC=0.920). These biomarkers were specifically validated in the EOC nude mouse model, and five of the biomarkers (lysophospholipids, adrenoyl ethanolamide et al.) were highly suspected of being associated with EOC because they were differentially expressed with the same tendency in the EOC nude mice versus normal controls. In conclusion, the selected metabolic biomarkers have considerable utility and significant potential for diagnosing early ovarian cancer and investigating its underlying mechanisms. PMID:27390602

  12. Alcohol Consumption in Demographic Subpopulations

    PubMed Central

    Delker, Erin; Brown, Qiana; Hasin, Deborah S.

    2016-01-01

    Alcohol consumption is common across subpopulations in the United States. However, the health burden associated with alcohol consumption varies across groups, including those defined by demographic characteristics such as age, race/ethnicity, and gender. Large national surveys, such as the National Epidemiologic Survey on Alcohol and Related Conditions and the National Survey on Drug Use and Health, found that young adults ages 18–25 were at particularly high risk of alcohol use disorder and unintentional injury caused by drinking. These surveys furthermore identified significant variability in alcohol consumption and its consequences among racial/ethnic groups. White respondents reported the highest prevalence of current alcohol consumption, whereas alcohol abuse and dependence were most prevalent among Native Americans. Native Americans and Blacks also were most vulnerable to alcohol-related health consequences. Even within ethnic groups, there was variability between and among different subpopulations. With respect to gender, men reported more alcohol consumption and binge drinking than women, especially in older cohorts. Men also were at greater risk of alcohol abuse and dependence, liver cirrhosis, homicide after alcohol consumption, and drinking and driving. Systematic identification and measurement of the variability across demographics will guide prevention and intervention efforts, as well as future research. PMID:27159807

  13. The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells

    PubMed Central

    Ferrell, Patrick I; Xi, Jiafei; Ma, Chao; Adlakha, Mitali; Kaufman, Dan S.

    2016-01-01

    Derivation of hematopoietic stem cells from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom+ hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of tdTom+ population. Notably, RUNX1c/tdTom+ cells represent only a limited subpopuation of CD34+CD45+ and CD34+CD43+ cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom+ cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom+ population to CD34+CD45+ umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs. PMID:25546363

  14. Phenotypic chemical screening using a zebrafish neural crest EMT reporter identifies retinoic acid as an inhibitor of epithelial morphogenesis.

    PubMed

    Jimenez, Laura; Wang, Jindong; Morrison, Monique A; Whatcott, Clifford; Soh, Katherine K; Warner, Steven; Bearss, David; Jette, Cicely A; Stewart, Rodney A

    2016-04-01

    The epithelial-to-mesenchymal transition (EMT) is a highly conserved morphogenetic program essential for embryogenesis, regeneration and cancer metastasis. In cancer cells, EMT also triggers cellular reprogramming and chemoresistance, which underlie disease relapse and decreased survival. Hence, identifying compounds that block EMT is essential to prevent or eradicate disseminated tumor cells. Here, we establish a whole-animal-based EMT reporter in zebrafish for rapid drug screening, calledTg(snai1b:GFP), which labels epithelial cells undergoing EMT to producesox10-positive neural crest (NC) cells. Time-lapse and lineage analysis ofTg(snai1b:GFP)embryos reveal that cranial NC cells delaminate from two regions: an early population delaminates adjacent to the neural plate, whereas a later population delaminates from within the dorsal neural tube. TreatingTg(snai1b:GFP)embryos with candidate small-molecule EMT-inhibiting compounds identified TP-0903, a multi-kinase inhibitor that blocked cranial NC cell delamination in both the lateral and medial populations. RNA sequencing (RNA-Seq) analysis and chemical rescue experiments show that TP-0903 acts through stimulating retinoic acid (RA) biosynthesis and RA-dependent transcription. These studies identify TP-0903 as a new therapeutic for activating RAin vivoand raise the possibility that RA-dependent inhibition of EMT contributes to its prior success in eliminating disseminated cancer cells. PMID:26794130

  15. Phenotypic chemical screening using a zebrafish neural crest EMT reporter identifies retinoic acid as an inhibitor of epithelial morphogenesis

    PubMed Central

    Jimenez, Laura; Wang, Jindong; Morrison, Monique A.; Whatcott, Clifford; Soh, Katherine K.; Warner, Steven; Bearss, David; Jette, Cicely A.; Stewart, Rodney A.

    2016-01-01

    ABSTRACT The epithelial-to-mesenchymal transition (EMT) is a highly conserved morphogenetic program essential for embryogenesis, regeneration and cancer metastasis. In cancer cells, EMT also triggers cellular reprogramming and chemoresistance, which underlie disease relapse and decreased survival. Hence, identifying compounds that block EMT is essential to prevent or eradicate disseminated tumor cells. Here, we establish a whole-animal-based EMT reporter in zebrafish for rapid drug screening, called Tg(snai1b:GFP), which labels epithelial cells undergoing EMT to produce sox10-positive neural crest (NC) cells. Time-lapse and lineage analysis of Tg(snai1b:GFP) embryos reveal that cranial NC cells delaminate from two regions: an early population delaminates adjacent to the neural plate, whereas a later population delaminates from within the dorsal neural tube. Treating Tg(snai1b:GFP) embryos with candidate small-molecule EMT-inhibiting compounds identified TP-0903, a multi-kinase inhibitor that blocked cranial NC cell delamination in both the lateral and medial populations. RNA sequencing (RNA-Seq) analysis and chemical rescue experiments show that TP-0903 acts through stimulating retinoic acid (RA) biosynthesis and RA-dependent transcription. These studies identify TP-0903 as a new therapeutic for activating RA in vivo and raise the possibility that RA-dependent inhibition of EMT contributes to its prior success in eliminating disseminated cancer cells. PMID:26794130

  16. Can Villin be Used to Identify Malignant and Undifferentiated Normal Digestive Epithelial Cells?

    NASA Astrophysics Data System (ADS)

    Robine, S.; Huet, C.; Moll, R.; Sahuquillo-Merino, C.; Coudrier, E.; Zweibaum, A.; Louvard, D.

    1985-12-01

    We have investigated the presence of villin (a Ca2+-regulated actin binding protein) in various tissues (normal or malignant) and in established cell lines by using sensitive immunochemical techniques on cell extracts and immunofluorescence analysis on frozen sections. Our results show that villin is a marker that can be used to distinguish normal differentiated epithelial cells from the simple epithelia lining the gastrointestinal tract and renal tubules. Villin is found in the absorptive cells of the small and large intestines, in the duct cells of pancreas and biliary system, and in the cells of kidney proximal tubules. Furthermore, undifferentiated normal and tumoral cells of intestinal origin in vivo and in cell culture express villin. Therefore, expression of villin is seen in cells that do not necessarily display the morphological features characteristic of their terminally differentiated state, such as the microvilli-lined brush border. We suggest the possible clinical implications of using villin as a marker in the diagnosis of metastatic adenocarcinomas.

  17. An RNA interference lethality screen of the human druggable genome to identify molecular vulnerabilities in epithelial ovarian cancer.

    PubMed

    Sethi, Geetika; Pathak, Harsh B; Zhang, Hong; Zhou, Yan; Einarson, Margret B; Vathipadiekal, Vinod; Gunewardena, Sumedha; Birrer, Michael J; Godwin, Andrew K

    2012-01-01

    Targeted therapies have been used to combat many tumor types; however, few have effectively improved the overall survival in women with epithelial ovarian cancer, begging for a better understanding of this deadly disease and identification of essential drivers of tumorigenesis that can be targeted effectively. Therefore, we used a loss-of-function screening approach to help identify molecular vulnerabilities that may represent key points of therapeutic intervention. We employed an unbiased high-throughput lethality screen using a 24,088 siRNA library targeting over 6,000 druggable genes and studied their effects on growth and/or survival of epithelial ovarian cancer (EOC) cell lines. The top 300 "hits" affecting the viability of A1847 cells were rescreened across additional EOC cell lines and non-tumorigenic, human immortalized ovarian epithelial cell lines. Fifty-three gene candidates were found to exhibit effects in all tumorigenic cell lines tested. Extensive validation of these hits refined the list to four high quality candidates (HSPA5, NDC80, NUF2, and PTN). Mechanistic studies show that silencing of three genes leads to increased apoptosis, while HSPA5 silencing appears to alter cell growth through G1 cell cycle arrest. Furthermore, two independent gene expression studies show that NDC80, NUF2 and PTN were significantly aberrantly overexpressed in serous adenocarcinomas. Overall, our functional genomics results integrated with the genomics data provide an important unbiased avenue towards the identification of prospective therapeutic targets for drug discovery, which is an urgent and unmet clinical need for ovarian cancer. PMID:23056589

  18. Microcell-Mediated Chromosome Transfer Identifies EPB41L3 as a Functional Suppressor of Epithelial Ovarian Cancers12

    PubMed Central

    Dafou, Dimitra; Grun, Barbara; Sinclair, John; Lawrenson, Kate; Benjamin, Elizabeth C; Hogdall, Estrid; Kruger-Kjaer, Susanne; Christensen, Lise; Sowter, Heidi M; Al-Attar, Ahmed; Edmondson, Richard; Darby, Stephen; Berchuck, Andrew; Laird, Peter W; Pearce, C Leigh; Ramus, Susan J; Jacobs, Ian J; Gayther, Simon A

    2010-01-01

    We used a functional complementation approach to identify tumor-suppressor genes and putative therapeutic targets for ovarian cancer. Microcell-mediated transfer of chromosome 18 in the ovarian cancer cell line TOV21G induced in vitro and in vivo neoplastic suppression. Gene expression microarray profiling in TOV21G+18 hybrids identified 14 candidate genes on chromosome 18 that were significantly overexpressed and therefore associated with neoplastic suppression. Further analysis of messenger RNA and protein expression for these genes in additional ovarian cancer cell lines indicated that EPB41L3 (erythrocyte membrane protein band 4.1-like 3, alternative names DAL-1 and 4.1B) was a candidate ovarian cancer-suppressor gene. Immunoblot analysis showed that EPB41L3 was activated in TOV21G+18 hybrids, expressed in normal ovarian epithelial cell lines, but was absent in 15 (78%) of 19 ovarian cancer cell lines. Using immunohistochemistry, 66% of 794 invasive ovarian tumors showed no EPB41L3 expression compared with only 24% of benign ovarian tumors and 0% of normal ovarian epithelial tissues. EPB41L3 was extensively methylated in ovarian cancer cell lines and primary ovarian tumors compared with normal tissues (P = .00004), suggesting this may be the mechanism of gene inactivation in ovarian cancers. Constitutive reexpression of EPB41L3 in a three-dimensional multicellular spheroid model of ovarian cancer caused significant growth suppression and induced apoptosis. Transmission and scanning electron microscopy demonstrated many similarities between EPB41L3-expressing cells and chromosome 18 donor-recipient hybrids, suggesting that EPB41L3 is the gene responsible for neoplastic suppression after chromosome 18 transfer. Finally, an inducible model of EPB41L3 expression in three-dimensional spheroids confirmed that reexpression of EPB41L3 induces extensive apoptotic cell death in ovarian cancers. PMID:20651987

  19. Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. III. Growth conditions of human thymic epithelial cells and immunomodulatory activities in their culture supernatant.

    PubMed Central

    Schreiber, L; Eshel, I; Meilin, A; Sharabi, Y; Shoham, J

    1991-01-01

    We report here on a new approach to the cultivation of human thymic epithelial (HTE) cells, which apparently allows more faithful preservation of cell function. This approach, previously developed by us for mouse thymic epithelial (MTE) cells, is based on the use of culture plates coated with extracellular matrix (ECM), and on the use of serum-free, growth factor-supplemented medium. The nutritional requirements of HTE and MTE are somewhat different. Although both are critically dependent on ECM and insulin, they differ in their dependency on other growth factors: selenium and transferrin are much more important for HTE cells, whereas epidermal growth factor and hydrocortisone play a more essential role in MTE cultures. The epithelial nature of the cultured cells is indicated by positive staining with anti-keratin antibodies and by the presence of desmosomes and tonofilaments. The ultrastructural appearance of the cells further suggests high metabolic and secretory activities, not usually found in corresponding cell lines. The culture supernatant (CS) of HTE cells exhibited a strong enhancing effect on thymocyte response to Con A stimulation, as measured by cell proliferation and lymphokine production. The effect was observed on both human and mouse thymocytes, but was much stronger in the homologous combination. Thymic factors tested in parallel did not have such a differential effect. The dose-effect relationships were in the form of a bell-shaped curve, with fivefold enhancement of response at the peak and a measurable effect even with 1:1000 dilution, when human thymocytes were used. The responding thymocytes were those which do not bind peanut agglutinin and are resistant to hydrocortisone. The culture system described here may have advantages for the in vitro study of thymic stromal cell function. Images Figure 1 Figure 3 Figure 4 PMID:1783421

  20. Characterization of circulating tumor cell aggregates identified in patients with epithelial tumors

    NASA Astrophysics Data System (ADS)

    Cho, Edward H.; Wendel, Marco; Luttgen, Madelyn; Yoshioka, Craig; Marrinucci, Dena; Lazar, Daniel; Schram, Ethan; Nieva, Jorge; Bazhenova, Lyudmila; Morgan, Alison; Ko, Andrew H.; Korn, W. Michael; Kolatkar, Anand; Bethel, Kelly; Kuhn, Peter

    2012-02-01

    Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis and venous thromboembolism (VTE), two major causes of mortality in cancer patients. Thus far, existing CTC detection technologies have been unable to reproducibly detect CTC aggregates in order to address what contribution CTC aggregates may make to metastasis or VTE. We report here an enrichment-free immunofluorescence detection method that can reproducibly detect and enumerate homotypic CTC aggregates in patient samples. We identified CTC aggregates in 43% of 86 patient samples. The fraction of CTC aggregation was investigated in blood draws from 24 breast, 14 non-small cell lung, 18 pancreatic, 15 prostate stage IV cancer patients and 15 normal blood donors. Both single CTCs and CTC aggregates were measured to determine whether differences exist in the physical characteristics of these two populations. Cells contained in CTC aggregates had less area and length, on average, than single CTCs. Nuclear to cytoplasmic ratios between single CTCs and CTC aggregates were similar. This detection method may assist future studies in determining which population of cells is more physically likely to contribute to metastasis and VTE.

  1. Malnutrition Identified by the Nutritional Risk Index and Poor Prognosis in Advanced Epithelial Ovarian Carcinoma.

    PubMed

    Yim, Ga Won; Eoh, Kyung Jin; Kim, Sang Wun; Nam, Eun Ji; Kim, Young Tae

    2016-07-01

    Ovarian cancer is a chronic disease with a risk of malnutrition. Nutritional Risk Index (NRI) has been reported as a simple and accurate tool to assess the nutritional status. We sought to explore the prevalence of malnutrition and its association with survival in ovarian cancer. A retrospective study was conducted in 213 advanced ovarian cancer patients. NRI was calculated before and at the end of treatment using patients' body weight and serum albumin level. Overall survival (OS) and progression-free survival (PFS) were estimated by Kaplan-Meier method, and associations were assessed using a Cox proportional hazards analysis adjusted for known prognostic variables. Moderate to severely malnourished patients had lower 5-yr OS (45.3%) compared to normal to mild group (64.0%), respectively (P = 0.024). Adjusted for covariates, the relative risk of death was 5.8 times higher in moderate/severely malnourished group identified at the last course of chemotherapy (HR = 5.896, 95% CI = 2.723-12.764, P < 0.001). Similarly, this cohort had shorter PFS compared with normal to mild risk group (median 15 vs. 28 months, P = 0.011). Malnutrition is prevalent among ovarian cancer patients and is found to be a significant predictor for mortality. PMID:27044606

  2. Osteoarthritis subpopulations and implications for clinical trial design

    PubMed Central

    2011-01-01

    Treatment guidelines for osteoarthritis have stressed the need for research on clinical predictors of response to different treatments. However, identifying such clinical predictors of response is less easy than it seems, and there is not a given classification of osteoarthritis subpopulations. This review article highlights the key methodical issues when analyzing and designing clinical studies to detect important subgroups with respect to treatment effect. In addition, we discuss the main osteoarthritis subpopulations and give examples of how specific treatment effects in these subpopulations have been assessed. PMID:21470393

  3. Functional genomics identifies five distinct molecular subtypes with clinical relevance and pathways for growth control in epithelial ovarian cancer

    PubMed Central

    Tan, Tuan Zea; Miow, Qing Hao; Huang, Ruby Yun-Ju; Wong, Meng Kang; Ye, Jieru; Lau, Jieying Amelia; Wu, Meng Chu; Bin Abdul Hadi, Luqman Hakim; Soong, Richie; Choolani, Mahesh; Davidson, Ben; Nesland, Jahn M; Wang, Ling-Zhi; Matsumura, Noriomi; Mandai, Masaki; Konishi, Ikuo; Goh, Boon-Cher; Chang, Jeffrey T; Thiery, Jean Paul; Mori, Seiichi

    2013-01-01

    Epithelial ovarian cancer (EOC) is hallmarked by a high degree of heterogeneity. To address this heterogeneity, a classification scheme was developed based on gene expression patterns of 1538 tumours. Five, biologically distinct subgroups — Epi-A, Epi-B, Mes, Stem-A and Stem-B — exhibited significantly distinct clinicopathological characteristics, deregulated pathways and patient prognoses, and were validated using independent datasets. To identify subtype-specific molecular targets, ovarian cancer cell lines representing these molecular subtypes were screened against a genome-wide shRNA library. Focusing on the poor-prognosis Stem-A subtype, we found that two genes involved in tubulin processing, TUBGCP4 and NAT10, were essential for cell growth, an observation supported by a pathway analysis that also predicted involvement of microtubule-related processes. Furthermore, we observed that Stem-A cell lines were indeed more sensitive to inhibitors of tubulin polymerization, vincristine and vinorelbine, than the other subtypes. This subtyping offers new insights into the development of novel diagnostic and personalized treatment for EOC patients. PMID:23666744

  4. Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

    PubMed Central

    Celià-Terrassa, Toni; Meca-Cortés, Óscar; Mateo, Francesca; Martínez de Paz, Alexia; Rubio, Nuria; Arnal-Estapé, Anna; Ell, Brian J.; Bermudo, Raquel; Díaz, Alba; Guerra-Rebollo, Marta; Lozano, Juan José; Estarás, Conchi; Ulloa, Catalina; ρlvarez-Simón, Daniel; Milà, Jordi; Vilella, Ramón; Paciucci, Rosanna; Martínez-Balbás, Marian; García de Herreros, Antonio; Gomis, Roger R.; Kang, Yibin; Blanco, Jerónimo; Fernández, Pedro L.; Thomson, Timothy M.

    2012-01-01

    Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs. PMID:22505459

  5. Chemodynamic subpopulations of the Carina dwarf galaxy

    NASA Astrophysics Data System (ADS)

    Kordopatis, G.; Amorisco, N. C.; Evans, N. W.; Gilmore, G.; Koposov, S. E.

    2016-04-01

    We study the chemodynamical properties of the Carina dwarf spheroidal by combining an intermediate spectroscopic resolution data set of more than 900 red giant and red clump stars, with high-precision photometry to derive the atmospheric parameters, metallicities and age estimates for our targets. Within the red giant branch population, we find evidence for the presence of three distinct stellar subpopulations with different metallicities, spatial distributions, kinematics and ages. As in the Fornax and Sculptor dwarf spheroidals, the subpopulation with the lowest average metallicity is more extended and kinematically hotter than all other populations. However, we identify an inversion in the parallel ordering of metallicity, kinematics and characteristic length-scale in the two most metal-rich subpopulations, which therefore do not contribute to a global negative chemical gradient. Contrary to common trends in the chemical properties with radius, the metal richest population is more extended and mildly kinematically hotter than the main component of intermediate metallicity. More investigations are required to ascertain the nature of this inversion, but we comment on the mechanisms that might have caused it.

  6. A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells.

    PubMed

    Trevino, Victor; Cassese, Alberto; Nagy, Zsuzsanna; Zhuang, Xiaodong; Herbert, John; Antzack, Philipp; Clarke, Kim; Davies, Nicholas; Rahman, Ayesha; Campbell, Moray J; Guindani, Michele; Bicknell, Roy; Vannucci, Marina; Falciani, Francesco

    2016-04-01

    The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication networks

  7. A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells

    PubMed Central

    Trevino, Victor; Cassese, Alberto; Nagy, Zsuzsanna; Zhuang, Xiaodong; Herbert, John; Antzack, Philipp; Clarke, Kim; Davies, Nicholas; Rahman, Ayesha; Campbell, Moray J.; Bicknell, Roy; Vannucci, Marina; Falciani, Francesco

    2016-01-01

    Abstract The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication

  8. Live Imaging and Gene Expression Analysis in Zebrafish Identifies a Link between Neutrophils and Epithelial to Mesenchymal Transition

    PubMed Central

    Freisinger, Christina M.; Huttenlocher, Anna

    2014-01-01

    Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRasV12. Live imaging revealed that expression of HRasV12 in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRasV12 using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRasV12 transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRasV12 in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells. PMID:25372289

  9. Next-Generation Sequencing Identifies TGF-β1-Associated Gene Expression Profiles in Renal Epithelial Cells Reiterated in Human Diabetic Nephropathy

    PubMed Central

    Brennan, Eoin P.; Morine, Melissa J.; Walsh, David W.; Roxburgh, Sarah A.; Lindenmeyer, Maja T.; Brazil, Derek P.; Gaora, Peadar Ó.; Roche, Helen M.; Sadlier, Denise M.; Cohen, Clemens D.; Godson, Catherine; Martin, Finian

    2012-01-01

    Transforming growth factor-beta (TGF-β1) is implicated in the onset and progression of renal fibrosis and diabetic nephropathy (DN), leading to a loss of epithelial characteristics of tubular cells. The transcriptional profile of renal tubular epithelial cells stimulated with TGF-β1 was assessed using RNA-Seq, with 2027 differentially expressed genes identified. Promoter analysis of transcription factor binding sites in the TGF-β1 responsive gene set predicted activation of multiple transcriptional networks, including NFκB. Comparison of RNA-Seq with microarray data from identical experimental conditions identified low abundance transcripts exclusive to RNA-Seq data. We compared these findings to human disease by analyzing transcriptomic data from renal biopsies of patients with DN versus control groups, identifying a shared subset of 179 regulated genes. ARK5, encoding an AMP-related kinase, and TGFBI - encoding transforming growth factor, beta-induced protein were induced by TGF-β1 and also upregulated in human DN. Suppression of ARK5 attenuated fibrotic responses of renal epithelia to TGF-β1 exposure; and silencing of TGFBI induced expression of the epithelial cell marker – E-cadherin. We identified low abundance transcripts in sequence data and validated expression levels of several transcripts (ANKRD56, ENTPD8) in tubular enriched kidney biopsies of DN patients versus living donors. In conclusion, we have defined a TGF-β1-driven pro-fibrotic signal in renal epithelial cells that is also evident in the DN renal transcriptome. PMID:22266139

  10. Lipidome and Transcriptome Profiling of Pneumolysin Intoxication Identifies Networks Involved in Statin-Conferred Protection of Airway Epithelial Cells

    PubMed Central

    Statt, Sarah; Ruan, Jhen-Wei; Huang, Chih-Ting; Wu, Reen; Kao, Cheng-Yuan

    2015-01-01

    Pneumonia remains one of the leading causes of death in both adults and children worldwide. Despite the adoption of a wide variety of therapeutics, the mortality from community-acquired pneumonia has remained relatively constant. Although viral and fungal acute airway infections can result in pneumonia, bacteria are the most common cause of community-acquired pneumonia, with Streptococcus pneumoniae isolated in nearly 50% of cases. Pneumolysin is a cholesterol-dependent cytolysin or pore-forming toxin produced by Streptococcus pneumonia and has been shown to play a critical role in bacterial pathogenesis. Airway epithelium is the initial site of many bacterial contacts and its barrier and mucosal immunity functions are central to infectious lung diseases. In our studies, we have shown that the prior exposure to statins confers significant resistance of airway epithelial cells to the cytotoxicity of pneumolysin. We decided to take this study one step further, assessing changes in both the transcriptome and lipidome of human airway epithelial cells exposed to toxin, statin or both. Our current work provides the first global view in human airway epithelial cells of both the transcriptome and the lipid interactions that result in cellular protection from pneumolysin. PMID:26023727

  11. Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties

    PubMed Central

    Gudjonsson, Thorarinn; Villadsen, René; Nielsen, Helga Lind; Rønnov-Jessen, Lone; Bissell, Mina J.; Petersen, Ole William

    2002-01-01

    The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA+) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC−/ESA+). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA+ epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC−/ESA+ epithelial cell line was able to generate itself as well as MUC+/ESA+ epithelial cells and Thy-1+/α-smooth muscle actin+ (ASMA+) myoepithelial cells. The MUC−/ESA+ epithelial cell line further differed from the MUC+/ESA+ epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA+ epithelial cell line formed acinus-like spheres. In contrast, the MUC−/ESA+ epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by

  12. Monoclonal antibodies to equine CD23 identify the low-affinity receptor for IgE on subpopulations of IgM+ and IgG1+ B-cells in horses.

    PubMed

    Wagner, Bettina; Hillegas, Julia M; Babasyan, Susanna

    2012-04-15

    CD23, also called FcεRII, is the low-affinity receptor for IgE and has first been described as a major receptor regulating IgE responses. In addition, CD23 also binds to CD21, integrins and MHC class II molecules and thus has a much wider functional role in immune regulation ranging from involvement in antigen-presentation to multiple cytokine-like functions of soluble CD23. The role of CD23 during immune responses of the horse is less well understood. Here, we expressed equine CD23 in mammalian cells using a novel IL-4 expression system. Expression resulted in high yield of recombinant IL-4/CD23 fusion protein which was purified and used for the generation of monoclonal antibodies (mAbs) to equine CD23. Seven anti-CD23 mAbs were further characterized. The expression of the low-affinity IgE receptor on equine peripheral blood mononuclear cells was analyzed by flow cytometric analysis. Cell surface staining showed that CD23 is mainly expressed by a subpopulation of equine B-cells. Only a very few equine T-cells or monocytes expressed CD23. CD23(+) B-cells were either IgM(+) or IgG1(+) cells. All CD23(+) cells were also positive for cell surface IgE staining suggesting in vivo IgE binding by the receptor. Two of the CD23 mAbs detected either the complete extracellular region of CD23 or a 22kDa cleavage product of CD23 by Western blotting. The new anti-CD23 mAbs provide valuable reagents to further analyze the roles of CD23 during immune responses of the horse in health and disease. PMID:22405681

  13. Housekeeping Gene Sequencing and Multilocus Variable-Number Tandem-Repeat Analysis To Identify Subpopulations within Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. tomato That Correlate with Host Specificity

    PubMed Central

    Gironde, S.

    2012-01-01

    Pseudomonas syringae pv. maculicola causes bacterial spot on Brassicaceae worldwide, and for the last 10 years severe outbreaks have been reported in the Loire Valley, France. P. syringae pv. maculicola resembles P. syringae pv. tomato in that it is also pathogenic for tomato and causes the same types of symptoms. We used a collection of 106 strains of P. syringae to characterize the relationships between P. syringae pv. maculicola and related pathovars, paying special attention to P. syringae pv. tomato. Phylogenetic analysis of gyrB and rpoD gene sequences showed that P. syringae pv. maculicola, which causes diseases in Brassicaceae, forms six genetic lineages within genomospecies 3 of P. syringae strains as defined by L. Gardan et al. (Int. J. Syst. Bacteriol. 49[Pt 2]:469–478, 1999), whereas P. syringae pv. tomato forms two distinct genetic lineages. A multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) conducted with eight minisatellite loci confirmed the genetic structure obtained with rpoD and gyrB sequence analyses. These results provide promising tools for fine-scale epidemiological studies on diseases caused by P. syringae pv. maculicola and P. syringae pv. tomato. The two pathovars had distinct host ranges; only P. syringae pv. maculicola strains were pathogenic for Brassicaceae. A subpopulation of P. syringae pv. maculicola strains that are pathogenic for Pto-expressing tomato plants were shown to lack avrPto1 and avrPtoB or to contain a disrupted avrPtoB homolog. Taking phylogenetic and pathological features into account, our data suggest that the DC3000 strain belongs to P. syringae pv. maculicola. This study shows that P. syringae pv. maculicola and P. syringae pv. tomato appear multiclonal, as they did not diverge from a single common ancestral group within the ancestral P. syringae genomospecies 3, and suggests that pathovar specificity within P. syringae may be due to independent genetic events. PMID:22389364

  14. Demographic and traditional knowledge perspectives on the current status of Canadian polar bear subpopulations.

    PubMed

    York, Jordan; Dowsley, Martha; Cornwell, Adam; Kuc, Miroslaw; Taylor, Mitchell

    2016-05-01

    Subpopulation growth rates and the probability of decline at current harvest levels were determined for 13 subpopulations of polar bears (Ursus maritimus) that are within or shared with Canada based on mark-recapture estimates of population numbers and vital rates, and harvest statistics using population viability analyses (PVA). Aboriginal traditional ecological knowledge (TEK) on subpopulation trend agreed with the seven stable/increasing results and one of the declining results, but disagreed with PVA status of five other declining subpopulations. The decline in the Baffin Bay subpopulation appeared to be due to over-reporting of harvested numbers from outside Canada. The remaining four disputed subpopulations (Southern Beaufort Sea, Northern Beaufort Sea, Southern Hudson Bay, and Western Hudson Bay) were all incompletely mark-recapture (M-R) sampled, which may have biased their survival and subpopulation estimates. Three of the four incompletely sampled subpopulations were PVA identified as nonviable (i.e., declining even with zero harvest mortality). TEK disagreement was nonrandom with respect to M-R sampling protocols. Cluster analysis also grouped subpopulations with ambiguous demographic and harvest rate estimates separately from those with apparently reliable demographic estimates based on PVA probability of decline and unharvested subpopulation growth rate criteria. We suggest that the correspondence between TEK and scientific results can be used to improve the reliability of information on natural systems and thus improve resource management. Considering both TEK and scientific information, we suggest that the current status of Canadian polar bear subpopulations in 2013 was 12 stable/increasing and one declining (Kane Basin). We do not find support for the perspective that polar bears within or shared with Canada are currently in any sort of climate crisis. We suggest that monitoring the impacts of climate change (including sea ice decline) on polar bear

  15. Integrated long non-coding RNA analyses identify novel regulators of epithelial-mesenchymal transition in the mouse model of pulmonary fibrosis.

    PubMed

    Sun, Hao; Chen, Junjie; Qian, Wenyi; Kang, Jiang; Wang, Jun; Jiang, Lei; Qiao, Li; Chen, Wei; Zhang, Jinsong

    2016-07-01

    Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease characterized by aberrant accumulation of fibroblast population and deposition of extra cellular matrix. Increasing evidence support that epithelial-mesenchymal transition (EMT) of alveolar epithelial cells is a critical process in the pathogenesis of IPF. Although delivery of bleomycin to induce acute lung injury is the most well-studied animal model of pulmonary fibrosis, there is considerable interest to pursue other models to understand the common and/or specific pathological mechanisms. In this study, we established a mouse model of pulmonary injury and progressive interstitial fibrosis via intraperitoneal injection of paraquat, a widely used herbicide known to cause pulmonary fibrosis in human. Using transcriptome sequencing and microarray analysis, we profiled expression of long non-coding RNAs (lncRNAs) and identified 513 up-regulated and 204 down-regulated lncRNAs in paraquat-induced fibrotic lung tissues. Gene ontology analysis revealed that the differentially expressed lncRNAs are implicated in cell differentiation, epithelium morphogenesis and wound healing, pathways closely associated with EMT. Furthermore, we identified the evolutionally conserved target genes of two up-regulated lncRNAs, uc.77 and 2700086A05Rik, as Zeb2 and Hoxa3, respectively, both of which are important modulators of EMT. Consistently, overexpression of uc.77 or 2700086A05Rik in human lung epithelial cells induced EMT as demonstrated by changes in gene and protein expression of various EMT markers and cell morphology. Collectively, our results uncovered a crucial role of lncRNA in the regulation of EMT during lung fibrosis and provide potential avenues for the discovery of novel molecular markers and therapeutic targets for IPF. PMID:26824344

  16. Characterization of corneal stromal stem cells with the potential for epithelial transdifferentiation

    PubMed Central

    2013-01-01

    Introduction The corneal stroma is being increasingly recognized as a repository for stem cells. Like the limbal and endothelial niches, stromal stem cells often reside in the peripheral cornea and limbus. These peripheral and limbal corneal stromal cells (PLCSCs) are known to produce mesenchymal stem cells in vitro. Recently, a common corneal stromal and epithelial progenitor was hinted at. This study aims to examine the stem cell potential of corneal stromal cells and to investigate their epithelial transdifferentiation ability. Methods PLCSCs were grown in traditional Dulbecco modified Eagle medium (DMEM)-based keratocyte culture medium and an M199-based medium and analyzed for a profile of cell-surface markers by using flow cytometry and differentiated into mesenchymal phenotypes analyzed with quantitative polymerase chain reaction (qPCR) and histologic staining. PLCSCs in M199 were subsequently divided into subpopulations based on CD34 and CD105 expression by using fluorescence- activated cell sorting (FACS). Subpopulations were characterized by marker profile and mesenchymal differentiation ability. Both whole PLCSCs and subpopulations were also cultured for epithelial transdifferentiation. Results Cells cultured in M199 demonstrated a more stem-like cell-surface marker profile, and the keratocyte marker CD34 was retained for several passages but absent in cells cultured in DMEM. Cells cultured in M199 also exhibited a greater mesenchymal differentiation potential, compared with DMEM. PLCSCs could be divided into CD34+CD105+, CD34-CD105+, and CD34-CD105- subpopulations, of which CD34+CD105+ cells were the most stemlike with regard to marker expression and mesenchymal differentiation potential. Subpopulations of PLCSCs exhibited differing abilities to transdifferentiate into epithelial phenotypes. Cells that were initially CD34+CD105+ showed the greatest differentiation potential, producing CK3+ and CK19+ cells, and expressed a range of both epithelial

  17. Genetic differentiation of sockeye salmon subpopulations from a geologically young Alaskan lake system

    USGS Publications Warehouse

    Burger, C.V.; Spearman, W.J.; Cronin, M.A.

    1997-01-01

    The Tustumena lake drainage in southcentral Alaska is glacially turbid and geologically young (<2,000 years old). Previous field studies identified at least three subpopulations of sockeye salmon Oncorhynchus nerka at Tustumena Lake, based on the distribution and timing of spawners. The subpopulations included early-run salmon that spawned in six clearwater tributaries of the lake (mid August), lake shoreline spawners (late August), and late-run fish that spawned in the lake's outlet, the Kasilof River (late September). Our objective was to determine the degree of genetic differentiation among these subpopulations based on restriction enzyme analyses of the cytochrome b gene of mitochondrial DNA and analyses of four polymorphic allozyme loci. Mitochondrial DNA haplotype frequencies for outlet-spawning sockeye salmon differed significantly from those of all other subpopulations. The most common (36%) haplotype in the outlet subpopulation did not occur elsewhere, thus suggesting little or no gene flow between outlet spawners and other spatially close subpopulations at Tustumena Lake. Allele frequencies at two allozyme loci also indicated a degree of differentiation of the outlet subpopulation from the shoreline and tributary subpopulations. Allele frequencies for three tributary subpopulations were temporally stable over approximately 20 years (based on a comparison to previously published results) despite initiation of a hatchery program in two of the tributaries during the intervening period. Collectively, our results are consistent with the hypothesis that significant genetic differentiation has occurred within the Tustumena Lake drainage since deglaciation approximately 2,000 years ago.

  18. Intestinal Epithelial Cell-Intrinsic Deletion of Setd7 Identifies Role for Developmental Pathways in Immunity to Helminth Infection.

    PubMed

    Oudhoff, Menno J; Antignano, Frann; Chenery, Alistair L; Burrows, Kyle; Redpath, Stephen A; Braam, Mitchell J; Perona-Wright, Georgia; Zaph, Colby

    2016-09-01

    The intestine is a common site for a variety of pathogenic infections. Helminth infections continue to be major causes of disease worldwide, and are a significant burden on health care systems. Lysine methyltransferases are part of a family of novel attractive targets for drug discovery. SETD7 is a member of the Suppressor of variegation 3-9-Enhancer of zeste-Trithorax (SET) domain-containing family of lysine methyltransferases, and has been shown to methylate and alter the function of a wide variety of proteins in vitro. A few of these putative methylation targets have been shown to be important in resistance against pathogens. We therefore sought to study the role of SETD7 during parasitic infections. We find that Setd7-/- mice display increased resistance to infection with the helminth Trichuris muris but not Heligmosomoides polygyrus bakeri. Resistance to T. muris relies on an appropriate type 2 immune response that in turn prompts intestinal epithelial cells (IECs) to alter differentiation and proliferation kinetics. Here we show that SETD7 does not affect immune cell responses during infection. Instead, we found that IEC-specific deletion of Setd7 renders mice resistant to T. muris by controlling IEC turnover, an important aspect of anti-helminth immune responses. We further show that SETD7 controls IEC turnover by modulating developmental signaling pathways such as Hippo/YAP and Wnt/β-Catenin. We show that the Hippo pathway specifically is relevant during T. muris infection as verteporfin (a YAP inhibitor) treated mice became susceptible to T. muris. We conclude that SETD7 plays an important role in IEC biology during infection. PMID:27598373

  19. Subpopulations in purified platelets adhering on glass.

    PubMed

    Donati, Alessia; Gupta, Swati; Reviakine, Ilya

    2016-01-01

    Understanding how platelet activation is regulated is important in the context of cardiovascular disorders and their management with antiplatelet therapy. Recent evidence points to different platelet subpopulations performing different functions. In particular, procoagulant and aggregating subpopulations have been reported in the literature in platelets treated with the GPVI agonists. How the formation of platelet subpopulations upon activation is regulated remains unclear. Here, it is shown that procoagulant and aggregating platelet subpopulations arise spontaneously upon adhesion of purified platelets on clean glass surfaces. Calcium ionophore treatment of the adhering platelets resulted in one platelet population expressing both the procoagulant and the adherent population markers phosphatidylserine and the activated form of GPIIb/IIIa, while all of the platelets expressed CD62P independently of the ionophore treatment. Therefore, all platelets have the capacity to express all three activation markers. It is concluded that platelet subpopulations observed in various studies reflect the dynamics of the platelet activation process. PMID:27338300

  20. CHARACTERIZING AND PROVIDING ADEQUATE PROTECTION FOR SUSCEPTIBLE SUBPOPULATIONS: SUSCEPTIBLE POPULATIONS: SUSCEPTIBILITY ASSOCIATED WITH THE AGED

    EPA Science Inventory

    SUMMARY: An important Agency mandate is to identify subpopulations particularly susceptible to adverse effects associated with exposure to environmental contaminants. For example, a critical public health question is the extent to which exposure to environmental contaminants cont...

  1. Highly variable cancer subpopulations that exhibit enhanced transcriptome variability and metastatic fitness.

    PubMed

    Nguyen, Alexander; Yoshida, Mitsukuni; Goodarzi, Hani; Tavazoie, Sohail F

    2016-01-01

    Individual cells within a tumour can exhibit distinct genetic and molecular features. The impact of such diversification on metastatic potential is unknown. Here we identify clonal human breast cancer subpopulations that display different levels of morphological and molecular diversity. Highly variable subpopulations are more proficient at metastatic colonization and chemotherapeutic survival. Through single-cell RNA-sequencing, inter-cell transcript expression variability is identified as a defining feature of the highly variable subpopulations that leads to protein-level variation. Furthermore, we identify high variability in the spliceosomal machinery gene set. Engineered variable expression of the spliceosomal gene SNRNP40 promotes metastasis, attributable to cells with low expression. Clinically, low SNRNP40 expression is associated with metastatic relapse. Our findings reveal transcriptomic variability generation as a mechanism by which cancer subpopulations can diversify gene expression states, which may allow for enhanced fitness under changing environmental pressures encountered during cancer progression. PMID:27138336

  2. Highly variable cancer subpopulations that exhibit enhanced transcriptome variability and metastatic fitness

    PubMed Central

    Nguyen, Alexander; Yoshida, Mitsukuni; Goodarzi, Hani; Tavazoie, Sohail F.

    2016-01-01

    Individual cells within a tumour can exhibit distinct genetic and molecular features. The impact of such diversification on metastatic potential is unknown. Here we identify clonal human breast cancer subpopulations that display different levels of morphological and molecular diversity. Highly variable subpopulations are more proficient at metastatic colonization and chemotherapeutic survival. Through single-cell RNA-sequencing, inter-cell transcript expression variability is identified as a defining feature of the highly variable subpopulations that leads to protein-level variation. Furthermore, we identify high variability in the spliceosomal machinery gene set. Engineered variable expression of the spliceosomal gene SNRNP40 promotes metastasis, attributable to cells with low expression. Clinically, low SNRNP40 expression is associated with metastatic relapse. Our findings reveal transcriptomic variability generation as a mechanism by which cancer subpopulations can diversify gene expression states, which may allow for enhanced fitness under changing environmental pressures encountered during cancer progression. PMID:27138336

  3. Connectivity among subpopulations of Louisiana black bears as estimated by a step selection function

    USGS Publications Warehouse

    Clark, Joseph D.; Jared S. Laufenberg; Maria Davidson; Jennifer L. Murrow

    2015-01-01

    Habitat fragmentation is a fundamental cause of population decline and increased risk of extinction for many wildlife species; animals with large home ranges and small population sizes are particularly sensitive. The Louisiana black bear (Ursus americanus luteolus) exists only in small, isolated subpopulations as a result of land clearing for agriculture, but the relative potential for inter-subpopulation movement by Louisiana black bears has not been quantified, nor have characteristics of effective travel routes between habitat fragments been identified. We placed and monitored global positioning system (GPS) radio collars on 8 female and 23 male bears located in 4 subpopulations in Louisiana, which included a reintroduced subpopulation located between 2 of the remnant subpopulations. We compared characteristics of sequential radiolocations of bears (i.e., steps) with steps that were possible but not chosen by the bears to develop step selection function models based on conditional logistic regression. The probability of a step being selected by a bear increased as the distance to natural land cover and agriculture at the end of the step decreased and as distance from roads at the end of a step increased. To characterize connectivity among subpopulations, we used the step selection models to create 4,000 hypothetical correlated random walks for each subpopulation representing potential dispersal events to estimate the proportion that intersected adjacent subpopulations (hereafter referred to as successful dispersals). Based on the models, movement paths for males intersected all adjacent subpopulations but paths for females intersected only the most proximate subpopulations. Cross-validation and genetic and independent observation data supported our findings. Our models also revealed that successful dispersals were facilitated by a reintroduced population located between 2 distant subpopulations. Successful dispersals for males were dependent on natural land

  4. Systematic CpG Islands Methylation Profiling of Genes in the Wnt Pathway in Epithelial Ovarian Cancer Identifies Biomarkers of Progression-Free Survival

    PubMed Central

    Dai, Wei; Teodoridis, Jens M.; Zeller, Constanze; Graham, Janet; Hersey, Jenny; Flanagan, James M.; Stronach, Euan; Millan, David W.; Siddiqui, Nadeem; Paul, Jim; Brown, Robert

    2011-01-01

    Purpose Wnt pathways control key biological processes that potentially impact on tumour progression and patient survival. We aimed to evaluate DNA methylation at promoter CpG islands (CGIs) of Wnt pathway genes in ovarian tumours at presentation and identify biomarkers of patient progression-free survival (PFS). Experimental Design Epithelial ovarian tumours (screening study n=120, validation study n=61) prospectively collected through a cohort study, were analysed by differential methylation hybridisation (DMH) at 302 loci spanning 189 promoter CGIs at 137 genes in Wnt pathways. The association of methylation and progression free survival was examined by Cox proportional hazards model. Results DNA methylation is associated with PFS at 20/302 loci (p<0.05, n=111), with 5 loci significant at FDR<10%. 11/20 loci retain significance in an independent validation cohort (n=48,p≤0.05,FDR≤10%), and 7 of these loci, at FZD4, DVL1, NFATC3, ROCK1, LRP5, AXIN1 and NKD1 genes, are independent from clinical parameters (adjusted p<0.05). Increased methylation at these loci associates with increased hazard of disease progression. A multivariate Cox model incorporates only NKD1 and DVL1, identifying two groups differing in PFS (HR=2.09; 95%CI (1.39, 3.15); permutation test p<0.005). Methylation at DVL1 and NFATC3 show significant association with response. Consistent with their epigenetic regulation, reduced expression of FZD4, DVL1 and ROCK1 is an indicator of early disease relapse in an independent ovarian tumour cohort (n=311, adjusted p<0.05). Conclusions The data highlights the importance of epigenetic regulation of multiple promoter CGIs of Wnt pathway genes in ovarian cancer and identifies methylation at NKD1 and DVL1 as independent predictors of PFS. PMID:21459799

  5. MCA: Multiresolution Correlation Analysis, a graphical tool for subpopulation identification in single-cell gene expression data

    PubMed Central

    2014-01-01

    Background Biological data often originate from samples containing mixtures of subpopulations, corresponding e.g. to distinct cellular phenotypes. However, identification of distinct subpopulations may be difficult if biological measurements yield distributions that are not easily separable. Results We present Multiresolution Correlation Analysis (MCA), a method for visually identifying subpopulations based on the local pairwise correlation between covariates, without needing to define an a priori interaction scale. We demonstrate that MCA facilitates the identification of differentially regulated subpopulations in simulated data from a small gene regulatory network, followed by application to previously published single-cell qPCR data from mouse embryonic stem cells. We show that MCA recovers previously identified subpopulations, provides additional insight into the underlying correlation structure, reveals potentially spurious compartmentalizations, and provides insight into novel subpopulations. Conclusions MCA is a useful method for the identification of subpopulations in low-dimensional expression data, as emerging from qPCR or FACS measurements. With MCA it is possible to investigate the robustness of covariate correlations with respect subpopulations, graphically identify outliers, and identify factors contributing to differential regulation between pairs of covariates. MCA thus provides a framework for investigation of expression correlations for genes of interests and biological hypothesis generation. PMID:25015590

  6. Identifying Metabolic Subpopulations from Population Level Mass Spectrometry

    PubMed Central

    Springer, Michael

    2016-01-01

    Metabolism underlies many important cellular decisions, such as the decisions to proliferate and differentiate, and defects in metabolic signaling can lead to disease and aging. In addition, metabolic heterogeneity can have biological consequences, such as differences in outcomes and drug susceptibilities in cancer and antibiotic treatments. Many approaches exist for characterizing the metabolic state of a population of cells, but technologies for measuring metabolism at the single cell level are in the preliminary stages and are limited. Here, we describe novel analysis methodologies that can be applied to established experimental methods to measure metabolic variability within a population. We use mass spectrometry to analyze amino acid composition in cells grown in a mixture of 12C- and 13C-labeled sugars; these measurements allow us to quantify the variability in sugar usage and thereby infer information about the behavior of cells within the population. The methodologies described here can be applied to a large range of metabolites and macromolecules and therefore have the potential for broad applications. PMID:26986964

  7. Transcriptional regulators transforming growth factor-beta 1 and estrogen-related receptor-alpha identified as putative mediators of calf rumen epithelial tissue development and function during weaning

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Molecular mechanisms controlling rumen epithelial development at weaning remain largely unknown. To identify gene networks and regulatory factors responsive to concentrate versus forage feeding at weaning, Holstein bull calves (n = 18) were fed commercial milk replacer only (MRO) until 42 d of age. ...

  8. T and B Lymphocyte Subpopulations

    ERIC Educational Resources Information Center

    Seeger, Robert C.; Stiehm, E. Richard

    1975-01-01

    Reviewed are diagnostic tests of symphocyte subgroups which identify immuno deficiency disorders (such as DiGeorge's Syndrome) and malignant cells in lymphoproliferative disorders (such as lumphoid leukemia). (CL)

  9. CD44 variant 6 is correlated with peritoneal dissemination and poor prognosis in patients with advanced epithelial ovarian cancer

    PubMed Central

    Tjhay, Francisca; Motohara, Takeshi; Tayama, Shingo; Narantuya, Dashdemberel; Fujimoto, Koichi; Guo, Jianying; Sakaguchi, Isao; Honda, Ritsuo; Tashiro, Hironori; Katabuchi, Hidetaka

    2015-01-01

    Cancer stem cells (CSCs) drive tumor initiation and metastasis in several types of human cancer. However, the contribution of ovarian CSCs to peritoneal metastasis remains unresolved. The cell adhesion molecule CD44 has been identified as a major marker for CSCs in solid tumors, including epithelial ovarian cancer. CD44 exists as a standard form (CD44s) and also as numerous variant isoforms (CD44v) generated by alternative mRNA splicing. Here we show that disseminated ovarian tumors in the pelvic peritoneum contain highly enriched CD44v6-positive cancer cells, which drive tumor metastasis and are responsible for tumor resistance to chemotherapy. Clinically, an increased number of CD44v6-positive cancer cells in primary tumors was associated with a shortened overall survival in stage III–IV ovarian cancer patients. Furthermore, a subpopulation of CD44v6-positive cancer cells manifested the ability to initiate tumor metastasis in the pelvic peritoneum in an in vivo mouse model, suggesting that CD44v6-positive cells show the potential to serve as metastasis-initiating cells. Thus, the peritoneal disseminated metastasis of epithelial ovarian cancer is initiated by the CD44v6-positive subpopulation, and CD44v6 expression is a biomarker for the clinical outcome of advanced ovarian cancer patients. Given that a distinct subpopulation of CD44v6-positive cancer cells plays a critical role in peritoneal metastasis, definitive treatment should target this subpopulation of CD44v6-positive cells in epithelial ovarian cancer. PMID:26250934

  10. A Synthetic Lethality Screen Using a Focused siRNA Library to Identify Sensitizers to Dasatinib Therapy for the Treatment of Epithelial Ovarian Cancer

    PubMed Central

    Pathak, Harsh B.; Zhou, Yan; Sethi, Geetika; Hirst, Jeff; Schilder, Russell J.; Golemis, Erica A.; Godwin, Andrew K.

    2015-01-01

    Molecular targeted therapies have been the focus of recent clinical trials for the treatment of patients with recurrent epithelial ovarian cancer (EOC). The majority have not fared well as monotherapies for improving survival of these patients. Poor bioavailability, lack of predictive biomarkers, and the presence of multiple survival pathways can all diminish the success of a targeted agent. Dasatinib is a tyrosine kinase inhibitor of the Src-family kinases (SFK) and in preclinical studies shown to have substantial activity in EOC. However, when evaluated in a phase 2 clinical trial for patients with recurrent or persistent EOC, it was found to have minimal activity. We hypothesized that synthetic lethality screens performed using a cogently designed siRNA library would identify second-site molecular targets that could synergize with SFK inhibition and improve dasatinib efficacy. Using a systematic approach, we performed primary siRNA screening using a library focused on 638 genes corresponding to a network centered on EGFR, HER2, and the SFK-scaffolding proteins BCAR1, NEDD9, and EFS to screen EOC cells in combination with dasatinib. We followed up with validation studies including deconvolution screening, quantitative PCR to confirm effective gene silencing, correlation of gene expression with dasatinib sensitivity, and assessment of the clinical relevance of hits using TCGA ovarian cancer data. A refined list of five candidates (CSNK2A1, DAG1, GRB2, PRKCE, and VAV1) was identified as showing the greatest potential for improving sensitivity to dasatinib in EOC. Of these, CSNK2A1, which codes for the catalytic alpha subunit of protein kinase CK2, was selected for additional evaluation. Synergistic activity of the clinically relevant inhibitor of CK2, CX-4945, with dasatinib in reducing cell proliferation and increasing apoptosis was observed across multiple EOC cell lines. This overall approach to improving drug efficacy can be applied to other targeted agents

  11. Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells.

    PubMed

    Fan, Y-H; Ding, J; Nguyen, S; Liu, X-J; Xu, G; Zhou, H-Y; Duan, N-N; Yang, S-M; Zern, M A; Wu, J

    2016-01-01

    Hepatoma exhibits a series of heterogeneous subpopulations in its cell surface markers, tumorigenicity, invasion and metastatic capability. We previously demonstrated that the CD133(-)/EpCAM(-) hepatoma subpopulation was more metastatic than its counterpart; however, the controlling mechanisms are unexplored. The present study aimed to delineate the significance of aberrant hedgehog (Hh) signaling in the mediation of metastases. Fluorescence-activated cell sorting-enriched CD133(-)/EpCAM(-) (double negative, DN), Huh-7 cells underwent a transwell selection for metastatic cells (transwell-selected, TS). The TS cells displayed much greater metastatic activity as evidenced by an increased invasion rate, extremely upregulated expression of matrix metalloproteinase (MMP)-1/2/9 genes compared with DN and double-positive (DP) subpopulations. In contrast to DP cells, TS cells lost E-cadherin and were all vimentin-positive as shown by immunocytochemistry. There was a transitional increase in Gli-1/2 gene expression levels from DP, DN to TS subpopulations, which was consistent with elevated Gli-1/2 or Twist-1 protein levels in the nuclear fraction. Furthermore, truncated Gli-1 (tGli-1), which transactivates molecules involved in metastasis, was detected in the highly invasive Huh-7 cell subpopulation, but not in less metastatic hepatoma cells or normal hepatocytes. The enhanced metastatic features with increased expression of MMPs as well as the presence of twist and snail genes in TS Huh-7 cells were reversed by LDE225, a potent Smoothened antagonist. In conclusion, the highly metastatic capability of a unique TS subpopulation was highly attributed to significant epithelial-mesenchymal transition, enhanced Hh activity and aberrant occurrence of a tGli-1 variant, which appears to be responsible for the highly invasive behavior. PMID:25772244

  12. Injury induces direct lineage segregation of functionally distinct airway basal stem/progenitor cell subpopulations.

    PubMed

    Pardo-Saganta, Ana; Law, Brandon M; Tata, Purushothama Rao; Villoria, Jorge; Saez, Borja; Mou, Hongmei; Zhao, Rui; Rajagopal, Jayaraj

    2015-02-01

    Following injury, stem cells restore normal tissue architecture by producing the proper number and proportions of differentiated cells. Current models of airway epithelial regeneration propose that distinct cytokeratin 8-expressing progenitor cells, arising from p63(+) basal stem cells, subsequently differentiate into secretory and ciliated cell lineages. We now show that immediately following injury, discrete subpopulations of p63(+) airway basal stem/progenitor cells themselves express Notch pathway components associated with either secretory or ciliated cell fate commitment. One basal cell population displays intracellular Notch2 activation and directly generates secretory cells; the other expresses c-myb and directly yields ciliated cells. Furthermore, disrupting Notch ligand activity within the basal cell population at large disrupts the normal pattern of lineage segregation. These non-cell-autonomous effects demonstrate that effective airway epithelial regeneration requires intercellular communication within the broader basal stem/progenitor cell population. These findings have broad implications for understanding epithelial regeneration and stem cell heterogeneity. PMID:25658372

  13. Gain-of-function microRNA screens identify miR-193a regulating proliferation and apoptosis in epithelial ovarian cancer cells

    PubMed Central

    NAKANO, HARUO; YAMADA, YOJI; MIYAZAWA, TATSUYA; YOSHIDA, TETSUO

    2013-01-01

    MicroRNAs (miRNAs) are a small class of non-coding RNAs that negatively regulate gene expression, and are considered as new therapeutic targets for treating cancer. In this study, we performed a gain-of-function screen using miRNA mimic library (319 miRNA species) to identify those affecting cell proliferation in human epithelial ovarian cancer cells (A2780). We discovered a number of miRNAs that increased or decreased the cell viability of A2780 cells. Pro-proliferative and anti-proliferative miRNAs include oncogenic miR-372 and miR-373, and tumor suppressive miR-124a, miR-7, miR-192 and miR-193a, respectively. We found that overexpression of miR-124a, miR-192, miR-193a and miR-193b inhibited BrdU incorporation in A2780 cells, indicating that these miRNAs affected the cell cycle. Overexpression of miR-193a and miR-193b induced an activation of caspase 3/7, and resulted in apoptotic cell death in A2780 cells. A genome-wide gene expression analysis with miR-193a-transfected A2780 cells led to identification of ARHGAP19, CCND1, ERBB4, KRAS and MCL1 as potential miR-193a targets. We demonstrated that miR-193a decreased the amount of MCL1 protein by binding 3′UTR of its mRNA. Our study suggests the potential of miRNA screens to discover miRNAs as therapeutic tools to treat ovarian cancer. PMID:23588298

  14. Morphologically and Functionally Distinct Lipid Droplet Subpopulations

    PubMed Central

    Zhang, Shuyan; Wang, Yang; Cui, Liujuan; Deng, Yaqin; Xu, Shimeng; Yu, Jinhai; Cichello, Simon; Serrero, Ginette; Ying, Yunshu; Liu, Pingsheng

    2016-01-01

    Lipid droplet (LD), a multi-functional organelle, is often found to associate with other cellular membranous structures and vary in size in a given cell, which may be related to their functional diversity. Here we established a method to separate LD subpopulations from isolated CHO K2 LDs into three different size categories. The subpopulation with smallest LDs was nearly free of ER and other membranous structures while those with larger LDs contained intact ER. These distinct subpopulations of LDs differed in their protein composition and ability to recruit proteins. This method was also applicable to LDs obtained from other sources, such as Huh7 cells, mouse liver and brown adipose tissue, et al. We developed an in vitro assay requiring only isolated LDs, Coenzyme A, and ATP to drive lipid synthesis. The LD subpopulation nearly depleted of ER was able to incorporate fatty acids into triacylglycerol and phospholipids. Together, our data demonstrate that LDs in a given cell are heterogeneous in size and function, and suggest that LDs are one of cellular lipid synthetic organelles. PMID:27386790

  15. STR profiling of epithelial cells identified by X/Y-FISH labelling and laser microdissection using standard and elevated PCR conditions.

    PubMed

    Lynch, Laura; Gamblin, Amelia; Vintiner, Sue; Simons, Joanne L

    2015-05-01

    During the investigation of allegations of sexual assault, samples are frequently encountered that contain DNA from a female and a male donor. These may represent contributions of DNA from the complainant and potentially, the offender. Many semen stained samples successfully undergo DNA analysis and interpretation using a differential extraction method that separates sperm from the epithelial cells present in the stain. However, for those mixed cell samples that contain only epithelial cells, separation of any male cells from female cells is problematic. This paper describes the application of fluorescent in situ hybridisation (FISH) for the gender identification of epithelial cells and subsequent recovery of target cells using laser microdissection (LMD). The profiling results obtained from samples of known cell numbers using the Identifiler™ multiplex at standard 28-cycle PCR conditions and, when cell numbers are low, the SGM Plus™ multiplex at elevated 34-cycle PCR conditions (also known as Low Copy Number DNA analysis (LCN)) are described. PMID:25555139

  16. Sub-population analysis based on temporal features of high content images

    PubMed Central

    2009-01-01

    Background High content screening techniques are increasingly used to understand the regulation and progression of cell motility. The demand of new platforms, coupled with availability of terabytes of data has challenged the traditional technique of identifying cell populations by manual methods and resulted in development of high-dimensional analytical methods. Results In this paper, we present sub-populations analysis of cells at the tissue level by using dynamic features of the cells. We used active contour without edges for segmentation of cells, which preserves the cell morphology, and autoregressive modeling to model cell trajectories. The sub-populations were obtained by clustering static, dynamic and a combination of both features. We were able to identify three unique sub-populations in combined clustering. Conclusion We report a novel method to identify sub-populations using kinetic features and demonstrate that these features improve sub-population analysis at the tissue level. These advances will facilitate the application of high content screening data analysis to new and complex biological problems. PMID:19958514

  17. Epithelial ovarian cancer stem cells: underlying complexity of a simple paradigm.

    PubMed

    Garson, Kenneth; Vanderhyden, Barbara C

    2015-02-01

    The lack of significant progress in the treatment of epithelial ovarian cancer (EOC) underscores the need to gain a better understanding of the processes that lead to chemoresistance and recurrence. The cancer stem cell (CSC) hypothesis offers an attractive explanation of how a subpopulation of cells within a patient's tumour might remain refractory to treatment and subsequently form the basis of recurrent chemoresistant disease. This review examines the literature defining somatic stem cells of the ovary and fallopian tube, two tissues that give rise to EOC. In addition, considerable research has been reviewed, that has identified subpopulations of EOC cells, based on marker expression (CD133, CD44, CD117, CD24, epithelial cell adhesion molecule, LY6A, ALDH1 and side population (SP)), which are enriched for tumour initiating cells (TICs). While many studies identified either CD133 or CD44 as markers useful for enriching for TICs, there is little consensus. This suggests that EOC cells may have a phenotypic plasticity that may preclude the identification of universal markers defining a CSC. The assay that forms the basis of quantifying TICs is the xenograft assay. Considerable controversy surrounds the xenograft assay and it is essential that some of the potential limitations be examined in this review. Highlighting such limitations or weaknesses is required to properly evaluate data and broaden our interpretation of potential mechanisms that might be contributing to the pathogenesis of ovarian cancer. PMID:25301968

  18. CYTOCHROME C OXIDASE SEQUENCE DATA DISTINGUISH A THELYTOKOUS SUBPOPULATION OF ODONTOSEMA ANASTREPHAE FROM A MORPHOLOGICALLY IDENTICAL ARRENOTOKOUS SUBPOPULATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A thelytokous subpopulation of Odontosema anastrephae was collected from Vera Cruz State, Mexico, and compared to an arrenotokous subpopulation of O. anastrephae. The two subpopulations were unable to be distinguished on the basis of morphology. Mitochondrial sequence analysis, however, revealed t...

  19. Alcohol Consumption in Demographic Subpopulations: An Epidemiologic Overview.

    PubMed

    Delker, Erin; Brown, Qiana; Hasin, Deborah S

    2016-01-01

    Alcohol consumption is common across subpopulations in the United States. However, the health burden associated with alcohol consumption varies across groups, including those defined by demographic characteristics such as age, race/ ethnicity, and gender. Large national surveys, such as the National Epidemiologic Survey on Alcohol and Related Conditions and the National Survey on Drug Use and Health, found that young adults ages 18-25 were at particularly high risk of alcohol use disorder and unintentional injury caused by drinking. These surveys furthermore identified significant variability in alcohol consumption and its consequences among racial/ethnic groups. White respondents reported the highest prevalence of current alcohol consumption, whereas alcohol abuse and dependence were most prevalent among Native Americans. Native Americans and Blacks also were most vulnerable to alcohol-related health consequences. Even within ethnic groups, there was variability between and among different subpopulations. With respect to gender, men reported more alcohol consumption and binge drinking than women, especially in older cohorts. Men also were at greater risk of alcohol abuse and dependence, liver cirrhosis, homicide after alcohol consumption, and drinking and driving. Systematic identification and measurement of the variability across demographics will guide prevention and intervention efforts, as well as future research. PMID:27159807

  20. Redox subpopulations and the risk of cancer progression: a new method for characterizing redox heterogeneity

    NASA Astrophysics Data System (ADS)

    Xu, He N.; Li, Lin Z.

    2016-02-01

    It has been shown that a malignant tumor is akin to a complex organ comprising of various cell populations including tumor cells that are genetically, metabolically and functionally different. Our redox imaging data have demonstrated intra-tumor redox heterogeneity in all mouse xenografts derived from human melanomas, breast, prostate, and colon cancers. Based on the signals of NADH and oxidized flavoproteins (Fp, including flavin adenine dinucleotide (FAD)) and their ratio, i.e., the redox ratio, which is an indicator of mitochondrial metabolic status, we have discovered several distinct redox subpopulations in xenografts of breast tumors potentially recapitulating functional/metabolic heterogeneity within the tumor. Furthermore, xenografts of breast tumors with higher metastatic potential tend to have a redox subpopulation whose redox ratio is significantly different from that of tumors with lower metastatic potential and usually have a bi-modal distribution of the redox ratio. The redox subpopulations from human breast cancer samples can also be very complex with multiple subpopulations as determined by fitting the redox ratio histograms with multi- Gaussian functions. In this report, we present a new method for identifying the redox subpopulations within individual breast tumor xenografts and human breast tissues, which may be used to differentiate between breast cancer and normal tissue and among breast cancer with different risks of progression.

  1. Genetic screening of targeted subpopulations: the role of communal discourse in evaluating sociocultural implications.

    PubMed

    Foster, M W; Eisenbraun, A J; Carter, T H

    Targeting socially identifiable subpopulations for genetic screening entails the risk of stigmatizing them. The potential for such harm should be considered before programs are initiated. There is an emerging consensus that targeted subpopulations should be actively involved in evaluating these risks. A process of communal discourse engages the community in discussions that reflect both public and private sociocultural contexts in which individual decisions about screening will be made. This allows the subpopulation to address the collective implications of testing in a culturally appropriate way. Communal discourse was used to evaluate the collective implications of genetic testing in two Native American communities. We found that private social units were more influential than public units in reaching communal consensus, that local sociocultural issues were of more concern than were general issues such as employment and insurance discrimination, and that heterogeneity within a subpopulation may be just as significant a consideration in designing a targeted screening program as diversity between subpopulations. Heterogeneity is constructed by using a dichotomy between community-specific and biomedical health representations and practices. How genetic screening is socially constructed using a community's existing dichotomy may be central to its success. PMID:10464656

  2. A cluster analysis method for identification of subpopulations of cells in flow cytometric list-mode arrays

    NASA Technical Reports Server (NTRS)

    Li, Z. K.

    1985-01-01

    A specialized program was developed for flow cytometric list-mode data using an heirarchical tree method for identifying and enumerating individual subpopulations, the method of principal components for a two-dimensional display of 6-parameter data array, and a standard sorting algorithm for characterizing subpopulations. The program was tested against a published data set subjected to cluster analysis and experimental data sets from controlled flow cytometry experiments using a Coulter Electronics EPICS V Cell Sorter. A version of the program in compiled BASIC is usable on a 16-bit microcomputer with the MS-DOS operating system. It is specialized for 6 parameters and up to 20,000 cells. Its two-dimensional display of Euclidean distances reveals clusters clearly, as does its 1-dimensional display. The identified subpopulations can, in suitable experiments, be related to functional subpopulations of cells.

  3. Improving removal-based estimates of abundance by sampling a population of spatially distinct subpopulations

    USGS Publications Warehouse

    Dorazio, R.M.; Jelks, H.L.; Jordan, F.

    2005-01-01

     A statistical modeling framework is described for estimating the abundances of spatially distinct subpopulations of animals surveyed using removal sampling. To illustrate this framework, hierarchical models are developed using the Poisson and negative-binomial distributions to model variation in abundance among subpopulations and using the beta distribution to model variation in capture probabilities. These models are fitted to the removal counts observed in a survey of a federally endangered fish species. The resulting estimates of abundance have similar or better precision than those computed using the conventional approach of analyzing the removal counts of each subpopulation separately. Extension of the hierarchical models to include spatial covariates of abundance is straightforward and may be used to identify important features of an animal's habitat or to predict the abundance of animals at unsampled locations.

  4. A “Rice Diversity Panel” evaluated for genetic and agro-morphological variation between subpopulations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since ancient times, Indica and Japonica have been recognized as the two major subspecies of Asian rice (Oryza sativa L.). First with isozymes and subsequently with DNA markers, five subpopulations indica, aus, temperate japonica, tropical japonica and aromatic/GroupV were identified. A “Rice Diver...

  5. The Gulliver Effect: The Impact of Error in an Elephantine Subpopulation on Estimates for Lilliputian Subpopulations

    ERIC Educational Resources Information Center

    Micceri, Theodore; Parasher, Pradnya; Waugh, Gordon W.; Herreid, Charlene

    2009-01-01

    An extensive review of the research literature and a study comparing over 36,000 survey responses with archival true scores indicated that one should expect a minimum of at least three percent random error for the least ambiguous of self-report measures. The Gulliver Effect occurs when a small proportion of error in a sizable subpopulation exerts…

  6. Elevated T cell subpopulations in dental students

    SciTech Connect

    Eedy, D.J.; Burrows, D.; Clifford, T.; Fay, A. )

    1990-05-01

    The absolute numbers of circulating white cells and lymphocyte subpopulations were studied in 25 final-year dental students and compared with a control group of 28 medical students. The total lymphocyte count, total T cell numbers (CD3), T helper/inducer (CD4), and T suppressor/cytotoxic (CD8) numbers were significantly elevated in the dental students as compared with the control group. There was no significant difference in the T helper/inducer to T suppressor/cytotoxic cell ratios or the circulating B cell (CD21) and natural killer cell (CD16) numbers between the study and control groups. Patch testing to mercury and mercuric compounds in both the study and control groups showed no evidence of cutaneous hypersensitivity to mercury. The reason for the observed elevations in T cell subpopulations in dental students is not clear. However, one possible explanation is the dental student's occupational exposure to mercury. Further work is underway to examine this possible relationship and it is suggested that dental personnel take adequate measures to reduce their exposure to mercury until the results of these studies are available.

  7. Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression.

    PubMed

    Chabot, Andréanne; Hertig, Vanessa; Boscher, Elena; Nguyen, Quang Trinh; Boivin, Benoît; Chebli, Jasmine; Bissonnette, Lyse; Villeneuve, Louis; Brochiero, Emmanuelle; Dupuis, Jocelyn; Calderone, Angelino

    2016-07-01

    Endothelial and epithelial cell transition to a mesenchymal phenotype was identified as cellular paradigms implicated in the appearance of fibroblasts and development of reactive fibrosis in interstitial lung disease. The intermediate filament protein nestin was highly expressed in fibrotic tissue, detected in fibroblasts and participated in proliferation and migration. The present study tested the hypothesis that the transition of endothelial and epithelial cells to a mesenchymal phenotype was delineated by nestin expression. Three weeks following hypobaric hypoxia, adult male Sprague-Dawley rats characterized by alveolar and perivascular lung fibrosis were associated with increased nestin protein and mRNA levels and marked appearance of nestin/collagen type I((+))-fibroblasts. In the perivascular region of hypobaric hypoxic rats, displaced CD31((+))-endothelial cells were detected, exhibited a mesenchymal phenotype and co-expressed nestin. Likewise, epithelial cells in the lungs of hypobaric hypoxic rats transitioned to a mesenchymal phenotype distinguished by the co-expression of E-cadherin and collagen. Following the removal of FBS from primary passage rat alveolar epithelial cells, TGF-β1 was detected in the media and a subpopulation acquired a mesenchymal phenotype characterized by E-cadherin downregulation and concomitant induction of collagen and nestin. Bone morphogenic protein-7 treatment of alveolar epithelial cells prevented E-cadherin downregulation, suppressed collagen induction but partially inhibited nestin expression. These data support the premise that the transition of endothelial and epithelial cells to a mesenchymal cell may have contributed in part to the appearance nestin/collagen type I((+))-fibroblasts and the reactive fibrotic response in the lungs of hypobaric hypoxic rats. PMID:26574905

  8. Quantifying landscape linkages among giant panda subpopulations in regional scale conservation.

    PubMed

    Qi, Dunwu; Hu, Yibo; Gu, Xiaodong; Yang, Xuyi; Yang, Guang; Wei, Fuwen

    2012-06-01

    Understanding habitat requirements and identifying landscape linkages are essential for the survival of isolated populations of endangered species. Currently, some of the giant panda populations are isolated, which threatens their long-term survival, particularly in the Xiaoxiangling mountains. In the present study, we quantified niche requirements and then identified potential linkages of giant panda subpopulations in the most isolated region, using ecological niche factor analysis and a least-cost path model. Giant pandas preferred habitat with conifer forest and gentle slopes (>20 to ≤30°). Based on spatial distribution of suitable habitat, linkages were identified for the Yele subpopulation to 4 other subpopulations (Liziping, Matou, Xinmin and Wanba). Their lengths ranged from 15 to 54 km. The accumulated cost ranged from 693 to 3166 and conifer forest covered over 31%. However, a variety of features (e.g. major roads, human settlements and large unforested areas) might act as barriers along the linkages for giant panda dispersal. Our analysis quantified giant panda subpopulation connectivity to ensure long-term survival. PMID:22691200

  9. Evaluation of Candidate Stromal Epithelial Cross-Talk Genes Identifies Association between Risk of Serous Ovarian Cancer and TERT, a Cancer Susceptibility “Hot-Spot”

    PubMed Central

    Chen, Xiaoqing; Macgregor, Stuart; Duffy, David L.; Spurdle, Amanda B.; deFazio, Anna; Gava, Natalie; Webb, Penelope M.; Rossing, Mary Anne; Doherty, Jennifer Anne; Goodman, Marc T.; Lurie, Galina; Thompson, Pamela J.; Wilkens, Lynne R.; Ness, Roberta B.; Moysich, Kirsten B.; Chang-Claude, Jenny; Wang-Gohrke, Shan; Cramer, Daniel W.; Terry, Kathryn L.; Hankinson, Susan E.; Tworoger, Shelley S.; Garcia-Closas, Montserrat; Yang, Hannah; Lissowska, Jolanta; Chanock, Stephen J.; Pharoah, Paul D.; Song, Honglin; Whitemore, Alice S.; Pearce, Celeste L.; Stram, Daniel O.; Wu, Anna H.; Pike, Malcolm C.; Gayther, Simon A.; Ramus, Susan J.; Menon, Usha; Gentry-Maharaj, Aleksandra; Anton-Culver, Hoda; Ziogas, Argyrios; Hogdall, Estrid; Kjaer, Susanne K.; Hogdall, Claus; Berchuck, Andrew; Schildkraut, Joellen M.; Iversen, Edwin S.; Moorman, Patricia G.; Phelan, Catherine M.; Sellers, Thomas A.; Cunningham, Julie M.; Vierkant, Robert A.; Rider, David N.; Goode, Ellen L.; Haviv, Izhak; Chenevix-Trench, Georgia

    2010-01-01

    We hypothesized that variants in genes expressed as a consequence of interactions between ovarian cancer cells and the host micro-environment could contribute to cancer susceptibility. We therefore used a two-stage approach to evaluate common single nucleotide polymorphisms (SNPs) in 173 genes involved in stromal epithelial interactions in the Ovarian Cancer Association Consortium (OCAC). In the discovery stage, cases with epithelial ovarian cancer (n = 675) and controls (n = 1,162) were genotyped at 1,536 SNPs using an Illumina GoldenGate assay. Based on Positive Predictive Value estimates, three SNPs—PODXL rs1013368, ITGA6 rs13027811, and MMP3 rs522616—were selected for replication using TaqMan genotyping in up to 3,059 serous invasive cases and 8,905 controls from 16 OCAC case-control studies. An additional 18 SNPs with Pper-allele<0.05 in the discovery stage were selected for replication in a subset of five OCAC studies (n = 1,233 serous invasive cases; n = 3,364 controls). The discovery stage associations in PODXL, ITGA6, and MMP3 were attenuated in the larger replication set (adj. Pper-allele≥0.5). However genotypes at TERT rs7726159 were associated with ovarian cancer risk in the smaller, five-study replication study (Pper-allele = 0.03). Combined analysis of the discovery and replication sets for this TERT SNP showed an increased risk of serous ovarian cancer among non-Hispanic whites [adj. ORper-allele 1.14 (1.04–1.24) p = 0.003]. Our study adds to the growing evidence that, like the 8q24 locus, the telomerase reverse transcriptase locus at 5p15.33, is a general cancer susceptibility locus. PMID:20628624

  10. Mesenchymal stem cell subpopulations: phenotype, property and therapeutic potential.

    PubMed

    Mo, Miaohua; Wang, Shan; Zhou, Ying; Li, Hong; Wu, Yaojiong

    2016-09-01

    Mesenchymal stem cells (MSC) are capable of differentiating into cells of multiple cell lineages and have potent paracrine effects. Due to their easy preparation and low immunogenicity, MSC have emerged as an extremely promising therapeutic agent in regenerative medicine for diverse diseases. However, MSC are heterogeneous with respect to phenotype and function in current isolation and cultivation regimes, which often lead to incomparable experimental results. In addition, there may be specific stem cell subpopulations with definite differentiation capacity toward certain lineages in addition to stem cells with multi-differentiation potential. Recent studies have identified several subsets of MSC which exhibit distinct features and biological activities, and enhanced therapeutic potentials for certain diseases. In this review, we give an overview of these subsets for their phenotypic, biological and functional properties. PMID:27141940

  11. Identification of drug-resistant subpopulations in canine hemangiosarcoma.

    PubMed

    Khammanivong, A; Gorden, B H; Frantz, A M; Graef, A J; Dickerson, E B

    2016-09-01

    Canine hemangiosarcoma is a rapidly progressive disease that is poorly responsive to conventional chemotherapy. Despite numerous attempts to advance treatment options and improve outcomes, drug resistance remains a hurdle to successful therapy. To address this problem, we used recently characterized progenitor cell populations derived from canine hemangiosarcoma cell lines and grown as non-adherent spheres to identify potential drug resistance mechanisms as well as drug-resistant cell populations. Cells from sphere-forming cultures displayed enhanced resistance to chemotherapy drugs, expansion of dye-excluding side populations and altered ATP-binding cassette (ABC) transporter expression. Invasion studies demonstrated variability between cell lines as well as between sphere and monolayer cell populations. Collectively, our results suggest that sphere cell populations contain distinct subpopulations of drug-resistant cells that utilize multiple mechanisms to evade cytotoxic drugs. Our approach represents a new tool for the study of drug resistance in hemangiosarcoma, which could alter approaches for treating this disease. PMID:25112808

  12. Analysis of marker-defined HNSCC subpopulations reveals a dynamic regulation of tumor initiating properties.

    PubMed

    Bragado, Paloma; Estrada, Yeriel; Sosa, Maria Soledad; Avivar-Valderas, Alvaro; Cannan, David; Genden, Eric; Teng, Marita; Ranganathan, Aparna C; Wen, Huei-Chi; Kapoor, Avnish; Bernstein, Emily; Aguirre-Ghiso, Julio A

    2012-01-01

    Head and neck squamous carcinoma (HNSCC) tumors carry dismal long-term prognosis and the role of tumor initiating cells (TICs) in this cancer is unclear. We investigated in HNSCC xenografts whether specific tumor subpopulations contributed to tumor growth. We used a CFSE-based label retentions assay, CD49f (α6-integrin) surface levels and aldehyde dehydrogenase (ALDH) activity to profile HNSCC subpopulations. The tumorigenic potential of marker-positive and -negative subpopulations was tested in nude (Balb/c nu/nu) and NSG (NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ) mice and chicken embryo chorioallantoic membrane (CAM) assays. Here we identified in HEp3, SQ20b and FaDu HNSCC xenografts a subpopulation of G0/G1-arrested slow-cycling CD49f(high)/ALDH1A1(high)/H3K4/K27me3(low) subpopulation (CD49f+) of tumor cells. A strikingly similar CD49f(high)/H3K27me3(low) subpopulation is also present in primary human HNSCC tumors and metastases. While only sorted CD49f(high)/ALDH(high), label retaining cells (LRC) proliferated immediately in vivo, with time the CD49f(low)/ALDH(low), non-LRC (NLRC) tumor cell subpopulations were also able to regain tumorigenic capacity; this was linked to restoration of CD49f(high)/ALDH(high), label retaining cells. In addition, CD49f is required for HEp3 cell tumorigenicity and to maintain low levels of H3K4/K27me3. CD49f+ cells also displayed reduced expression of the histone-lysine N-methyltransferase EZH2 and ERK1/2 phosphorylation. This suggests that although transiently quiescent, their unique chromatin structure is poised for rapid transcriptional activation. CD49f- cells can "reprogram" and also achieve this state eventually. We propose that in HNSCC tumors, epigenetic mechanisms likely driven by CD49f signaling dynamically regulate HNSCC xenograft phenotypic heterogeneity. This allows multiple tumor cell subpopulations to drive tumor growth suggesting that their dynamic nature renders them a "moving target" and their eradication might

  13. Analysis of Marker-Defined HNSCC Subpopulations Reveals a Dynamic Regulation of Tumor Initiating Properties

    PubMed Central

    Bragado, Paloma; Estrada, Yeriel; Sosa, Maria Soledad; Avivar-Valderas, Alvaro; Cannan, David; Genden, Eric; Teng, Marita; Ranganathan, Aparna C.; Wen, Huei-Chi; Kapoor, Avnish; Bernstein, Emily; Aguirre-Ghiso, Julio A.

    2012-01-01

    Head and neck squamous carcinoma (HNSCC) tumors carry dismal long-term prognosis and the role of tumor initiating cells (TICs) in this cancer is unclear. We investigated in HNSCC xenografts whether specific tumor subpopulations contributed to tumor growth. We used a CFSE-based label retentions assay, CD49f (α6-integrin) surface levels and aldehyde dehydrogenase (ALDH) activity to profile HNSCC subpopulations. The tumorigenic potential of marker-positive and -negative subpopulations was tested in nude (Balb/c nu/nu) and NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice and chicken embryo chorioallantoic membrane (CAM) assays. Here we identified in HEp3, SQ20b and FaDu HNSCC xenografts a subpopulation of G0/G1-arrested slow-cycling CD49fhigh/ALDH1A1high/H3K4/K27me3low subpopulation (CD49f+) of tumor cells. A strikingly similar CD49fhigh/H3K27me3low subpopulation is also present in primary human HNSCC tumors and metastases. While only sorted CD49fhigh/ALDHhigh, label retaining cells (LRC) proliferated immediately in vivo, with time the CD49flow/ALDHlow, non-LRC (NLRC) tumor cell subpopulations were also able to regain tumorigenic capacity; this was linked to restoration of CD49fhigh/ALDHhigh, label retaining cells. In addition, CD49f is required for HEp3 cell tumorigenicity and to maintain low levels of H3K4/K27me3. CD49f+ cells also displayed reduced expression of the histone-lysine N-methyltransferase EZH2 and ERK1/2phosphorylation. This suggests that although transiently quiescent, their unique chromatin structure is poised for rapid transcriptional activation. CD49f− cells can “reprogram” and also achieve this state eventually. We propose that in HNSCC tumors, epigenetic mechanisms likely driven by CD49f signaling dynamically regulate HNSCC xenograft phenotypic heterogeneity. This allows multiple tumor cell subpopulations to drive tumor growth suggesting that their dynamic nature renders them a “moving target” and their eradication might require more

  14. A Multi-Omics Approach Identifies Key Hubs Associated with Cell Type-Specific Responses of Airway Epithelial Cells to Staphylococcal Alpha-Toxin

    PubMed Central

    Richter, Erik; Harms, Manuela; Ventz, Katharina; Gierok, Philipp; Chilukoti, Ravi Kumar; Hildebrandt, Jan-Peter; Mostertz, Jörg; Hochgräfe, Falko

    2015-01-01

    Responsiveness of cells to alpha-toxin (Hla) from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects. PMID:25816343

  15. Harnessing 3D models of mammary epithelial morphogenesis: An off the beaten path approach to identify candidate biomarkers of early stage breast cancer.

    PubMed

    Rossetti, Stefano; Bshara, Wiam; Reiners, Johanna A; Corlazzoli, Francesca; Miller, Austin; Sacchi, Nicoletta

    2016-10-01

    Regardless of the etiological factor, an aberrant morphology is the common hallmark of ductal carcinoma in situ (DCIS), which is a highly heterogeneous disease. To test if critical core morphogenetic mechanisms are compromised by different mutations, we performed proteomics analysis of five mammary epithelial HME1 mutant lines that develop a DCIS-like morphology in three dimensional (3D) culture. Here we show first, that all HME1 mutant lines share a common protein signature highlighting an inverse deregulation of two annexins, ANXA2 and ANXA8. Either ANXA2 downregulation or ANXA8 upregulation in the HME1 cell context are per se sufficient to confer a 3D DCIS-like morphology. Seemingly, different mutations impinged on a common mechanism that differentially regulates the two annexins. Second, we show that ANXA8 expression is significantly higher in DCIS tissue samples versus normal breast tissue and atypical ductal hyperplasia (ADH). Apparently, ANXA8 expression is significantly more upregulated in ER-negative versus ER-positive cases, and significantly correlates with tumor stage, grade and positive lymph node. Based on our study, 3D mammary morphogenesis models can be an alternate/complementary strategy for unraveling new DCIS mechanisms and biomarkers. PMID:27422542

  16. Transcriptional regulators transforming growth factor-β1 and estrogen-related receptor-α identified as putative mediators of calf rumen epithelial tissue development and function during weaning.

    PubMed

    Connor, E E; Baldwin, R L; Walker, M P; Ellis, S E; Li, C; Kahl, S; Chung, H; Li, R W

    2014-07-01

    Molecular mechanisms regulating rumen epithelial development remain largely unknown. To identify gene networks and regulatory factors controlling rumen development, Holstein bull calves (n=18) were fed milk replacer only (MRO) until 42 d of age. Three calves each were euthanized at 14 and 42 d of age for tissue collection to represent preweaning, and the remaining calves were provided diets of either milk replacer + orchard grass hay (MH; n=6) to initiate weaning without development of rumen papillae, or milk replacer + calf starter (MG; n=6) to initiate weaning and development of rumen papillae. At 56 and 70 d of age, 3 calves from the MH and MG groups were euthanized for collection of rumen epithelium. Total RNA and protein were extracted for microarray analysis and to validate detected changes in selected protein expression, respectively. As expected, calves fed MRO had no rumen papillae and development of papillae was greater in MG versus MH calves. Differentially expressed genes between the MRO diet at d 42 (preweaning) versus the MG or MH diets at d 56 (during weaning) were identified using permutation analysis of differential expression. Expression of 345 and 519 transcripts was uniquely responsive to MG and MH feeding, respectively. Ingenuity Pathway Analysis (Qiagen, Redwood City, CA) indicated that the top-ranked biological function affected by the MG diet was the cell cycle, and TFGB1, FBOX01, and PPARA were identified as key transcriptional regulators of genes responsive to the MG diet and associated with development of rumen papillae. Increased expressions of TGFB1 mRNA and protein in response to the MG diet were confirmed by subsequent analyses. The top-ranking biological function affected by the MH diet was energy production. Receptors for IGF-1 and insulin, ESRRA, and PPARD were identified by ingenuity pathway analysis as transcriptional regulators of genes responsive to the MH diet. Further analysis of TGFB1 and ESRRA mRNA expression in rumen

  17. CHILDREN AS A SENSITIVE SUBPOPULATION FOR THE RISK ASSESSMENT PROCESS

    EPA Science Inventory

    Children as a sensitive subpopulation for the risk assessment process
    Abstract
    For cancer risk assessment purposes, it is necessary to consider how to incorporate sensitive subpopulations into the process to ensure that they are appropriately protected. Children represent o...

  18. Metabolic history impacts mammary tumor epithelial hierarchy and early drug response in mice.

    PubMed

    Montales, Maria Theresa E; Melnyk, Stepan B; Liu, Shi J; Simmen, Frank A; Liu, Y Lucy; Simmen, Rosalia C M

    2016-09-01

    The emerging links between breast cancer and metabolic dysfunctions brought forth by the obesity pandemic predict a disproportionate early disease onset in successive generations. Moreover, sensitivity to chemotherapeutic agents may be influenced by the patient's metabolic status that affects the disease outcome. Maternal metabolic stress as a determinant of drug response in progeny is not well defined. Here, we evaluated mammary tumor response to doxorubicin in female mouse mammary tumor virus-Wnt1 transgenic offspring exposed to a metabolically compromised environment imposed by maternal high-fat diet. Control progeny were from dams consuming diets with regular fat content. Maternal high-fat diet exposure increased tumor incidence and reduced tumor latency but did not affect tumor volume response to doxorubicin, compared with control diet exposure. However, doxorubicin-treated tumors from high-fat-diet-exposed offspring demonstrated higher proliferation status (Ki-67), mammary stem cell-associated gene expression (Notch1, Aldh1) and basal stem cell-like (CD29(hi)CD24(+)) epithelial subpopulation frequencies, than tumors from control diet progeny. Notably, all epithelial subpopulations (CD29(hi)CD24(+), CD29(lo)CD24(+), CD29(hi)CD24(+)Thy1(+)) in tumors from high-fat-diet-exposed offspring were refractory to doxorubicin. Further, sera from high-fat-diet-exposed offspring promoted sphere formation of mouse mammary tumor epithelial cells and of human MCF7 cells. Untargeted metabolomics analyses identified higher levels of kynurenine and 2-hydroxyglutarate in plasma of high-fat diet than control diet offspring. Kynurenine/doxorubicin co-treatment of MCF7 cells enhanced the ability to form mammosphere and decreased apoptosis, relative to doxorubicin-only-treated cells. Maternal metabolic dysfunctions during pregnancy and lactation may be targeted to reduce breast cancer risk and improve early drug response in progeny, and may inform clinical management of disease

  19. Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae.

    PubMed

    Band, Victor I; Crispell, Emily K; Napier, Brooke A; Herrera, Carmen M; Tharp, Greg K; Vavikolanu, Kranthi; Pohl, Jan; Read, Timothy D; Bosinger, Steven E; Trent, M Stephen; Burd, Eileen M; Weiss, David S

    2016-01-01

    Antibiotic resistance is a major public health threat, further complicated by unexplained treatment failures caused by bacteria that appear antibiotic susceptible. We describe an Enterobacter cloacae isolate harbouring a minor subpopulation that is highly resistant to the last-line antibiotic colistin. This subpopulation was distinct from persisters, became predominant in colistin, returned to baseline after colistin removal and was dependent on the histidine kinase PhoQ. During murine infection, but in the absence of colistin, innate immune defences led to an increased frequency of the resistant subpopulation, leading to inefficacy of subsequent colistin therapy. An isolate with a lower-frequency colistin-resistant subpopulation similarly caused treatment failure but was misclassified as susceptible by current diagnostics once cultured outside the host. These data demonstrate the ability of low-frequency bacterial subpopulations to contribute to clinically relevant antibiotic resistance, elucidating an enigmatic cause of antibiotic treatment failure and highlighting the critical need for more sensitive diagnostics. PMID:27572838

  20. Transcriptome Sequencing of Tumor Subpopulations Reveals a Spectrum of Therapeutic Options for Squamous Cell Lung Cancer

    PubMed Central

    Barrett, Christian L.; Schwab, Richard B.; Jung, HyunChul; Crain, Brian; Goff, Daniel J.; Jamieson, Catriona H. M.; Thistlethwaite, Patricia A.; Harismendy, Olivier; Carson, Dennis A.; Frazer, Kelly A.

    2013-01-01

    Background The only therapeutic options that exist for squamous cell lung carcinoma (SCC) are standard radiation and cytotoxic chemotherapy. Cancer stem cells (CSCs) are hypothesized to account for therapeutic resistance, suggesting that CSCs must be specifically targeted. Here, we analyze the transcriptome of CSC and non-CSC subpopulations by RNA-seq to identify new potential therapeutic strategies for SCC. Methods We sorted a SCC into CD133− and CD133+ subpopulations and then examined both by copy number analysis (CNA) and whole genome and transcriptome sequencing. We analyzed The Cancer Genome Atlas (TCGA) transcriptome data of 221 SCCs to determine the generality of our observations. Results Both subpopulations highly expressed numerous mRNA isoforms whose protein products are active drug targets for other cancers; 31 (25%) correspond to 18 genes under active investigation as mAb targets and an additional 4 (3%) are of therapeutic interest. Moreover, we found evidence that both subpopulations were proliferatively driven by very high levels of c-Myc and the TRAIL long isoform (TRAILL) and that normal apoptotic responses to high expression of these genes was prevented through high levels of Mcl-1L and Bcl-xL and c-FlipL—isoforms for which drugs are now in clinical development. SCC RNA-seq data (n = 221) from TCGA supported our findings. Our analysis is inconsistent with the CSC concept that most cells in a cancer have lost their proliferative potential. Furthermore, our study suggests how to target both the CSC and non-CSC subpopulations with one treatment strategy. Conclusions Our study is relevant to SCC in particular for it presents numerous potential options to standard therapy that target the entire tumor. In so doing, it demonstrates how transcriptome sequencing provides insights into the molecular underpinnings of cancer propagating cells that, importantly, can be leveraged to identify new potential therapeutic options for cancers beyond what is

  1. Effects of repeated electroejaculations on kinematic sperm subpopulations and quality markers of Mexican creole goats.

    PubMed

    Vázquez, A J F; Cedillo, M J; Quezada, V J; Rivas, A C; Morales, E C L; Ayala, E M E; Hernández, M J; González, R A; Aragón, M A

    2015-03-01

    Here we show the effects of repeated electroejaculation (EE) on mean values of motility, mitochondrial functionality, and expression of active caspases on goat sperm obtained by EE. Evaluations were done using CASA and flow cytometry. A strategy for identification of kinematic sperm subpopulations, when individual data of sperm are not provided by the CASA system, is provided. Fifty semen samples, five of each of ten adult creole goats, were obtained by electroejaculation. Mean values of total motility, progressive motility and flow cytometry evaluations were compared among EEs. Relationships among mean values of variables were investigated using Spearman correlation and principal component analysis (PCA). For identification of kinematic sperm subpopulations, PCA followed by hierarchical clustering was applied on data of the intervals provided automatically by the CASA system. Total motility does no change after repeated EE. Mean values of motility parameters and molecular markers were unrelated in multivariate space, but bivariate correlations were found. Values in upper and lower intervals defined clearly the sperm subpopulations, which had motility parameters changing over time. Taken together, our results show that repeated EE does not affect mean values of total motility, that molecular markers are not related with motility parameters, and that it is possible to identify kinematic sperm subpopulations when individual data, of motility parameters, are not provided by the CASA system. PMID:25600145

  2. Screening of an E. coli O157:H7 Bacterial Artificial Chromosome Library by Comparative Genomic Hybridization to Identify Genomic Regions Contributing to Growth in Bovine Gastrointestinal Mucus and Epithelial Cell Colonization

    PubMed Central

    Bai, Jianing; McAteer, Sean P.; Paxton, Edith; Mahajan, Arvind; Gally, David L.; Tree, Jai J.

    2011-01-01

    Enterohemorrhagic E. coli (EHEC) O157:H7 can cause serious gastrointestinal and systemic disease in humans following direct or indirect exposure to ruminant feces containing the bacterium. The main colonization site of EHEC O157:H7 in cattle is the terminal rectum where the bacteria intimately attach to the epithelium and multiply in the intestinal mucus. This study aimed to identify genomic regions of EHEC O157:H7 that contribute to colonization and multiplication at this site. A bacterial artificial chromosome (BAC) library was generated from a derivative of the sequenced E. coli O157:H7 Sakai strain. The library contains 1152 clones averaging 150 kbp. To verify the library, clones containing a complete locus of enterocyte effacement (LEE) were identified by DNA hybridization. In line with a previous report, these did not confer a type III secretion (T3S) capacity to the K-12 host strain. However, conjugation of one of the BAC clones into a strain containing a partial LEE deletion restored T3S. Three hundred eighty-four clones from the library were subjected to two different selective screens; one involved three rounds of adherence assays to bovine primary rectal epithelial cells while the other competed the clones over three rounds of growth in bovine rectal mucus. The input strain DNA was then compared with the selected strains using comparative genomic hybridization (CGH) on an E. coli microarray. The adherence assay enriched for pO157 DNA indicating the importance of this plasmid for colonization of rectal epithelial cells. The mucus assay enriched for multiple regions involved in carbohydrate utilization, including hexuronate uptake, indicating that these regions provide a competitive growth advantage in bovine mucus. This BAC-CGH approach provides a positive selection screen that complements negative selection transposon-based screens. As demonstrated, this may be of particular use for identifying genes with redundant functions such as adhesion and carbon

  3. The Rotation of Subpopulations in ω Centauri

    NASA Astrophysics Data System (ADS)

    Pancino, E.; Galfo, A.; Ferraro, F. R.; Bellazzini, M.

    2007-06-01

    We present the first result of the Ital-FLAMES survey of red giant branch (RGB) stars in ω Cen. Radial velocities with a precision of ~0.5 km s-1 are presented for 650 members of ω Cen observed with FLAMES-GIRAFFE at the Very Large Telescope. We found that stars belonging to the metal-poor (RGB-MP), metal-intermediate (RGB-MInt), and metal-rich (RGB-a) subpopulations of ω Cen are all compatible with having the same rotational pattern. Our results appear to contradict past findings by Norris et al., who could not detect any rotational signature for metal-rich stars. The slightly higher precision of the present measurements and the much larger sample size, especially for the stars richer in metals, appear as the most likely explanations for this discrepancy. The result presented here weakens the body of evidence in favor of a merger event in the past history of ω Cen. Based on data obtained with the Giraffe-FLAMES facility of ESO Very Large Telescope during the Ital-FLAMES GTO program 71.D-0217(A). Also based on data from the VALD and GEISA databases.

  4. Distinct transcriptome profiles identified in normal human bronchial epithelial cells after exposure to γ-rays and different elemental particles of high Z and energy

    PubMed Central

    2013-01-01

    Background Ionizing radiation composed of accelerated ions of high atomic number (Z) and energy (HZE) deposits energy and creates damage in cells in a discrete manner as compared to the random deposition of energy and damage seen with low energy radiations such as γ- or x-rays. Such radiations can be highly effective at cell killing, transformation, and oncogenesis, all of which are concerns for the manned space program and for the burgeoning field of HZE particle radiotherapy for cancer. Furthermore, there are differences in the extent to which cells or tissues respond to such exposures that may be unrelated to absorbed dose. Therefore, we asked whether the energy deposition patterns produced by different radiation types would cause different molecular responses. We performed transcriptome profiling using human bronchial epithelial cells (HBECs) after exposure to γ-rays and to two different HZE particles (28Si and 56Fe) with different energy transfer properties to characterize the molecular response to HZE particles and γ-rays as a function of dose, energy deposition pattern, and time post-irradiation. Results Clonogenic assay indicated that the relative biological effectiveness (RBE) for 56Fe was 3.91 and for 28Si was 1.38 at 34% cell survival. Unsupervised clustering analysis of gene expression segregated samples according to the radiation species followed by the time after irradiation, whereas dose was not a significant parameter for segregation of radiation response. While a subset of genes associated with p53-signaling, such as CDKN1A, TRIM22 and BTG2 showed very similar responses to all radiation qualities, distinct expression changes were associated with the different radiation species. Gene enrichment analysis categorized the differentially expressed genes into functional groups related to cell death and cell cycle regulation for all radiation types, while gene pathway analysis revealed that the pro-inflammatory Acute Phase Response Signaling was

  5. Bcl-xL mediates therapeutic resistance of a mesenchymal breast cancer cell subpopulation

    PubMed Central

    Keitel, Ulrike; Scheel, Andreas; Thomale, Jürgen; Halpape, Rovena; Kaulfuß, Silke; Scheel, Christina; Dobbelstein, Matthias

    2014-01-01

    The transition from an epithelial to a mesenchymal phenotype (EMT) confers increased invasiveness and clonogenic potential to tumor cells. We used a breast epithelium-derived cell culture model to evaluate the impact of EMT on the cellular sensitivity towards chemotherapeutics and apoptotic stimuli. Cells that had passed through an EMT acquired resistance towards chemotherapeutics and death ligands. Mechanistically, we found that the levels of the apoptosis inhibitor Bcl-xL were strongly enhanced in mesenchymal versus epithelial cells, whereas the pro-apoptotic proteins Bim and Puma were diminished. Clinical samples from breast cancer showed enhanced Bcl-xL staining in cells that had dispersed into the desmoplastic stroma, as compared to cells that were part of large tumor cell aggregates, suggesting increased Bcl-xL expression when cells invade the stroma. Bcl-xL was necessary for apoptotic resistance in mesenchymal cells, and its expression was sufficient to confer such resistance to epithelial cells. To antagonize Bcl-xL, BH3-mimetics were used. They successfully interfered with the proliferation and survival of mesenchymal cells, and also inhibited the growth of xenograft tumors raised from the mesenchymal subpopulation. We conclude that enhanced Bcl-xL levels confer resistance to cells upon EMT, and that Bcl-xL represents a promising target for therapy directed against invasive cancer cells. PMID:25473892

  6. Identification of a Bipotent Epithelial Progenitor Population in the Adult Thymus

    PubMed Central

    Ulyanchenko, Svetlana; O’Neill, Kathy E.; Medley, Tanya; Farley, Alison M.; Vaidya, Harsh J.; Cook, Alistair M.; Blair, Natalie F.; Blackburn, C. Clare

    2016-01-01

    Summary Thymic epithelial cells (TECs) are critically required for T cell development, but the cellular mechanisms that maintain adult TECs are poorly understood. Here, we show that a previously unidentified subpopulation, EpCam+UEA1−Ly-51+PLET1+MHC class IIhi, which comprises <0.5% of adult TECs, contains bipotent TEC progenitors that can efficiently generate both cortical (c) TECs and medullary (m) TECs. No other adult TEC population tested in this study contains this activity. We demonstrate persistence of PLET1+Ly-51+ TEC-derived cells for 9 months in vivo, suggesting the presence of thymic epithelial stem cells. Additionally, we identify cTEC-restricted short-term progenitor activity but fail to detect high efficiency mTEC-restricted progenitors in the adult thymus. Our data provide a phenotypically defined adult thymic epithelial progenitor/stem cell that is able to generate both cTECs and mTECs, opening avenues for improving thymus function in patients. PMID:26997270

  7. Identification of a Bipotent Epithelial Progenitor Population in the Adult Thymus.

    PubMed

    Ulyanchenko, Svetlana; O'Neill, Kathy E; Medley, Tanya; Farley, Alison M; Vaidya, Harsh J; Cook, Alistair M; Blair, Natalie F; Blackburn, C Clare

    2016-03-29

    Thymic epithelial cells (TECs) are critically required for T cell development, but the cellular mechanisms that maintain adult TECs are poorly understood. Here, we show that a previously unidentified subpopulation, EpCam(+)UEA1(-)Ly-51(+)PLET1(+)MHC class II(hi), which comprises <0.5% of adult TECs, contains bipotent TEC progenitors that can efficiently generate both cortical (c) TECs and medullary (m) TECs. No other adult TEC population tested in this study contains this activity. We demonstrate persistence of PLET1(+)Ly-51(+) TEC-derived cells for 9 months in vivo, suggesting the presence of thymic epithelial stem cells. Additionally, we identify cTEC-restricted short-term progenitor activity but fail to detect high efficiency mTEC-restricted progenitors in the adult thymus. Our data provide a phenotypically defined adult thymic epithelial progenitor/stem cell that is able to generate both cTECs and mTECs, opening avenues for improving thymus function in patients. PMID:26997270

  8. Separation and identification of equine leukocyte populations and subpopulations.

    PubMed

    Dutta, S K; Bumgardner, M K; Scott, J C; Myrup, A C

    1981-06-01

    Various methods of separation and identification of major equine leukocyte populations and subpopulations were used. The purity of T and B lymphocytes separated in Sephadex anti-equine F(ab')2 columns was 87% to 99% and 83% of 97%, respectively. The purity of T lymphocytes separated in nylon-wool columns was 89% to 98%. Preparations of B lymphocytes separated in glass-bead columns were 68% to 79% pure. The presence (or absence) of surface immunoglobulin by immunofluorescence was the most consistent and reliable method for the identification of B or T lymphocytes, respectively. However, the erythrocyte-antibody-complement-rosette method for the identification of B cells and the erythrocyte-rosette method for the identification of T cells were not suitable. Monocytes were separated by the adherence method, and the purity, as identified by the latex particle ingestion procedure, was 70% to 78%. Electron microscopy of monocytes stained by peroxidase activity did not identify these cells. The purity of neutrophils obtained by the Ficoll-Hypaque separation method was 95% to 97%. The merits and usefulness of these methods were discussed. PMID:6974519

  9. Sub-populations among the Jupiter Trojans

    NASA Astrophysics Data System (ADS)

    Wong, I.; Brown, M.

    2014-07-01

    observed bimodalities are self-consistent and categorize 220 of the 842 Trojans with absolute magnitudes in the range H<12.3 into the two color populations. We demonstrate that the magnitude distributions of the two color populations are distinct to a high confidence level, suggesting that the red and less-red Trojans were created in different locations and/or experienced different evolutionary histories. This observation has broad implications for the formation and composition of the Trojans as well as the details of their purported inward migration. Much can be learned about the evolution of the Trojans since formation by examining the faint objects, which have undergone significant collisional alteration. To explore this, we have collected color measurements of a large number of Trojans using the Suprime-Cam instrument on the Subaru telescope. The new data extend the known magnitude-color distributions of both Trojan sub-populations to much smaller sizes. This enables a fuller comparison between the two sub- populations as well as with attested sub-populations within the Kuiper Belt, thereby giving insight into the formation and evolutionary history of minor bodies in the outer Solar System.

  10. Three-Dimensional Telomeric Analysis of Isolated Circulating Tumor Cells (CTCs) Defines CTC Subpopulations12

    PubMed Central

    Adebayo Awe, Julius; Xu, Mark Chu; Wechsler, Janine; Benali-Furet, Naoual; Cayre, Yvon E; Saranchuk, Jeff; Drachenberg, Darrel; Mai, Sabine

    2013-01-01

    Circulating tumor cells (CTCs) have been identified with the potential to serve as suitable biomarkers for tumor stage and progression, but the availability of effective isolation technique(s) coupled with detailed molecular characterization have been the challenges encountered in making CTCs clinically relevant. For the first time, we combined isolation of CTCs using the ScreenCell filtration technique with quantitative analysis of CTC telomeres by TeloView. This resulted in the identification and molecular characterization of different subpopulations of CTCs in the same patient. Three-dimensional (3D) telomeric analysis was carried out on isolated CTCs of 19 patients that consisted of four different tumor types, namely, prostate, colon, breast, melanoma, and one lung cancer cell line. With telomeric analysis of the filter-isolated CTCs, the level of chromosomal instability (CIN) of the CTCs can be determined. Our study shows that subpopulations of CTCs can be identified on the basis of their 3D telomeric properties. PMID:23418617

  11. Adipogenic Potential of Adipose Stem Cell Subpopulations

    PubMed Central

    Li, Han; Zimmerlin, Ludovic; Marra, Kacey G.; Donnenberg, Vera S.; Donnenberg, Albert D.; Rubin, J. Peter

    2014-01-01

    Background Adipose stem cells represent a heterogenous population. Understanding the functional characteristics of subpopulations will be useful in developing adipose stem cell–based therapies for regenerative medicine applications. The aim of this study was to define distinct populations within the stromal vascular fraction based on surface marker expression, and to evaluate the ability of each cell type to differentiate to mature adipocytes. Methods Subcutaneous whole adipose tissue was obtained by abdominoplasty from human patients. The stromal vascular fraction was separated and four cell populations were isolated by flow cytometry and studied. Candidate perivascular cells (pericytes) were defined as CD146+/CD31−/CD34−. Two CD31+ endothelial populations were detected and differentiated by CD34 expression. These were tentatively designated as mature endothelial (CD 31+/CD34−), and immature endothelial (CD31+/CD34+). Both endothelial populations were heterogeneous with respect to CD146. The CD31−/CD34+ fraction (preadipocyte candidate) was also CD90+ but lacked CD146 expression. Results Proliferation was greatest in the CD31−/CD34+ group and slowest in the CD146+ group. Expression of adipogenic genes, peroxisome proliferator-activated receptor-γ, and fatty acid binding protein 4, were significantly higher in the CD31−/CD34+ group compared with all other populations after in vitro adipogenic differentiation. This group also demonstrated the highest proportion of AdipoRed lipid staining. Conclusions The authors have isolated four distinct stromal populations from human adult adipose tissue and characterized their adipogenic potential. Of these four populations, the CD31/CD34+ group is the most prevalent and has the greatest potential for adipogenic differentiation. This cell type appears to hold the most promise for adipose tissue engineering. PMID:21572381

  12. Electronic Sorting of Immune Cell Subpopulations Based on Highly Plastic Genes.

    PubMed

    Wang, Pingzhang; Han, Wenling; Ma, Dalong

    2016-07-15

    Immune cells are highly heterogeneous and plastic with regard to gene expression and cell phenotype. In this study, we categorized genes into those with low and high gene plasticity, and those categories revealed different functions and applications. We proposed that highly plastic genes could be suited for the labeling of immune cell subpopulations; thus, novel immune cell subpopulations could be identified by gene plasticity analysis. For this purpose, we systematically analyzed highly plastic genes in human and mouse immune cells. In total, 1,379 human and 883 mouse genes were identified as being extremely plastic. We also expanded our previous immunoinformatic method, electronic sorting, which surveys big data to perform virtual analysis. This approach used correlation analysis and took dosage changes into account, which allowed us to identify the differentially expressed genes. A test with human CD4(+) T cells supported the method's feasibility, effectiveness, and predictability. For example, with the use of human nonregulatory T cells, we found that FOXP3(hi)CD4(+) T cells were highly expressive of certain known molecules, such as CD25 and CTLA4, and that this process of investigation did not require isolating or inducing these immune cells in vitro. Therefore, the sorting process helped us to discover the potential signature genes or marker molecules and to conduct functional evaluations for immune cell subpopulations. Finally, in human CD4(+) T cells, 747 potential immune cell subpopulations and their candidate signature genes were identified, which provides a useful resource for big data-driven knowledge discoveries. PMID:27288532

  13. Isolation of melanoma cell subpopulations using negative selection

    PubMed Central

    Slipicevic, Ana; Somasundaram, Rajasekharan; Sproesser, Katrin; Herlyn, Meenhard

    2014-01-01

    Melanomas are phenotypically and functiwonally heterogeneous tumors comprising of distinct subpopulations that drive disease progression and are responsible for resistance to therapy. Identification and characterization of such subpopulations are highly important to develop novel targeted therapies. However, this can be a challenging task as there is a lack of clearly defined markers to distinguish the melanoma subpopulations from a general tumor cell population. Also, there is a lack of optimal isolation methods and functional assays that can fully recapitulate their phenotype. Here we describe a method for isolating tumor cells from fresh human tumor tissue specimens using an antibody coupled magnetic bead sorting technique that is well established in our laboratory. Thus, melanoma cells are enriched by negative cell sorting and elimination of non-tumor cell population such as erythrocytes, leukocytes, and endothelial cells. Enriched unmodified tumor cells can be further used for phenotypic and functional characterization of melanoma subpopulations. PMID:24258995

  14. Could Killing Bacterial Subpopulations Hit Tuberculosis out of the Park?

    PubMed

    Baranowski, Catherine; Rubin, Eric J

    2016-07-14

    One hurdle to treating tuberculosis could be that it is so difficult to kill nonreplicating subpopulations of the causative pathogens. This work describes two new cephalosporin derivatives that specifically target this population of Mycobacterium tuberculosis. PMID:27322073

  15. Drosophila Wnt and STAT Define Apoptosis-Resistant Epithelial Cells for Tissue Regeneration after Irradiation.

    PubMed

    Verghese, Shilpi; Su, Tin Tin

    2016-09-01

    Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR) that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. We have identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1) and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog) activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. Our findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue. PMID:27584613

  16. Applications of mouse airway epithelial cell culture for asthma research.

    PubMed

    Horani, Amjad; Dickinson, John D; Brody, Steven L

    2013-01-01

    Primary airway epithelial cell culture provides a valuable tool for studying cell differentiation, cell-cell interactions, and the role of immune system factors in asthma pathogenesis. In this chapter, we discuss the application of mouse tracheal epithelial cell cultures for the study of asthma biology. A major advantage of this system is the ability to use airway epithelial cells from mice with defined genetic backgrounds. The in vitro proliferation and differentiation of mouse airway epithelial cells uses the air-liquid interface condition to generate well-differentiated epithelia with characteristics of native airways. Protocols are provided for manipulation of differentiation, induction of mucous cell metaplasia, genetic modification, and cell and pathogen coculture. Assays for the assessment of gene expression, responses of cells, and analysis of specific cell subpopulations within the airway epithelium are included. PMID:23943446

  17. Expressions of Machismo in Colorectal Cancer Screening Among New Mexico Hispanic Subpopulations

    PubMed Central

    Getrich, Christina M.; Sussman, Andrew L.; Helitzer, Deborah L.; Hoffman, Richard M.; Warner, Teddy D.; Sánchez, Victoria; Solares, Angélica; Rhyne, Robert L.

    2013-01-01

    Although national colorectal cancer (CRC) incidence rates have steadily decreased, the rate for New Mexico Hispanics has been increasing and screening rates are low. We conducted an exploratory qualitative study to determine barriers to CRC screening for New Mexico Hispanics. We found that machismo served as a dynamic influence on men’s health seeking behaviors; however, it was conceptualized differently by two distinct Hispanic subpopulations and therefore appeared to play a different role in shaping their screening attitudes and behaviors. Machismo emerged as more of an influence for Mexican men, who expressed concern over colonoscopies being potentially transformative and/or stigmatizing, but was not as salient for Hispanos, who viewed the colonoscopy as “strictly medical” and were more concerned with discomfort and pain. This study highlights the importance of identifying varying characteristics among subpopulations to better understand screening barriers and provide optimal CRC screening counseling in primary care settings. PMID:22138258

  18. An infrared spectral signature of human lymphocyte subpopulations from peripheral blood.

    PubMed

    Wald, N; Legat, A; Meyer, C; Speiser, D E; Goormaghtigh, E

    2015-04-01

    Metastatic melanomas are frequently refractory to most adjuvant therapies such as chemotherapies and radiotherapies. Recently, immunotherapies have shown good results in the treatment of some metastatic melanomas. Immune cell infiltration in the tumor has been associated with successful immunotherapy. More generally, tumor infiltrating lymphocytes (TILs) in the primary tumor and in metastases of melanoma patients have been demonstrated to correlate positively with favorable clinical outcomes. Altogether, these findings suggest the importance of being able to identify, quantify and characterize immune infiltration at the tumor site for a better diagnostic and treatment choice. In this paper, we used Fourier Transform Infrared (FTIR) imaging to identify and quantify different subpopulations of T cells: the cytotoxic T cells (CD8+), the helper T cells (CD4+) and the regulatory T cells (T reg). As a proof of concept, we investigated pure populations isolated from human peripheral blood from 6 healthy donors. These subpopulations were isolated from blood samples by magnetic labeling and purities were assessed by Fluorescence Activated Cell Sorting (FACS). The results presented here show that Fourier Transform Infrared (FTIR) imaging followed by supervised Partial Least Square Discriminant Analysis (PLS-DA) allows an accurate identification of CD4+ T cells and CD8+ T cells (>86%). We then developed a PLS regression allowing the quantification of T reg in a different mix of immune cells (e.g. Peripheral Blood Mononuclear Cells (PBMCs)). Altogether, these results demonstrate the sensitivity of infrared imaging to detect the low biological variability observed in T cell subpopulations. PMID:25553786

  19. Epithelial Cell Adhesion Molecule

    PubMed Central

    Trzpis, Monika; McLaughlin, Pamela M.J.; de Leij, Lou M.F.H.; Harmsen, Martin C.

    2007-01-01

    The epithelial cell adhesion molecule (EpCAM, CD326) is a glycoprotein of ∼40 kd that was originally identified as a marker for carcinoma, attributable to its high expression on rapidly proliferating tumors of epithelial origin. Normal epithelia express EpCAM at a variable but generally lower level than carcinoma cells. In early studies, EpCAM was proposed to be a cell-cell adhesion molecule. However, recent insights revealed a more versatile role for EpCAM that is not limited only to cell adhesion but includes diverse processes such as signaling, cell migration, proliferation, and differentiation. Cell surface expression of EpCAM may actually prevent cell-cell adhesion. Here, we provide a comprehensive review of the current knowledge on EpCAM biology in relation to other cell adhesion molecules. We discuss the implications of the newly identified functions of EpCAM in view of its prognostic relevance in carcinoma, inflammatory pathophysiology, and tissue development and regeneration as well as its role in normal epithelial homeostasis. PMID:17600130

  20. Identifiability, stratification and minimum variance estimation of causal effects.

    PubMed

    Tong, Xingwei; Zheng, Zhongguo; Geng, Zhi

    2005-10-15

    The weakest sufficient condition for the identifiability of causal effects is the weakly ignorable treatment assignment, which implies that potential responses are independent of treatment assignment in each fine subpopulation stratified by a covariate. In this paper, we expand the independence that holds in fine subpopulations to the case that the independence may also hold in several coarse subpopulations, each of which consists of several fine subpopulations and may have overlaps with other coarse subpopulations. We first show that the identifiability of causal effects occurs if and only if the coarse subpopulations partition the whole population. We then propose a principle, called minimum variance principle, which says that the estimator possessing the minimum variance is preferred, in dealing with the stratification and the estimation of the causal effects. The simulation results with the detail programming and a practical example demonstrate that it is a feasible and reasonable way to achieve our goals. PMID:16149123

  1. Regulatory T Cells in Melanoma Revisited by a Computational Clustering of FOXP3+ T Cell Subpopulations

    PubMed Central

    Fujii, Hiroko; Josse, Julie; Tanioka, Miki; Miyachi, Yoshiki; Husson, François

    2016-01-01

    CD4+ T cells that express the transcription factor FOXP3 (FOXP3+ T cells) are commonly regarded as immunosuppressive regulatory T cells (Tregs). FOXP3+ T cells are reported to be increased in tumor-bearing patients or animals and are considered to suppress antitumor immunity, but the evidence is often contradictory. In addition, accumulating evidence indicates that FOXP3 is induced by antigenic stimulation and that some non-Treg FOXP3+ T cells, especially memory-phenotype FOXP3low cells, produce proinflammatory cytokines. Accordingly, the subclassification of FOXP3+ T cells is fundamental for revealing the significance of FOXP3+ T cells in tumor immunity, but the arbitrariness and complexity of manual gating have complicated the issue. In this article, we report a computational method to automatically identify and classify FOXP3+ T cells into subsets using clustering algorithms. By analyzing flow cytometric data of melanoma patients, the proposed method showed that the FOXP3+ subpopulation that had relatively high FOXP3, CD45RO, and CD25 expressions was increased in melanoma patients, whereas manual gating did not produce significant results on the FOXP3+ subpopulations. Interestingly, the computationally identified FOXP3+ subpopulation included not only classical FOXP3high Tregs, but also memory-phenotype FOXP3low cells by manual gating. Furthermore, the proposed method successfully analyzed an independent data set, showing that the same FOXP3+ subpopulation was increased in melanoma patients, validating the method. Collectively, the proposed method successfully captured an important feature of melanoma without relying on the existing criteria of FOXP3+ T cells, revealing a hidden association between the T cell profile and melanoma, and providing new insights into FOXP3+ T cells and Tregs. PMID:26864030

  2. Cell-cycle dependent localization of MELK and its new partner RACK1 in epithelial versus mesenchyme-like cells in Xenopus embryo.

    PubMed

    Chartrain, Isabelle; Le Page, Yann; Hatte, Guillaume; Körner, Roman; Kubiak, Jacek Z; Tassan, Jean-Pierre

    2013-01-01

    Maternal Embryonic Leucine zipper Kinase (MELK) was recently shown to be involved in cell division of Xenopus embryo epithelial cells. The cytokinetic furrow of these cells ingresses asymmetrically and is developmentally regulated. Two subpopulations of xMELK, the mMELK (for "mitotic" xMELK) and iMELK ("interphase" xMELK), which differ in their spatial and temporal regulation, are detected in Xenopus embryo. How cells regulate these two xMELK populations is unknown. In this study we show that, in epithelial cells, xMELK is present at a higher concentration at the apical junctional complex, in contrast to mesenchyme-like cells, which have uniform distribution of cortical MELK. Interestingly, mMELK and iMELK also differ by their requirements towards cell-cell contacts to establish their proper cortical localization both in epithelial and mesenchyme-like cells. Receptor for Activated protein Kinase C (RACK1), which we identified as an xMELK partner, co-localizes with xMELK at the tight junction. Moreover, a truncated RACK1 construct interferes with iMELK localization at cell-cell contacts. Collectively, our results suggest that iMELK and RACK1 are present in the same complex and that RACK1 is involved in the specific recruitment of iMELK at the apical junctional complex in epithelial cells of Xenopus embryos. PMID:24167714

  3. Characterization of the T-cell subpopulations in the granulation tissues of chronic suppurative otitis media

    PubMed Central

    WANG, BING; CHENG, YING; XU, MIN

    2016-01-01

    The present study aimed to investigate the potential involvement of specific T-cell subpopulations in granulation tissue formation in chronic suppurative otitis media (CSOM). Fifteen patients with CSOM were enrolled in this study. Granulation tissues were obtained from the middle ear cavity. Hematoxylin and eosin staining was performed for histopathological observation, and different T-cell subpopulations were characterized by immunohistochemistry. No evident association was identified between granulation tissue formation and disease course. The number of cluster of differentiation 8+ (CD8+) T cells, forkhead box P3+ (FOXP3+) regulatory T (Treg) cells and OX40+ T cells were significantly higher in granulation tissues from patients with ear discharge within the last 6 months compared to those without (P<0.05). Fresh granulation tissues had more CD8+ T cells and FOXP3+ Treg cells compared to the mature granulation tissues (P<0.05). There was a differential abundance of specific T-cell subpopulations in the granulation tissues in CSOM with different disease courses or with ear discharge, suggesting that T cell-mediated cellular immunity is involved in lesion formation of CSOM. PMID:27313854

  4. Immature oocyte quality and maturational competence of porcine cumulus-oocyte complexes subpopulations.

    PubMed

    Alvarez, Gabriel Martin; Dalvit, Gabriel Carlos; Achi, María Verónica; Miguez, Marcelo Sergio; Cetica, Pablo Daniel

    2009-12-01

    Porcine immature oocyte quality (i.e., that of live oocytes at the germinal vesicle stage) was evaluated according to features of the surrounding cumulus, aiming to establish maturational competence of different subpopulations of such cumulus-oocyte complexes. Six subpopulations were identified: A1 (with a dense cumulus), A2 (with a translucent cumulus), B1 (with the corona radiata), B2 (partly naked oocytes), C (naked oocytes), D (with a dark cumulus). The percent incidence of live oocyte in these subpopulations changed significantly as related to cumulus features, however the occurrence of oocytes in the germinal vesicle stage was lower in class D only. Similar metaphase II rates achieved in A1, A2, B1 and B2 classes after in vitro maturation suggest that the nucleus may in fact mature in vitro, in spite of the different accompanying cumulus features which are typical of these classes. In contrast, a higher cytoplasmic maturation rate obtained in class A may indicate a stronger dependence of this variable upon cumulus features than that shown by nuclear maturation. When different types of cumulus expansion after in vitro maturation were considered (i.e., fully expanded cumulus, partly expanded cumulus, and partly naked oocyte), no differences were found in the percent of oocytes reaching metaphase II or cytoplasmic maturation. It is concluded that morphological features of the collected porcine cumulus-oocyte complexes (rather than cumulus behavior during culture) may be useful for selection of potentially competent oocytes for in vitro fertilization and embryo production. PMID:20067032

  5. Particle Backtracking Improves Breeding Subpopulation Discrimination and Natal-Source Identification in Mixed Populations

    PubMed Central

    Fraker, Michael E.; Anderson, Eric J.; Brodnik, Reed M.; Carreon-Martinez, Lucia; DeVanna, Kristen M.; Fryer, Brian J.; Heath, Daniel D.; Reichert, Julie M.; Ludsin, Stuart A.

    2015-01-01

    We provide a novel method to improve the use of natural tagging approaches for subpopulation discrimination and source-origin identification in aquatic and terrestrial animals with a passive dispersive phase. Our method integrates observed site-referenced biological information on individuals in mixed populations with a particle-tracking model to retrace likely dispersal histories prior to capture (i.e., particle backtracking). To illustrate and test our approach, we focus on western Lake Erie’s yellow perch (Perca flavescens) population during 2006–2007, using microsatellite DNA and otolith microchemistry from larvae and juveniles as natural tags. Particle backtracking showed that not all larvae collected near a presumed hatching location may have originated there, owing to passive drift during the larval stage that was influenced by strong river- and wind-driven water circulation. Re-assigning larvae to their most probable hatching site (based on probabilistic dispersal trajectories from the particle backtracking model) improved the use of genetics and otolith microchemistry to discriminate among local breeding subpopulations. This enhancement, in turn, altered (and likely improved) the estimated contributions of each breeding subpopulation to the mixed population of juvenile recruits. Our findings indicate that particle backtracking can complement existing tools used to identify the origin of individuals in mixed populations, especially in flow-dominated systems. PMID:25799555

  6. Time-dependent release of extracellular vesicle subpopulations in tumor CABA I cells.

    PubMed

    Giusti, Ilaria; Di Francesco, Marianna; Cantone, Laura; D'Ascenzo, Sandra; Bollati, Valentina; Carta, Gaspare; Dolo, Vincenza

    2015-11-01

    Investigations into extracellular vesicles (EVs) have significantly increased since their role in physiological and pathological processes has become more clearly understood. Furthermore, it has become increasingly clear that several subpopulations of EVs exist, such as exosomes (EXOs) and microvesicles (MVs). Various methods and techniques used to identify and isolate the specific EVs subpopulations exist. However, these methods should be further elucidated. A deep understanding of the different factors that affect the EVs release may therefore be useful for the standardization of protocols and to establish guidelines for a more adequate analysis and correct inter‑laboratory comparison. In the present study, we investigated whether composition and molecular features of EVs altered over time following a trigger stimulus. Starved CABA I cells were stimulated with FBS and conditioned medium was collected after different time intervals (30 min and 4, 8 and 18 h). The dynamic of EVs release was time-dependent, as shown by the results of scanning electron microscopy. Additionally, the time elapsed from the stimulus affected the size distribution (as highlighted by transmission electron microscopy and NanoSight assay), amount (in terms of the number of particles and protein amount) and molecular composition (CD63, HLA, Ago-2, gelatinases, and plasminogen activators) suggesting that, different EVs subpopulations were released at different time intervals following cell stimulation. Collectively, the results suggested that, parameters useful to standardize procedures for EVs isolation, including stimulation time should be considered. PMID:26323210

  7. Identification of geographically distributed sub-populations of Leishmania (Leishmania) major by microsatellite analysis

    PubMed Central

    2008-01-01

    Background Leishmania (Leishmania) major, one of the agents causing cutaneous leishmaniasis (CL) in humans, is widely distributed in the Old World where different species of wild rodent and phlebotomine sand fly serve as animal reservoir hosts and vectors, respectively. Despite this, strains of L. (L.) major isolated from many different sources over many years have proved to be relatively uniform. To investigate the population structure of the species highly polymorphic microsatellite markers were employed for greater discrimination among it's otherwise closely related strains, an approach applied successfully to other species of Leishmania. Results Multilocus Microsatellite Typing (MLMT) based on 10 different microsatellite markers was applied to 106 strains of L. (L.) major from different regions where it is endemic. On applying a Bayesian model-based approach, three main populations were identified, corresponding to three separate geographical regions: Central Asia (CA); the Middle East (ME); and Africa (AF). This was congruent with phylogenetic reconstructions based on genetic distances. Re-analysis separated each of the populations into two sub-populations. The two African sub-populations did not correlate well with strains' geographical origin. Strains falling into the sub-populations CA and ME did mostly group according to their place of isolation although some anomalies were seen, probably, owing to human migration. Conclusion The model- and distance-based analyses of the microsatellite data exposed three main populations of L. (L.) major, Central Asia, the Middle East and Africa, each of which separated into two sub-populations. This probably correlates with the different species of rodent host. PMID:18577226

  8. Epithelial stem cells.

    PubMed

    Draheim, Kyle M; Lyle, Stephen

    2011-01-01

    It is likely that adult epithelial stem cells will be useful in the treatment of diseases, such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa, and alopecias. Additionally, other skin problems such as burn wounds, chronic wounds, and ulcers will benefit from stem cell-related therapies. However, there are many questions that need to be answered before this goal can be realized. The most important of these questions is what regulates the adhesion of stem cells to the niche versus migration to the site of injury. We have started to identify the mechanisms involved in this decision-making process. PMID:21618097

  9. An analysis of glucocorticoid receptor-mediated gene expression in BEAS-2B human airway epithelial cells identifies distinct, ligand-directed, transcription profiles with implications for asthma therapeutics

    PubMed Central

    Joshi, T; Johnson, M; Newton, R; Giembycz, M

    2015-01-01

    Background and Purpose International asthma guidelines recommend that inhaled glucocorticoids be used as a monotherapy in all patients with mild to moderate disease because of their ability to suppress airways inflammation. Current evidence suggests that the therapeutic benefit of glucocorticoids is due to the transactivation and transrepression of anti-inflammatory and pro-inflammatory genes respectively. However, the extent to which clinically relevant glucocorticoids are equivalent in their ability to modulate gene expression is unclear. Experimental Approach A pharmacodynamics investigation of glucocorticoid receptor (GR)-mediated gene transactivation in BEAS-2B human airway epithelial cells was performed using a glucocorticoid response element luciferase reporter coupled with an analysis of glucocorticoid-inducible genes encoding proteins with anti-inflammatory and adverse-effect potential. Key Results Using transactivation as a functionally relevant output, a given glucocorticoid displayed a unique, gene expression ‘fingerprint’ where intrinsic efficacy and GR density were essential determinants. We showed that depending on the gene selected for analysis, a given glucocorticoid can behave as an antagonist, partial agonist, full agonist or even ‘super agonist’. In the likely event that different, tissue-dependent gene expression profiles are reproduced in vivo, then the anti-inflammatory and adverse-effect potential of many glucocorticoids currently available as asthma therapeutics may not be equivalent. Conclusions and Implications The generation of gene expression ‘fingerprints’ in target and off-target human tissues could assist the rational design of GR agonists with improved therapeutic ratios. This approach could identify compounds that are useful in the management of severe asthma and other inflammatory disorders where systemic exposure is desirable. PMID:25393397

  10. Recognition of a CD4+ mouse medullary thymocyte subpopulation by Amaranthus leucocarpus lectin.

    PubMed

    Lascurain, R; Chávez, R; Gorocica, P; Pérez, A; Montaño, L F; Zenteno, E

    1994-11-01

    We have used the Gal beta(1-->3)GalNAc-specific Amaranthus leucocarpus lectin to isolate a thymus cell subpopulation which is different from that sorted with Arachis hypogaea lectin. The cells recognized by A. leucocarpus lectin were predominantly CD4+, whereas a minor proportion of CD8+ cells (approximately 11%) were also identified. The A. leucocarpus-positive cells were located in the thymus medulla and the cortico-medullary junction. The cortex was negative for A. leucocarpus cells. PMID:7835965

  11. Cancerous epithelial cell lines shed extracellular vesicles with a bimodal size distribution that is sensitive to glutamine inhibition

    NASA Astrophysics Data System (ADS)

    Santana, Steven Michael; Antonyak, Marc A.; Cerione, Richard A.; Kirby, Brian J.

    2014-12-01

    Extracellular shed vesicles (ESVs) facilitate a unique mode of cell-cell communication wherein vesicle uptake can induce a change in the recipient cell's state. Despite the intensity of ESV research, currently reported data represent the bulk characterization of concentrated vesicle samples with little attention paid to heterogeneity. ESV populations likely represent diversity in mechanisms of formation, cargo and size. To better understand ESV subpopulations and the signaling cascades implicated in their formation, we characterize ESV size distributions to identify subpopulations in normal and cancerous epithelial cells. We have discovered that cancer cells exhibit bimodal ESV distributions, one small-diameter and another large-diameter population, suggesting that two mechanisms may govern ESV formation, an exosome population and a cancer-specific microvesicle population. Altered glutamine metabolism in cancer is thought to fuel cancer growth but may also support metastatic niche formation through microvesicle production. We describe the role of a glutaminase inhibitor, compound 968, in ESV production. We have discovered that inhibiting glutamine metabolism significantly impairs large-diameter microvesicle production in cancer cells.

  12. CCAST: a model-based gating strategy to isolate homogeneous subpopulations in a heterogeneous population of single cells.

    PubMed

    Anchang, Benedict; Do, Mary T; Zhao, Xi; Plevritis, Sylvia K

    2014-07-01

    A model-based gating strategy is developed for sorting cells and analyzing populations of single cells. The strategy, named CCAST, for Clustering, Classification and Sorting Tree, identifies a gating strategy for isolating homogeneous subpopulations from a heterogeneous population of single cells using a data-derived decision tree representation that can be applied to cell sorting. Because CCAST does not rely on expert knowledge, it removes human bias and variability when determining the gating strategy. It combines any clustering algorithm with silhouette measures to identify underlying homogeneous subpopulations, then applies recursive partitioning techniques to generate a decision tree that defines the gating strategy. CCAST produces an optimal strategy for cell sorting by automating the selection of gating markers, the corresponding gating thresholds and gating sequence; all of these parameters are typically manually defined. Even though CCAST is optimized for cell sorting, it can be applied for the identification and analysis of homogeneous subpopulations among heterogeneous single cell data. We apply CCAST on single cell data from both breast cancer cell lines and normal human bone marrow. On the SUM159 breast cancer cell line data, CCAST indicates at least five distinct cell states based on two surface markers (CD24 and EPCAM) and provides a gating sorting strategy that produces more homogeneous subpopulations than previously reported. When applied to normal bone marrow data, CCAST reveals an efficient strategy for gating T-cells without prior knowledge of the major T-cell subtypes and the markers that best define them. On the normal bone marrow data, CCAST also reveals two major mature B-cell subtypes, namely CD123+ and CD123- cells, which were not revealed by manual gating but show distinct intracellular signaling responses. More generally, the CCAST framework could be used on other biological and non-biological high dimensional data types that are

  13. Differential distribution of sperm subpopulations and incidence of pleiomorphisms in ejaculates of captive howling monkeys (Alouatta caraya).

    PubMed

    Valle, R R; Carvalho, F M; Muniz, J A P C; Leal, C L V; García-Herreros, M

    2013-10-01

    The aim of this study was to develop an objective method to determine the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates of howling monkeys (Alouatta caraya) by using a combination of computerized analysis system (ASMA) and principal component analysis (PCA) methods. Ejaculates were collected by electroejaculation methods on a regular basis from five individuals maintained under identical captive environmental, nutritional, and management conditions. Each sperm head was measured for dimensional parameters (Area [A, (square micrometers)], Perimeter [P, (micrometers)], Length [L, (micrometers)], and Width [W, (micrometers)]) and shape-derived parameters (Ellipticity [(L/W)], Elongation [(L - W)/(L + W)], and Rugosity [(4лA/P (2))]). PCA revealed two principal components explaining more than the 96 % of the variance. Clustering methods and discriminant analyzes were performed and seven separate subpopulations were identified. There were differences (P < 0.001) in the distribution of the seven subpopulations as well as in the incidence of abnormal pleiomorphisms (58.6 %, 49.8 %, 35.1 %, 66.4 %, and 55.1 %, P < 0.05) among the five donors tested. Our results indicated that differences among individuals related to the incidence of pleiomorphisms, and sperm subpopulational structure was not related to the captivity conditions or the sperm collection method, since all individuals were studied under identical conditions. In conclusion, the combination of ASMA and PCA is a useful clinical diagnostic resource for detecting deficiencies in sperm morphology and sperm subpopulations in A. caraya ejaculates that could be used in ex situ conservation programs of threatened species in Alouatta genus or even other endangered neotropical primate species. PMID:23975115

  14. Differential distribution of sperm subpopulations and incidence of pleiomorphisms in ejaculates of captive howling monkeys ( Alouatta caraya)

    NASA Astrophysics Data System (ADS)

    Valle, R. R.; Carvalho, F. M.; Muniz, J. A. P. C.; Leal, C. L. V.; García-Herreros, M.

    2013-10-01

    The aim of this study was to develop an objective method to determine the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates of howling monkeys ( Alouatta caraya) by using a combination of computerized analysis system (ASMA) and principal component analysis (PCA) methods. Ejaculates were collected by electroejaculation methods on a regular basis from five individuals maintained under identical captive environmental, nutritional, and management conditions. Each sperm head was measured for dimensional parameters (Area [ A, (square micrometers)], Perimeter [ P, (micrometers)], Length [ L, (micrometers)], and Width [ W, (micrometers)]) and shape-derived parameters (Ellipticity [( L/ W)], Elongation [( L - W)/( L + W)], and Rugosity [(4л A/ P 2)]). PCA revealed two principal components explaining more than the 96 % of the variance. Clustering methods and discriminant analyzes were performed and seven separate subpopulations were identified. There were differences ( P < 0.001) in the distribution of the seven subpopulations as well as in the incidence of abnormal pleiomorphisms (58.6 %, 49.8 %, 35.1 %, 66.4 %, and 55.1 %, P < 0.05) among the five donors tested. Our results indicated that differences among individuals related to the incidence of pleiomorphisms, and sperm subpopulational structure was not related to the captivity conditions or the sperm collection method, since all individuals were studied under identical conditions. In conclusion, the combination of ASMA and PCA is a useful clinical diagnostic resource for detecting deficiencies in sperm morphology and sperm subpopulations in A. caraya ejaculates that could be used in ex situ conservation programs of threatened species in Alouatta genus or even other endangered neotropical primate species.

  15. Emergence of a large pore subpopulation during electroporating pulses.

    PubMed

    Smith, Kyle C; Son, Reuben S; Gowrishankar, T R; Weaver, James C

    2014-12-01

    Electroporation increases ionic and molecular transport through cell membranes by creating transient aqueous pores. These pores cannot be directly observed experimentally, but cell system modeling with dynamic electroporation predicts pore populations that produce cellular responses consistent with experiments. We show a cell system model's response that illustrates the life cycle of a pore population in response to a widely used 1 kV/cm, 100 μs trapezoidal pulse. Rapid pore creation occurs early in the pulse, followed by the gradual emergence of a subpopulation of large pores reaching ~30 nm radius. After the pulse, pores rapidly contract to form a single thermally broadened distribution of small pores (~1 nm radius) that slowly decays. We also show the response of the same model to pulses of 100 ns to 1 ms duration, each with an applied field strength adjusted such that a total of 10,000±100 pores are created. As pulse duration is increased, the pore size distributions vary dramatically and a distinct subpopulation of large pores emerges for pulses of microsecond and longer duration. This subpopulation of transient large pores is relevant to understanding rapid transport of macromolecules into and out of cells during a pulse. PMID:24290730

  16. Relatedness calculations for linked loci incorporating subpopulation effects.

    PubMed

    Bright, Jo-Anne; Curran, James M; Buckleton, John S

    2013-05-01

    Often the loci in forensic multiplexes are selected to avoid linked loci. However linked loci have occurred in some recent commercially available multiplexes. Previously formulae have been given for both joint and conditional match probabilities for some relationships that did not account for subpopulation effects. In this paper we extend these works to include a subpopulation correction of the form first suggested by Balding and Nichols. We extend the work to grandparent/grandchild, first cousin, uncle/nephew and half uncle/nephew relationships and apply these to two different populations and STR multiplexes. Our model assumes that the two people have no relationship other that the one specified. That is, we assume their parents are neither related nor themselves inbred. The multiplications inherent in these formulae also assume that there is no linkage disequilibrium at the subpopulation level for these loci. We found that when taking into account linkage the match statistic decreases for all relationships, with siblings having the greatest effect. However, the effect was less than a factor of two decrease in match statistic. PMID:23537755

  17. Novel flow cytometric approach for the detection of adipocyte subpopulations during adipogenesis[S

    PubMed Central

    Durandt, Chrisna; van Vollenstee, Fiona A.; Dessels, Carla; Kallmeyer, Karlien; de Villiers, Danielle; Murdoch, Candice; Potgieter, Marnie; Pepper, Michael S.

    2016-01-01

    The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool of MSCs. Cluster of differentiation (CD)36 plays an important role in the formation of intracellular lipid droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to develop a reproducible quantitative method to study adipocyte differentiation by comparing two lipophilic dyes [Nile Red (NR) and Bodipy 493/503] in combination with CD36 surface marker staining. We identified a subpopulation of adipose-derived stromal cells that express CD36 at intermediate/high levels and show that combining CD36 cell surface staining with neutral lipid-specific staining allows us to monitor differentiation of adipose-derived stromal cells that express CD36intermediate/high during adipocyte differentiation in vitro. The gradual increase of CD36intermediate/high/NRpositive cells during the 21 day adipogenesis induction period correlated with upregulation of adipogenesis-associated gene expression. PMID:26830859

  18. Subpopulation-Specific Transcriptome Analysis of Competence-Stimulating-Peptide-Induced Streptococcus mutans▿†

    PubMed Central

    Lemme, André; Gröbe, Lothar; Reck, Michael; Tomasch, Jürgen; Wagner-Döbler, Irene

    2011-01-01

    Competence-stimulating-peptide (CSP)-mediated competence development in Streptococcus mutans is a transient and biphasic process, since only a subpopulation induces the expression of ComX in the presence of CSP, and the activation of the DNA uptake machinery in this fraction shuts down ∼3 to 4 h postinduction. Here, we combine for the first time, to our knowledge, the bacterial flow-cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and noncompetent fraction of CSP-treated S. mutans cells. Sorting was guided by a ComX-green fluorescent protein (ComX-GFP) reporter, and the transcriptome analysis demonstrated the successful combination of both methods, because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, and among them was ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. In contrast, the expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and CipB (mutacin V) confirmed this expression pattern on the single-cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, the uptake of DNA could be shown on the single-cell level. This study demonstrates that all cells in the population respond to CSP through the activation of bacteriocin-related genes. Some of these cells start to activate ComX expression but then segregate into two subpopulations, one becoming competent and another one that lyses, resulting in intrapopulation diversity. PMID:21317319

  19. Identification of subpopulations of prairie voles differentially susceptible to peer influence to decrease high alcohol intake.

    PubMed

    Anacker, Allison M J; Ryabinin, Andrey E

    2013-01-01

    Peer influences are critical in the decrease of alcohol (ethanol) abuse and maintenance of abstinence. We previously developed an animal model of inhibitory peer influences on ethanol drinking using prairie voles and here sought to understand whether this influential behavior was due to specific changes in drinking patterns and to variation in a microsatellite sequence in the regulatory region of the vasopressin receptor 1a gene (avpr1a). Adult prairie voles' drinking patterns were monitored in a lickometer apparatus that recorded each lick a subject exhibited during continuous access to water and 10% ethanol during periods of isolation, pair housing of high and low drinkers, and subsequent isolation. Analysis of fluid consumption confirmed previous results that high drinkers typically decrease ethanol intake when paired with low drinkers, but that a subset of voles do not decrease. Analysis of bout structure revealed differences in the number of ethanol drinking bouts in the subpopulations of high drinkers when paired with low drinkers. Lickometer drinking patterns analyzed by visual and by cross-correlation analyses demonstrated that pair housing did not increase the rate of subjects drinking in bouts occurring at the same time. The length of the avpr1a microsatellite did not predict susceptibility to peer influence or any other drinking behaviors. In summary, subpopulations of high drinkers were identified, by fluid intake and number of drinking bouts, which did or did not lower their ethanol intake when paired with a low drinking peer, and these subpopulations should be explored for testing the efficacy of treatments to decrease ethanol use in groups that are likely to be responsive to different types of therapy. PMID:23847535

  20. TECHNICAL BRIEF: Optimized pipeline for isolation of high-quality RNA from corneal cell subpopulations

    PubMed Central

    Bath, Chris; Fink, Trine; Vorum, Henrik; Hjortdal, Jesper

    2014-01-01

    Purpose: Attempts to determine the transcriptional profile of discrete subsets of limbal epithelial cells in situ using laser capture microdissection (LCM) face two major challenges. First, the transcriptional profile of cells within a tissue may rapidly change as the tissue is excised and exposed to cold ischemia. Second, there is a risk of degradation of the RNA as the cellular compartment is separated from the remaining tissue. An optimized protocol for LCM of corneal epithelium is presented to address these issues. Methods: Experiments using porcine eye globes were carried out to determine both optimal procedures and settings for tissue harvest, transport, storage, histology, LCM, and RNA isolation. The optimized protocol was validated using human corneal epithelium. Results: To facilitate preservation of the gene expression profile, we have developed a mechanical tool for dissection of cornea that, in combination with flash freezing, enables tissue to be stored within 5 min of enucleation of the eye. Furthermore, we describe how RNA from limbal crypt cells may be obtained using a procedure involving cryosectioning, histological staining, and LCM. Conclusion: In this paper, we describe an optimized method for isolating high-quality RNA from cellular subpopulations confined to the limbal crypts of the cornea. The procedure yields RNA in amounts and quality suitable for downstream gene expression analyses, such as microarrays or next generation sequencing. PMID:24940035

  1. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor.

    PubMed

    Aslakson, C J; Miller, F R

    1992-03-15

    To identify selective steps in metastasis, those that eliminate nonmetastatic tumor cells more efficiently than metastatic cells, we have evaluated the sequential dissemination of tumor cells from a mammary fatpad, using both metastatic (4T1 and 66cl4) and nonmetastatic (67NR, 168FARN, and 4TO7) subpopulations of a single mouse mammary tumor. Each of these variant subpopulations is resistant to one or more selective drugs so they could be quantitatively identified by colony formation in selective media. We found that the 2 metastatic cell lines metastasized by different routes and that the nonmetastatic tumor cell lines failed at different points in dissemination. Line 67NR did not leave the primary site; clonogenic tumor cells were not detected in the nodes, blood, or lungs during the experiment (7 weeks). Tumor line 168FARN disseminated from the primary tumor because clonogenic cells were cultured from the draining lymph nodes throughout the experiment. However, dissemination essentially stopped in the node as cells were rarely isolated from blood, lungs, or lives. Whether 168FARN cells failed to reach these tissues or were killed very rapidly after traversing the lymph node is unknown. Line 4TO7 cells disseminated via the blood and were consistently recovered from lungs by day 19 but failed to proliferate. This panel of 5 subpopulations thus identifies different points of selective failure in tumor cell dissemination and should be valuable in the assessment of antimetastatic therapies. PMID:1540948

  2. Can multi-subpopulation reference sets improve the genomic predictive ability for pigs?

    PubMed

    Fangmann, A; Bergfelder-Drüing, S; Tholen, E; Simianer, H; Erbe, M

    2015-12-01

    In most countries and for most livestock species, genomic evaluations are obtained from within-breed analyses. To achieve reliable breeding values, however, a sufficient reference sample size is essential. To increase this size, the use of multibreed reference populations for small populations is considered a suitable option in other species. Over decades, the separate breeding work of different pig breeding organizations in Germany has led to stratified subpopulations in the breed German Large White. Due to this fact and the limited number of Large White animals available in each organization, there was a pressing need for ascertaining if multi-subpopulation genomic prediction is superior compared with within-subpopulation prediction in pigs. Direct genomic breeding values were estimated with genomic BLUP for the trait "number of piglets born alive" using genotype data (Illumina Porcine 60K SNP BeadChip) from 2,053 German Large White animals from five different commercial pig breeding companies. To assess the prediction accuracy of within- and multi-subpopulation reference sets, a random 5-fold cross-validation with 20 replications was performed. The five subpopulations considered were only slightly differentiated from each other. However, the prediction accuracy of the multi-subpopulations approach was not better than that of the within-subpopulation evaluation, for which the predictive ability was already high. Reference sets composed of closely related multi-subpopulation sets performed better than sets of distantly related subpopulations but not better than the within-subpopulation approach. Despite the low differentiation of the five subpopulations, the genetic connectedness between these different subpopulations seems to be too small to improve the prediction accuracy by applying multi-subpopulation reference sets. Consequently, resources should be used for enlarging the reference population within subpopulation, for example, by adding genotyped females

  3. A subpopulation of itch-sensing neurons marked by Ret and somatostatin expression.

    PubMed

    Stantcheva, Kalina K; Iovino, Loredana; Dhandapani, Rahul; Martinez, Concepcion; Castaldi, Laura; Nocchi, Linda; Perlas, Emerald; Portulano, Carla; Pesaresi, Martina; Shirlekar, Kalyanee S; de Castro Reis, Fernanda; Paparountas, Triantafillos; Bilbao, Daniel; Heppenstall, Paul A

    2016-04-01

    Itch, the unpleasant sensation that elicits a desire to scratch, is mediated by specific subtypes of cutaneous sensory neuron. Here, we identify a subpopulation of itch-sensing neurons based on their expression of the receptor tyrosine kinase Ret. We apply flow cytometry to isolate Ret-positive neurons from dorsal root ganglia and detected a distinct population marked by low levels of Ret and absence of isolectin B4 binding. We determine the transcriptional profile of these neurons and demonstrate that they express neuropeptides such as somatostatin (Sst), the NGF receptor TrkA, and multiple transcripts associated with itch. We validate the selective expression of Sst using an Sst-Cre driver line and ablated these neurons by generating mice in which the diphtheria toxin receptor is conditionally expressed from the sensory neuron-specific Avil locus. Sst-Cre::Avil(iDTR) mice display normal nociceptive responses to thermal and mechanical stimuli. However, scratching behavior evoked by interleukin-31 (IL-31) or agonist at the 5HT1F receptor is significantly reduced. Our data provide a molecular signature for a subpopulation of neurons activated by multiple pruritogens. PMID:26929027

  4. A novel subpopulation of peripheral blood mononuclear cells presents in major burn patients.

    PubMed

    Liu, Hongbin; Ding, Jie; Ma, Zengshuan; Zhu, Zhenshen; Shankowsky, Heather A; Tredget, Edward E

    2015-08-01

    Hypertrophic scars (HTS) are generally believed to result from proliferation and activation of resident connective tissue fibroblasts after burns. To demonstrate a potential role of blood-borne cells, the peripheral blood mononuclear cells (PBMCs) and the effect of PBMCs on dermal fibroblast behavior was investigated. Flow cytometry was used to analyze the surface and intracellular protein expression of PBMCs and fibroblasts. Transwell migration assay, enzyme-linked immunosorbent assay and real-time reverse transcription polymerase chain reaction was performed to assess fibroblast functions. We identified a novel subpopulation of PBMCs in burn patients in vivo that appears at an early stage following major thermal injuries, which primarily express procollagen 1, leukocyte specific protein 1, CD204, toll-like receptor 4 and stromal cell-derived factor 1 (SDF-1) receptor CXCR4. In vitro, the conditioned media from burn patient PBMCs up-regulated the expression of fibrotic growth factors and extracellular matrix molecules, down-regulated antifibrotic factor decorin, enhanced cell chemotaxis and promoted cell differentiation into contractile myofibroblasts in dermal fibroblasts. After thermal injury, this novel subpopulation of PBMCs is systemically triggered and attracted to the wounds under SDF-1/CXCR4 signaling where they appear to modulate the functions of resident connective tissue cells and thus contribute to the development of HTS. PMID:25683215

  5. Mental disorders associated with subpopulations of women affected by violence and abuse.

    PubMed

    Cavanaugh, Courtenay E; Martins, Silvia S; Petras, Hanno; Campbell, Jacquelyn C

    2013-08-01

    Violence against women is a major public health problem associated with mental disorders. Few studies have examined the heterogeneity of interpersonal violence and abuse (IVA) among women and associated mental health problems. Latent class analysis was used to identify subpopulations of women with similar lifetime histories of IVA victimization and to examine 10 associated past-year mental disorders. Participants were 19,816 adult women who participated in Wave 2 of the National Epidemiologic Study on Alcohol and Related Conditions (NESARC). The 3-class model was best supported by the data. Class 1 (6.7%) had a high probability of witnessing domestic violence as a child. Class 2 (21.8%) had a low probability of all events except lifetime sexual assault. Class 3 (71.5%) had a low probability for all events. Mental disorders were more common among members of Classes 1 and 2 than Class 3. For example, members in Class 1 were approximately 8 and 9 times more likely than members in Class 3 to have had posttraumatic stress disorder or a drug use disorder, respectively, during the past year. Of the 10 mental disorders, 5 were more common among members of Class 1 than of Class 2. Findings suggest the mental health consequences of IVA among women are extensive and interventions should be tailored for distinct subpopulations affected by IVA. PMID:23813596

  6. Characterization of two distinct liver progenitor cell subpopulations of hematopoietic and hepatic origins

    SciTech Connect

    Corcelle, V.; Stieger, B.; Gjinovci, A.; Wollheim, C.B.; Gauthier, B.R. . E-mail: Benoit.Gauthier@medecine.unige.ch

    2006-09-10

    Despite extensive studies, the hematopoietic versus hepatic origin of liver progenitor oval cells remains controversial. The aim of this study was to determine the origin of such cells after liver injury and to establish an oval cell line. Rat liver injury was induced by subcutaneous insertion of 2-AAF pellets for 7 days with subsequent injection of CCl{sub 4}. Livers were removed 9 to 13 days post-CCl{sub 4} treatment. Immunohistochemistry was performed using anti-c-kit, OV6, Thy1, CK19, AFP, vWF and Rab3b. Isolated non-parenchymal cells were grown on mouse embryonic fibroblast, and their gene expression profile was characterized by RT-PCR. We identified a subpopulation of OV6/CK19/Rab3b-expressing cells that was activated in the periportal region of traumatized livers. We also characterized a second subpopulation that expressed the HSCs marker c-kit but not Thy1. Although we successfully isolated both cell types, OV6/CK19/Rab3b{sup +} cells fail to propagate while c-kit {sup +}-HSCs appeared to proliferate for up to 7 weeks. Cells formed clusters which expressed c-kit, Thy1 and albumin. Our results indicate that a bona fide oval progenitor cell population resides within the liver and is distinct from c-kit {sup +}-HSCs. Oval cells require the hepatic niche to proliferate, while cells mobilized from the circulation proliferate and transdifferentiate into hepatocytes without evidence of cell fusion.

  7. Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content

    PubMed Central

    Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit; Saari, Heikki; Ibañez, Elisa Lazaro; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

    2015-01-01

    Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level. PMID:26649679

  8. An Aggressive Hypoxia Related Subpopulation of Melanoma Cells is TRP-2 Negative.

    PubMed

    Lenggenhager, Daniela; Curioni-Fontecedro, Alessandra; Storz, Martina; Shakhova, Olga; Sommer, Lukas; Widmer, Daniel S; Seifert, Burkhardt; Moch, Holger; Dummer, Reinhard; Mihic-Probst, Daniela

    2014-04-01

    Despite existing vaccination strategies targeting TRP-2, its function is not yet fully understood. TRP-2 is an enzyme involved in melanin biosynthesis and therefore discussed as a differentiation antigen. However, in mice Trp-2 was shown to be expressed in melanocyte stem cells of the hair follicle and therefore also considered as an indicator of stemness. A proper understanding of the TRP-2 function is crucial, considering a vaccination targeting cells with stemness properties would be highly effective in contrast to a therapy targeting differentiated melanoma cells. Analysing over 200 melanomas including primaries, partly matched metastases and patients' cell cultures we show that TRP-2 is correlated with Melan A expression and decreases with tumor progression. In mice it is expressed in differentiated melanocytes as well as in stem cells. Furthermore, we identify a TRP-2 negative, proliferative, hypoxia related cell subpopulation which is significantly associated with tumor thickness and diseases progression. Patients with a higher percentage of those cells have a less favourable tumor specific survival. Our findings underline that TRP-2 is a differentiation antigen, highlighting the importance to combine TRP-2 vaccination with other strategies targeting the aggressive undifferentiated hypoxia related subpopulation. PMID:24746711

  9. Raman spectroscopy of single extracellular vesicles reveals subpopulations with varying membrane content (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit S.; Saari, Heikki; Lazaro Ibañez, Elisa; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

    2016-03-01

    Exosomes are small (~100nm) membrane bound vesicles excreted by cells as part of their normal biological processes. These extracellular vesicles are currently an area of intense research, since they were recently found to carry functional mRNA that allows transfer of proteins and other cellular instructions between cells. Exosomes have been implicated in a wide range of diseases, including cancer. Cancer cells are known to have increased exosome production, and may use those exosomes to prepare remote environments for metastasis. Therefore, there is a strong need to develop characterization methods to help understand the structure and function of these vesicles. However, current techniques, such as proteomics and genomics technologies, rely on aggregating a large amount of exosome material and reporting on chemical content that is averaged over many millions of exosomes. Here we report on the use of laser-tweezers Raman spectroscopy (LTRS) to probe individual vesicles, discovering distinct heterogeneity among exosomes both within a cell line, as well as between different cell lines. Through principal components analysis followed by hierarchical clustering, we have identified four "subpopulations" of exosomes shared across seven cell lines. The key chemical differences between these subpopulations, as determined by spectral analysis of the principal component loadings, are primarily related to membrane composition. Specifically, the differences can be ascribed to cholesterol content, cholesterol to phospholipid ratio, and surface protein expression. Thus, we have shown LTRS to be a powerful method to probe the chemical content of single extracellular vesicles.

  10. High-density lipoprotein subpopulations in pathologic conditions.

    PubMed

    Asztalos, Bela F; Schaefer, Ernst J

    2003-04-01

    The role of low-density lipoprotein (LDL) cholesterol in coronary artery disease (CAD) and the impact of therapeutic agents on LDL cholesterol are well established. Less is known about the role of high-density lipoprotein (HDL) cholesterol and even less about the role of the different HDL subspecies in CAD. HDL particles vary in size and density, mainly because of differences in the number of apolipoprotein (apo) particles and the amount of cholesterol ester in the core of HDL. Apo A-I is essential and, together with lipid, sufficient for the formation of HDL particles. Apo A-I-containing HDL particles play a primary role in cholesterol efflux from membranes, at least in part through interactions with the adenosine triphosphate-binding cassette transporter A1 (ABCA1). Patients with Tangier disease have mutations in the gene encoding ABCA1, which result in functionally impaired protein, a marked deficiency in HDL cholesterol, and a high risk of premature CAD. Our studies of apo A-I-containing HDL subpopulations in various patient populations reveal that patients homozygous for Tangier disease have only the pre-beta(1) HDL subspecies. Tangier heterozygotes are severely depleted in the larger alpha- and pre-alpha-mobility subspecies. Patients with low HDL cholesterol levels and those with CAD also show deficiencies in the alpha(1) and pre-alpha(1-3) HDL subspecies. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) increase the levels of the large alpha(1) and pre-alpha(1) subpopulations and decrease the level of the small alpha(3) subpopulation. Thus, atorvastatin, for example, significantly moves the distribution of HDL particles toward normal, followed by simvastatin, pravastatin, and lovastatin in decreasing order of efficiency. A new statin, rosuvastatin, produces greater increases in HDL cholesterol than atorvastatin, but its effect on HDL particle distribution is yet to be determined. PMID:12679198

  11. Mitochondrial oxidative stress as a novel therapeutic target to overcome intrinsic drug resistance in melanoma cell subpopulations

    PubMed Central

    Cierlitza, Monika; Chauvistré, Heike; Bogeski, Ivan; Zhang, Xin; Hauschild, Axel; Herlyn, Meenhard; Schadendorf, Dirk; Vogt, Thomas; Roesch, Alexander

    2015-01-01

    Despite recent success in melanoma therapy, most patients with metastatic disease still undergo deadly progression. We have identified a novel mechanism of multidrug resistance allowing a small subpopulation of slow-cycling melanoma cells to survive based on elevated oxidative bioenergy metabolism. In this study, we asked whether such slow-cycling cells could be eliminated by co-treatment with the copper-chelator elesclomol. Elesclomol–copper complexes can cause oxidative stress by disruption of the mitochondrial respiration chain or by indirect non-mitochondrial induction of reactive oxygen species. We have found that elesclomol effectively kills the slow-cycling subpopulation and prevents the selective enrichment for slow-cycling cells, which usually results after monotreatment. We hypothesize that elesclomol could overcome the multidrug resistance of slow-cycling melanoma cells and prevent tumor repopulation in melanoma patients in future. PMID:25453510

  12. Mitochondrial oxidative stress as a novel therapeutic target to overcome intrinsic drug resistance in melanoma cell subpopulations.

    PubMed

    Cierlitza, Monika; Chauvistré, Heike; Bogeski, Ivan; Zhang, Xin; Hauschild, Axel; Herlyn, Meenhard; Schadendorf, Dirk; Vogt, Thomas; Roesch, Alexander

    2015-02-01

    Despite recent success in melanoma therapy, most patients with metastatic disease still undergo deadly progression. We have identified a novel mechanism of multidrug resistance allowing a small subpopulation of slow-cycling melanoma cells to survive based on elevated oxidative bioenergy metabolism. In this study, we asked whether such slow-cycling cells could be eliminated by co-treatment with the copper-chelator elesclomol. Elesclomol-copper complexes can cause oxidative stress by disruption of the mitochondrial respiration chain or by indirect non-mitochondrial induction of reactive oxygen species. We have found that elesclomol effectively kills the slow-cycling subpopulation and prevents the selective enrichment for slow-cycling cells, which usually results after monotreatment. We hypothesize that elesclomol could overcome the multidrug resistance of slow-cycling melanoma cells and prevent tumor repopulation in melanoma patients in future. PMID:25453510

  13. Identifying mechanistic indicators of childhood asthma from blood gene expression

    EPA Science Inventory

    Asthmatic individuals have been identified as a susceptible subpopulation for air pollutants. However, asthma represents a syndrome with multiple probable etiologies, and the identification of these asthma endotypes is critical to accurately define the most susceptible subpopula...

  14. Subpopulation-proteomics reveal growth rate, but not cell cycling, as a major impact on protein composition in Pseudomonas putida KT2440.

    PubMed

    Lieder, Sarah; Jahn, Michael; Seifert, Jana; von Bergen, Martin; Müller, Susann; Takors, Ralf

    2014-01-01

    Population heterogeneity occurring in industrial microbial bioprocesses is regarded as a putative effector causing performance loss in large scale. While the existence of subpopulations is a commonly accepted fact, their appearance and impact on process performance still remains rather unclear. During cell cycling, distinct subpopulations differing in cell division state and DNA content appear which contribute individually to the efficiency of the bioprocess. To identify stressed or impaired subpopulations, we analyzed the interplay of growth rate, cell cycle and phenotypic profile of subpopulations by using flow cytometry and cell sorting in conjunction with mass spectrometry based global proteomics. Adjusting distinct growth rates in chemostats with the model strain Pseudomonas putida KT2440, cells were differentiated by DNA content reflecting different cell cycle stages. The proteome of separated subpopulations at given growth rates was found to be highly similar, while different growth rates caused major changes of the protein inventory with respect to e.g. carbon storage, motility, lipid metabolism and the translational machinery. In conclusion, cells in various cell cycle stages at the same growth rate were found to have similar to identical proteome profiles showing no significant population heterogeneity on the proteome level. In contrast, the growth rate clearly determines the protein composition and therefore the metabolic strategy of the cells. PMID:25401072

  15. Coevolution of neoplastic epithelial cells and multilineage stroma via polyploid giant cells during immortalization and transformation of mullerian epithelial cells

    PubMed Central

    Zhang, Shiwu; Mercado-Uribe, Imelda; Sood, Anil; Bast, Robert C.; Liu, Jinsong

    2016-01-01

    Stromal cells are generally considered to be derived primarily from the host's normal mesenchymal stromal cells or bone marrow. However, the origins of stromal cells have been quite controversial. To determine the role of polyploidy in tumor development, we examined the fate of normal mullerian epithelial cells during the immortalization and transformation process by tracing the expression of SV40 large T antigen. Here we show that immortalized or HRAS-transformed mullerian epithelial cells contain a subpopulation of polyploid giant cells that grow as multicellular spheroids expressing hematopoietic markers in response to treatment with CoCl2. The immortalized or transformed epithelial cells can transdifferentiate into stromal cells when transplanted into nude mice. Immunofluorescent staining revealed expression of stem cell factors OCT4, Nanog, and SOX-2 in spheroid, whereas expression of embryonic stem cell marker SSEA1 was increased in HRAS-transformed cells compared with their immortalized isogenic counterparts. These results suggest that normal mullerian epithelial cells are intrinsically highly plastic, via the formation of polyploid giant cells and activation of embryonic stem-like program, which work together to promote the coevolution of neoplastic epithelial cells and multiple lineage stromal cells. PMID:27382431

  16. The Identification of Mitogen Responding Subpopulations of Human Lymphocytes by Flow Polarimeter Fluorescence Measurements.

    NASA Astrophysics Data System (ADS)

    Chan, Sandra Lynn

    I have developed a method to identify the mitogen responding subpopulation of human peripheral blood lymphocytes. This method employs a flow polarimeter to measure the distribution of the intensity and the polarization of intracellular fluorescein fluorescence in suspensions of mononuclear cells isolated on density gradients from the peripheral blood of donors. I have used the change in the fluorescence of cells exposed to the mitogens PHA and Con A to identify the responding cells and to quantitate this number. I have found that for most donors, the responding cells constitute about 20-40% of the lymphocyte population. The percent of responding cells decreases to zero in patients with acute lymphocytic leukemia (2 patients) and chronic lymphocyte leukemia (10 patients). For a variety of patients with other types of cancer, the responding fraction was not significantly different from healthy controls. Moreover, the number of responding cells does not appear to be age dependent in the age range of 20-80 years. I also found that the change in fluorescence polarization correlated strongly with changes in fluorescence intensity induced by mitogens--the number of responding cells, therefore can be estimated either from the intensity or polarization distributions. The shapes of fluorescence distributions depend strongly on a number of variables including the composition and density of the lymphocyte isolating medium, the mitogen and dye concentrations, the length of incubation with mitogen or dye, and the potassium, calcium, and magnesium concentrations in the medium. In the case of fluorescein, I have worked out a methodology that allows a consistent estimate of the responding lymphocyte number. I have also investigated the use of the dye carbocyanine for the same purpose. This dye presumably identifies the mitogen responding lymphocytes on the basis of changes in membrane potential. The results with carbocyanine were found to depend on a number of variables and I could

  17. Automated identification of stratifying signatures in cellular subpopulations

    PubMed Central

    Bruggner, Robert V.; Bodenmiller, Bernd; Dill, David L.; Tibshirani, Robert J.; Nolan, Garry P.

    2014-01-01

    Elucidation and examination of cellular subpopulations that display condition-specific behavior can play a critical contributory role in understanding disease mechanism, as well as provide a focal point for development of diagnostic criteria linking such a mechanism to clinical prognosis. Despite recent advancements in single-cell measurement technologies, the identification of relevant cell subsets through manual efforts remains standard practice. As new technologies such as mass cytometry increase the parameterization of single-cell measurements, the scalability and subjectivity inherent in manual analyses slows both analysis and progress. We therefore developed Citrus (cluster identification, characterization, and regression), a data-driven approach for the identification of stratifying subpopulations in multidimensional cytometry datasets. The methodology of Citrus is demonstrated through the identification of known and unexpected pathway responses in a dataset of stimulated peripheral blood mononuclear cells measured by mass cytometry. Additionally, the performance of Citrus is compared with that of existing methods through the analysis of several publicly available datasets. As the complexity of flow cytometry datasets continues to increase, methods such as Citrus will be needed to aid investigators in the performance of unbiased—and potentially more thorough—correlation-based mining and inspection of cell subsets nested within high-dimensional datasets. PMID:24979804

  18. Alteration in T lymphocyte subpopulations in inflammatory bowel disease.

    PubMed Central

    Victorino, R M; Hodgson, H J

    1980-01-01

    Peripheral blood mononuclear cells from thirty-one patients with inflammatory bowel disease (ulcerative colitis and Crohn's disease) were analysed for the proportions and absolute numbers of total T cells, and for the T cell subpopulations carrying Fc receptors for either IgM (Tmu cells) or IgG (T gamma cells). Twenty-six control subjects were studied simultaneously. Total T cell numbers were normal in patients with inflammatory bowel disease but there was a marked reduction in the proportion and absolute numbers of Tmu cells in patients, whether their disease was active or in remission. T gamma cells were normal. Simultaneous assessment of lymphocyte response to mitogens in vitro was performed in a group of patients. Responses to phytohaemagglutinin, concanavalin A and pokeweed mitogen were decreased and a positive correlation was found between the number of circulating Tmu cells and the responses to mitogens in vitro. These studies demonstrate that despite the presence of normal numbers of total T cells in inflammatory bowel disease, there is a marked imbalance in T cell subpopulations that correlates with mitogen responsiveness. This imbalance provides a possible cellular basis for the defect in cell-mediated immunity seen in these patients. PMID:6969150

  19. Estimation after subpopulation selection in adaptive seamless trials.

    PubMed

    Kimani, Peter K; Todd, Susan; Stallard, Nigel

    2015-08-15

    During the development of new therapies, it is not uncommon to test whether a new treatment works better than the existing treatment for all patients who suffer from a condition (full population) or for a subset of the full population (subpopulation). One approach that may be used for this objective is to have two separate trials, where in the first trial, data are collected to determine if the new treatment benefits the full population or the subpopulation. The second trial is a confirmatory trial to test the new treatment in the population selected in the first trial. In this paper, we consider the more efficient two-stage adaptive seamless designs (ASDs), where in stage 1, data are collected to select the population to test in stage 2. In stage 2, additional data are collected to perform confirmatory analysis for the selected population. Unlike the approach that uses two separate trials, for ASDs, stage 1 data are also used in the confirmatory analysis. Although ASDs are efficient, using stage 1 data both for selection and confirmatory analysis introduces selection bias and consequently statistical challenges in making inference. We will focus on point estimation for such trials. In this paper, we describe the extent of bias for estimators that ignore multiple hypotheses and selecting the population that is most likely to give positive trial results based on observed stage 1 data. We then derive conditionally unbiased estimators and examine their mean squared errors for different scenarios. PMID:25903293

  20. Cleft lip and palate in a Brazilian subpopulation

    PubMed Central

    Cuozzo, Fernanda Dornelles Martins; Espinosa, Mariano Martínez; da Silva, Katia Tavares Serafim; de Barros, Yolanda Benedita Abadia Martins; Bandeca, Matheus Coelho; Aranha, Andreza Maria Fabio; Borges, Alvaro Henrique; Volpato, Luiz Evaristo Ricci

    2013-01-01

    Background: This work aimed to access the profile of cleft lip and palate patients of a sub-population in Mid-West Brazil. Materials & Methods: Research was carried out through a cross-sectional study at the Craniofacial Rehabilitation Center of the University General Hospital of the University of Cuiabá, Mato Grosso, Brazil. Variables related to oral cleft type, gender, race, age and presence or absence of associated congenital anomalies or syndromes were analyzed. Results: 313 patients treated at the institution from 2004 to 2007 were recruited. There were 54% male and 46% female patients with the mean age of 11.4 years. Cleft lip and palate was the most prevalent alteration in 49.6% of cases. Caucasians were the most affected in 54.6% of cases. 6.4% of patients had other anomalies or syndromes associated with cleft. Conclusion: More comprehensive surveys should be conducted in order to supply the lack of data on the occurrence and determinants of oral clefts in this region. How to cite this article:Cuozzo FD, Espinosa MM, Serafim da Silva KT, Martins de Barros YB, Bandeca MC, Aranha AM, Borges AH, Volpato LE. Cleft lip and palate in a Brazilian subpopulation. J Int Oral Health 2013; 5(4):15-20. PMID:24155614

  1. Vascular channels formed by subpopulations of PECAM1+ melanoma cells

    PubMed Central

    Dunleavey, James M.; Xiao, Lin; Thompson, Joshua; Kim, Mi Mi; Shields, Janiel M.; Shelton, Sarah E.; Irvin, David M.; Brings, Victoria E.; Ollila, David; Brekken, Rolf A.; Dayton, Paul A.; Melero-Martin, Juan M.; Dudley, Andrew C.

    2014-01-01

    Targeting the vasculature remains a promising approach for treating solid tumors; however, the mechanisms of tumor neovascularization are diverse and complex. Here we uncover a new subpopulation of melanoma cells that express the vascular cell adhesion molecule PECAM1, but not VEGFR-2, and participate in a PECAM1-dependent form of vasculogenic mimicry (VM). Clonally-derived PECAM1+ tumor cells coalesce to form PECAM1-dependent networks in vitro and they generate well-perfused, VEGF-independent channels in mice. The neural crest specifier AP-2α is diminished in PECAM1+ melanoma cells and is a transcriptional repressor of PECAM1. Reintroduction of AP-2α into PECAM1+ tumor cells represses PECAM1 and abolishes tube-forming ability whereas AP-2α knockdown in PECAM1− tumor cells up-regulates PECAM1 expression and promotes tube formation. Thus, VM-competent subpopulations, rather than all cells within a tumor, may instigate VM, supplant host-derived endothelium, and form PECAM1-dependent conduits that are not diminished by neutralizing VEGF. PMID:25335460

  2. Group sequential designs with prospectively planned rules for subpopulation enrichment.

    PubMed

    Rosenblum, Michael; Luber, Brandon; Thompson, Richard E; Hanley, Daniel

    2016-09-20

    We propose a class of randomized trial designs aimed at gaining the advantages of wider generalizability and faster recruitment while mitigating the risks of including a population for which there is greater a priori uncertainty. We focus on testing null hypotheses for the overall population and a predefined subpopulation. Our designs have preplanned rules for modifying enrollment criteria based on data accrued at interim analyses. For example, enrollment can be restricted if the participants from a predefined subpopulation are not benefiting from the new treatment. Our designs have the following features: the multiple testing procedure fully leverages the correlation among statistics for different populations; the asymptotic familywise Type I error rate is strongly controlled; for outcomes that are binary or normally distributed, the decision rule and multiple testing procedure are functions of the data only through minimal sufficient statistics. Our designs incorporate standard group sequential boundaries for each population of interest; this may be helpful in communicating the designs, because many clinical investigators are familiar with such boundaries, which can be summarized succinctly in a single table or graph. We demonstrate these designs through simulations of a Phase III trial of a new treatment for stroke. User-friendly, free software implementing these designs is described. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27076411

  3. Hypolactasia in Saami subpopulations of Russia and Finland.

    PubMed

    Kozlov, A; Lisitsyn, D

    1997-12-01

    Primary hypolactasia is a gene attributed condition of the inability of adult individuals to consume whole milk. Subpopulations of the Russia (Kildin) and Finland Saami are characterized by a large variability of the LAC*R (lactase restriction) gene frequencies (0.50-0.77). The distribution of primary hypolactasia among the Saami is ranging from 25% to 60%. The intensive reindeer breeding was developed by the Saami only 300-400 years ago. Reindeer milk is poor in lactose (2.4%) and is consumed by the Saami in small amounts. Thus, "milk behaviour" connected with reindeer breeding could not have influenced the trait evolution too much. The large between-group differences of the LAC*R gene frequencies in the Saami seem to reflect the level of genetic influence of neighbouring non-Saami populations. The role of gene inflow in reducing the level of primary hypolactasia in various Saami subpopulations is confirmed by historic data of various ethnoterritorial groups as well as by the reduction of the number of traditional family name bearers and the change of the AB0 blood group gene frequencies among the Kildin Saami in the last 30 years. PMID:9468755

  4. Lymphotoxin beta receptor signaling limits mucosal damage through driving IL-23 production by epithelial cells.

    PubMed

    Macho-Fernandez, E; Koroleva, E P; Spencer, C M; Tighe, M; Torrado, E; Cooper, A M; Fu, Y-X; Tumanov, A V

    2015-03-01

    The immune mechanisms regulating epithelial cell repair after injury remain poorly defined. We demonstrate here that lymphotoxin beta receptor (LTβR) signaling in intestinal epithelial cells promotes self-repair after mucosal damage. Using a conditional gene-targeted approach, we demonstrate that LTβR signaling in intestinal epithelial cells is essential for epithelial interleukin-23 (IL-23) production and protection against epithelial injury. We further show that epithelial-derived IL-23 promotes mucosal wound healing by inducing the IL-22-mediated proliferation and survival of epithelial cells and mucus production. Additionally, we identified CD4(-)CCR6(+)T-bet(-) RAR-related orphan receptor gamma t (RORγt)(+) lymphoid tissue inducer cells as the main producers of protective IL-22 after epithelial damage. Thus, our results reveal a novel role for LTβR signaling in epithelial cells in the regulation of intestinal epithelial cell homeostasis to limit mucosal damage. PMID:25183367

  5. Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

    SciTech Connect

    Hirai, H.; Lochter, A.; Galosy, S.; Koshida, S.; Niwa, S.; Bissell, M.J.

    1997-10-13

    Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

  6. Quantitative Morphology of Epithelial Folds.

    PubMed

    Štorgel, Nick; Krajnc, Matej; Mrak, Polona; Štrus, Jasna; Ziherl, Primož

    2016-01-01

    The shape of spatially modulated epithelial morphologies such as villi and crypts is usually associated with the epithelium-stroma area mismatch leading to buckling. We propose an alternative mechanical model based on intraepithelial stresses generated by differential tensions of apical, lateral, and basal sides of cells as well as on the elasticity of the basement membrane. We use it to theoretically study longitudinal folds in simple epithelia and we identify four types of corrugated morphologies: compact, invaginated, evaginated, and wavy. The obtained tissue contours and thickness profiles are compared to epithelial folds observed in invertebrates and vertebrates, and for most samples, the agreement is within the estimated experimental error. Our model establishes the groove-crest modulation of tissue thickness as a morphometric parameter that can, together with the curvature profile, be used to estimate the relative differential apicobasal tension in the epithelium. PMID:26745429

  7. DNA damage in different Eisenia andrei coelomocytes sub-populations after in vitro exposure to hydrogen peroxide.

    PubMed

    Mincarelli, Laura; Vischetti, Costantino; Craft, John; Tiano, Luca

    2016-01-01

    Earthworms play an essential role in providing soil fertility and may represent an important soil contamination bio-indicator. They are able to ingest soil particles, adsorb substances throughout the intestinal epithelium into the coelomic cavity, where chemicals can come in direct contact with coelomic fluid. Earthworm coelomic fluid shelters leucocytes (coelomocytes) that differ significantly both structurally and functionally. Cellular variability could lead to different susceptibility towards contaminants possibly present in soil ecosystem. In order to define population specific dose response to chemicals and to identify a homogeneous cell population to be used as a relevant biomarker, we investigated different coelomocytes subpopulation, obtained by Percoll density gradient centrifugation (5-35 %), exposed ex vivo to H2O2 in the range of concentration 15-120 µM. DNA damage levels were assessed by the comet assay on unseparated coelomocytes and on three enriched cellular fractions (light, medium and heavy density subpopulations). All tested samples showed a dose-response genotoxic effect following H2O2 exposure. Moreover, light density sub-population appeared more susceptible to oxidative insult highlighted by a significant increase in DNA damage indexes at lower concentrations of H2O2. Present data suggested that in these experimental condition coelomocytes light fraction may represent a more sensitive biomarker of genotoxic insult. PMID:27064673

  8. CXCR4(+)CD45(-) BMMNC subpopulation is superior to unfractionated BMMNCs for protection after ischemic stroke in mice.

    PubMed

    Wang, Jianping; Liu, Xi; Lu, Hong; Jiang, Chao; Cui, Xiaobing; Yu, Lie; Fu, Xiaojie; Li, Qian; Wang, Jian

    2015-03-01

    Cell-based therapy is considered to be a promising therapeutic strategy for stroke treatment. Although unfractionated bone marrow mononuclear cells (BMMNCs) have been tried in both preclinical and clinical trials, the effective subpopulations need to be identified. In this study, we used fluorescence-activated cell sorting to harvest the CXCR4(+)CD45(+) and CXCR4(+)CD45(-) BMMNC subpopulations from transgenic mice that express enhanced green fluorescent protein. We then allogeneically grafted unfractionated BMMNCs or a subpopulation into mice subjected to transient middle cerebral artery occlusion (tMCAO) and compared the effects on stroke outcomes. We found that CXCR4(+)CD45(-) BMMNCs, but not CXCR4(+)CD45(+) BMMNCs, more effectively reduced infarction volume and neurologic deficits than did unfractionated BMMNCs. Brain tissue from the ischemic hemisphere of mice treated with CXCR4(+)CD45(-) BMMNCs had higher levels of vascular endothelial growth factor and lower levels of TNF-α than did tissue from mice treated with unfractionated BMMNCs. In contrast, CXCR4(+)CD45(+) BMMNCs showed an increase in TNF-α. Additionally, CXCR4(+)CD45(+) and CXCR4(+)CD45(-) populations exhibited more robust migration into the lesion areas and were better able to express cell-specific markers of different linages than were the unfractionated BMMNCs. Endothelial and astrocyte cell markers did not colocalize with eGFP(+) cells in the brains of tMCAO mice that received CXCR4(+)CD45(+) BMMNCs. In vitro, the CXCR4(+)CD45(-) BMMNCs expressed significantly more Oct-4 and Nanog mRNA than did the unfractionated BMMNCs. However, we did not detect gene expression of these two pluripotent markers in CXCR4(+)CD45(+) BMMNCs. Taken together, our study shows for the first time that the CXCR4(+)CD45(-) BMMNC subpopulation is superior to unfractionated BMMNCs in ameliorating cerebral damage in a mouse model of tMCAO and could represent a new therapeutic approach for stroke treatment. PMID:25526817

  9. Specific polar subpopulations of astral microtubules control spindle orientation and symmetric neural stem cell division.

    PubMed

    Mora-Bermúdez, Felipe; Matsuzaki, Fumio; Huttner, Wieland B

    2014-01-01

    Mitotic spindle orientation is crucial for symmetric vs asymmetric cell division and depends on astral microtubules. Here, we show that distinct subpopulations of astral microtubules exist, which have differential functions in regulating spindle orientation and division symmetry. Specifically, in polarized stem cells of developing mouse neocortex, astral microtubules reaching the apical and basal cell cortex, but not those reaching the central cell cortex, are more abundant in symmetrically than asymmetrically dividing cells and reduce spindle orientation variability. This promotes symmetric divisions by maintaining an apico-basal cleavage plane. The greater abundance of apical/basal astrals depends on a higher concentration, at the basal cell cortex, of LGN, a known spindle-cell cortex linker. Furthermore, newly developed specific microtubule perturbations that selectively decrease apical/basal astrals recapitulate the symmetric-to-asymmetric division switch and suffice to increase neurogenesis in vivo. Thus, our study identifies a novel link between cell polarity, astral microtubules, and spindle orientation in morphogenesis. PMID:24996848

  10. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state

    PubMed Central

    Rotem, Assaf; Ram, Oren; Shoresh, Noam; Sperling, Ralph A.; Goren, Alon; Weitz, David A.; Bernstein, Bradley E.

    2015-01-01

    Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells, and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell is sparse, comprising on the order of 1000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of sub-populations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone. PMID:26458175

  11. Isolation and Characterization of Human Mesenchymal Stromal Cell Subpopulations: Comparison of Bone Marrow and Adipose Tissue.

    PubMed

    Busser, Hélène; Najar, Mehdi; Raicevic, Gordana; Pieters, Karlien; Velez Pombo, Rafael; Philippart, Pierre; Meuleman, Nathalie; Bron, Dominique; Lagneaux, Laurence

    2015-09-15

    Preparations of mesenchymal stromal cells (MSCs) are generally obtained from unfractionated tissue cells, resulting in heterogeneous cell mixtures. Several markers were proposed to enrich these cells, but the majority of these markers are defined for bone marrow (BM). Moreover, the surface markers of freshly isolated MSCs also differ from those of cultured MSCs in addition to a phenotypic variation depending on the MSC source. For tissue engineering applications, it is crucial to start with a well-defined cell population. In this study, we performed immunomagnetic selections with five single surface markers to isolate MSC subpopulations from BM and adipose tissue (AT): CD271, SUSD2, MSCA-1, CD44, and CD34. We determined the phenotype, the clonogenicity, the proliferation, the differentiation capacity, and the immunoregulatory profile of the subpopulations obtained in comparison with unselected cells. We showed that native BM-MSCs can be enriched from the positive fractions of MSCA-1, SUSD2, and CD271 selections. In contrast, we observed that SUSD2 and MSCA-1 were unable to identify MSCs from AT, meaning they are not expressed in situ. Only the CD34(+) selection successfully isolated MSCs from AT. Interestingly, we observed that CD271 selection can define AT cell subsets with particular abilities, but only in lipoaspiration samples and not in abdominoplasty samples. Importantly, we found a population of clear CD34(+) fresh BM-MSCs displaying different properties. A single marker-based selection for MSC enrichment should be more advantageous for cell therapy and would enable the standardization of efficient and safe therapeutic intervention through the use of a well-identified and homogeneous cell population. PMID:26086188

  12. Expected Shannon Entropy and Shannon Differentiation between Subpopulations for Neutral Genes under the Finite Island Model

    PubMed Central

    Chao, Anne; Jost, Lou; Hsieh, T. C.; Ma, K. H.; Sherwin, William B.; Rollins, Lee Ann

    2015-01-01

    Shannon entropy H and related measures are increasingly used in molecular ecology and population genetics because (1) unlike measures based on heterozygosity or allele number, these measures weigh alleles in proportion to their population fraction, thus capturing a previously-ignored aspect of allele frequency distributions that may be important in many applications; (2) these measures connect directly to the rich predictive mathematics of information theory; (3) Shannon entropy is completely additive and has an explicitly hierarchical nature; and (4) Shannon entropy-based differentiation measures obey strong monotonicity properties that heterozygosity-based measures lack. We derive simple new expressions for the expected values of the Shannon entropy of the equilibrium allele distribution at a neutral locus in a single isolated population under two models of mutation: the infinite allele model and the stepwise mutation model. Surprisingly, this complex stochastic system for each model has an entropy expressable as a simple combination of well-known mathematical functions. Moreover, entropy- and heterozygosity-based measures for each model are linked by simple relationships that are shown by simulations to be approximately valid even far from equilibrium. We also identify a bridge between the two models of mutation. We apply our approach to subdivided populations which follow the finite island model, obtaining the Shannon entropy of the equilibrium allele distributions of the subpopulations and of the total population. We also derive the expected mutual information and normalized mutual information (“Shannon differentiation”) between subpopulations at equilibrium, and identify the model parameters that determine them. We apply our measures to data from the common starling (Sturnus vulgaris) in Australia. Our measures provide a test for neutrality that is robust to violations of equilibrium assumptions, as verified on real world data from starlings. PMID

  13. Expected Shannon Entropy and Shannon Differentiation between Subpopulations for Neutral Genes under the Finite Island Model.

    PubMed

    Chao, Anne; Jost, Lou; Hsieh, T C; Ma, K H; Sherwin, William B; Rollins, Lee Ann

    2015-01-01

    Shannon entropy H and related measures are increasingly used in molecular ecology and population genetics because (1) unlike measures based on heterozygosity or allele number, these measures weigh alleles in proportion to their population fraction, thus capturing a previously-ignored aspect of allele frequency distributions that may be important in many applications; (2) these measures connect directly to the rich predictive mathematics of information theory; (3) Shannon entropy is completely additive and has an explicitly hierarchical nature; and (4) Shannon entropy-based differentiation measures obey strong monotonicity properties that heterozygosity-based measures lack. We derive simple new expressions for the expected values of the Shannon entropy of the equilibrium allele distribution at a neutral locus in a single isolated population under two models of mutation: the infinite allele model and the stepwise mutation model. Surprisingly, this complex stochastic system for each model has an entropy expressable as a simple combination of well-known mathematical functions. Moreover, entropy- and heterozygosity-based measures for each model are linked by simple relationships that are shown by simulations to be approximately valid even far from equilibrium. We also identify a bridge between the two models of mutation. We apply our approach to subdivided populations which follow the finite island model, obtaining the Shannon entropy of the equilibrium allele distributions of the subpopulations and of the total population. We also derive the expected mutual information and normalized mutual information ("Shannon differentiation") between subpopulations at equilibrium, and identify the model parameters that determine them. We apply our measures to data from the common starling (Sturnus vulgaris) in Australia. Our measures provide a test for neutrality that is robust to violations of equilibrium assumptions, as verified on real world data from starlings. PMID:26067448

  14. A variable immunoreceptor in a subpopulation of human neutrophils

    PubMed Central

    Puellmann, Kerstin; Kaminski, Wolfgang E.; Vogel, Mandy; Nebe, C. Thomas; Schroeder, Josef; Wolf, Hans; Beham, Alexander W.

    2006-01-01

    Neutrophils are thought to rely solely on nonspecific immune mechanisms. Here we provide molecular biological, immunological, ultrastructural, and functional evidence for the presence of a T cell receptor (TCR)-based variable immunoreceptor in a 5–8% subpopulation of human neutrophils. We demonstrate that these peripheral blood neutrophils express variable and individual-specific TCRαβ repertoires and the RAG1/RAG2 recombinase complex. The proinflammatory cytokine granulocyte colony-stimulating factor regulates expression of the neutrophil immunoreceptor and RAG1/RAG2 in vivo. Specific engagement of the neutrophil TCR complex protects from apoptosis and stimulates secretion of the neutrophil-activating chemokine IL-8. Our results, which also demonstrate the presence of the TCR in murine neutrophils, suggest the coexistence of a variable and an innate host defense system in mammalian neutrophils. PMID:16983085

  15. Chronic stress, leukocyte subpopulations, and humoral response to latent viruses

    SciTech Connect

    McKinnon, W.; Weisse, C.S.; Reynolds, C.P.; Bowles, C.A.; Baum, A. )

    1989-01-01

    Psychological stress has been shown to affect immune system status and function, but most studies of this relationship have focused on acute stress and/or laboratory situations. The present study compared total numbers of leukocytes and lymphocyte subpopulations (determined by flow cytometry) and antibody titers to latent and nonlatent viruses among a group of chronically stressed individuals living near the damaged Three Mile Island (TMI) nuclear power plant with those of a demographically comparable control group. Urinary catecholamine and cortisol levels were also examined. Residents of the TMI area exhibited greater numbers of neutrophils, which were positively correlated with epinephrine levels. The TMI group also exhibited fewer B lymphocytes, T-suppressor/cytotoxic lymphocytes, and natural killer cells. Antibody titers to herpes simplex were significantly different across groups as well, whereas titers to nonlatent rubella virus as well as IgG and IgM levels were comparable.

  16. Chronic stress, leukocyte subpopulations, and humoral response to latent viruses.

    PubMed

    McKinnon, W; Weisse, C S; Reynolds, C P; Bowles, C A; Baum, A

    1989-01-01

    Psychological stress has been shown to affect immune system status and function, but most studies of this relationship have focused on acute stress and/or laboratory situations. The present study compared total numbers of leukocytes and lymphocyte subpopulations (determined by flow cytometry) and antibody titers to latent and nonlatent viruses among a group of chronically stressed individuals living near the damaged Three Mile Island (TMI) nuclear power plant with those of a demographically comparable control group. Urinary catecholamine and cortisol levels were also examined. Residents of the TMI area exhibited greater numbers of neutrophils, which were positively correlated with epinephrine levels. The TMI group also exhibited fewer B lymphocytes, T-suppressor/cytotoxic lymphocytes, and natural killer cells. Antibody titers to herpes simplex were significantly different across groups as well, whereas titers to nonlatent rubella virus as well as IgG and IgM levels were comparable. PMID:2555149

  17. Osteogenic potential of sorted equine mesenchymal stem cell subpopulations

    PubMed Central

    Radtke, Catherine L.; Nino-Fong, Rodolfo; Rodriguez-Lecompte, Juan Carlos; Esparza Gonzalez, Blanca P.; Stryhn, Henrik; McDuffee, Laurie A.

    2015-01-01

    The objectives of this study were to use non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs), to sort equine muscle tissue-derived mesenchymal stem cells (MMSCs) and bone marrow-derived mesenchymal stem cells (BMSC) into subpopulations and to carry out assays in order to compare their osteogenic capabilities. Cells from 1 young adult horse were isolated from left semitendinosus muscle tissue and from bone marrow aspirates of the fourth and fifth sternebrae. Aliquots of 800 × 103 MSCs from each tissue source were sorted into 5 fractions using non-equilibrium GrFFF (GrFFF proprietary system). Pooled fractions were cultured and expanded for use in osteogenic assays, including flow cytometry, histochemistry, bone nodule assays, and real-time quantitative polymerase chain reaction (qPCR) for gene expression of osteocalcin (OCN), RUNX2, and osterix. Equine MMSCs and BMSCs were consistently sorted into 5 fractions that remained viable for use in further osteogenic assays. Statistical analysis confirmed strongly significant upregulation of OCN, RUNX2, and osterix for the BMSC fraction 4 with P < 0.00001. Flow cytometry revealed different cell size and granularity for BMSC fraction 4 and MMSC fraction 2 compared to unsorted controls and other fractions. Histochemisty and bone nodule assays revealed positive staining nodules without differences in average nodule area, perimeter, or stain intensity between tissues or fractions. As there are different subpopulations of MSCs with different osteogenic capacities within equine muscle- and bone marrow-derived sources, these differences must be taken into account when using equine stem cell therapy to induce bone healing in veterinary medicine. PMID:25852225

  18. Two subpopulations of stem cells for T cell lineage

    SciTech Connect

    Katsura, Y.; Amagai, T.; Kina, T.; Sado, T.; Nishikawa, S.

    1985-11-01

    An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells.

  19. CD161 expression characterizes a subpopulation of human regulatory T cells that produces IL-17 in a STAT3-dependent manner

    PubMed Central

    Afzali, Behdad; Mitchell, Peter J; Edozie, Francis C; Povoleri, Giovanni AM; Dowson, Sophie E; Demandt, Laura; Walter, Gina; Canavan, James B; Scotta, Cristiano; Menon, Bina; Chana, Prabhjoat S; Khamri, Wafa; Kordasti, Shahram Y; Heck, Susanne; Grimbacher, Bodo; Tree, Timothy; Cope, Andrew P; Taams, Leonie S; Lechler, Robert I; John, Susan; Lombardi, Giovanna

    2013-01-01

    Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell-based clinical therapy. However, human Treg cells are “plastic”, and are able to produce IL-17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL-17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL-17 in vitro when activated in the presence of IL-1β, but not IL-6. “IL-17 potential” is restricted to population III (CD4+CD25hiCD127loCD45RA−) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL-17 induction. Importantly, we find that IL-17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL-17. Finally, we show that CD161+ population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL-17-producing Treg-cell population at these sites. As IL-17 production from this Treg-cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary. PMID:23677517

  20. Prostate epithelial cell of origin determines cancer differentiation state in an organoid transformation assay

    PubMed Central

    Park, Jung Wook; Lee, John K.; Phillips, John W.; Huang, Patrick; Cheng, Donghui; Huang, Jiaoti; Witte, Owen N.

    2016-01-01

    The cell of origin for prostate cancer remains a subject of debate. Genetically engineered mouse models have demonstrated that both basal and luminal cells can serve as cells of origin for prostate cancer. Using a human prostate regeneration and transformation assay, our group previously demonstrated that basal cells can serve as efficient targets for transformation. Recently, a subpopulation of multipotent human luminal cells defined by CD26 expression that retains progenitor activity in a defined organoid culture was identified. We transduced primary human prostate basal and luminal cells with lentiviruses expressing c-Myc and activated AKT1 (myristoylated AKT1 or myrAKT1) to mimic the MYC amplification and PTEN loss commonly detected in human prostate cancer. These cells were propagated in organoid culture before being transplanted into immunodeficient mice. We found that c-Myc/myrAKT1–transduced luminal xenografts exhibited histological features of well-differentiated acinar adenocarcinoma, with strong androgen receptor (AR) and prostate-specific antigen (PSA) expression. In contrast, c-Myc/myrAKT1–transduced basal xenografts were histologically more aggressive, with a loss of acinar structures and low/absent AR and PSA expression. Our findings imply that distinct subtypes of prostate cancer may arise from luminal and basal epithelial cell types subjected to the same oncogenic insults. This study provides a platform for the functional evaluation of oncogenes in basal and luminal epithelial populations of the human prostate. Tumors derived in this fashion with defined genetics can be used in the preclinical development of targeted therapeutics. PMID:27044116

  1. Characterization of Distinct Macrophage Subpopulations during Nitrogen Mustard-Induced Lung Injury and Fibrosis.

    PubMed

    Venosa, Alessandro; Malaviya, Rama; Choi, Hyejeong; Gow, Andrew J; Laskin, Jeffrey D; Laskin, Debra L

    2016-03-01

    Nitrogen mustard (NM) is an alkylating agent known to cause extensive pulmonary injury progressing to fibrosis. This is accompanied by a persistent macrophage inflammatory response. In these studies, we characterized the phenotype of macrophages accumulating in the lung over time following NM exposure. Treatment of rats with NM (0.125 mg/kg, intratracheally) resulted in an increase in CD11b(+) macrophages in histologic sections. These cells consisted of inducible nitric oxide synthase(+) (iNOS) proinflammatory M1 macrophages, and CD68(+), CD163(+), CD206(+), YM-1(+), and arginase-II(+)antiinflammatory M2 macrophages. Although M1 macrophages were prominent 1-3 days after NM, M2 macrophages were most notable at 28 days. At this time, they were enlarged and vacuolated, consistent with a profibrotic phenotype. Flow cytometric analysis of isolated lung macrophages identified three phenotypically distinct subpopulations: mature CD11b(-), CD43(-), and CD68(+) resident macrophages, which decreased in numbers after NM; and two infiltrating (CD11b(+)) macrophage subsets: immature CD43(+) M1 macrophages and mature CD43(-) M2 macrophages, which increased sequentially. Time-related increases in M1 (iNOS, IL-12α, COX-2, TNF-α, matrix metalloproteinase-9, matrix metalloproteinase-10) and M2 (IL-10, pentraxin-2, connective tissue growth factor, ApoE) genes, as well as chemokines/chemokine receptors associated with trafficking of M1 (CCR2, CCR5, CCL2, CCL5) and M2 (CX3CR1, fractalkine) macrophages to sites of injury, were also noted in macrophages isolated from the lung after NM. The appearance of M1 and M2 macrophages in the lung correlated with NM-induced acute injury and the development of fibrosis, suggesting a potential role of these macrophage subpopulations in the pathogenic response to NM. PMID:26273949

  2. TH-E-BRF-08: Subpopulations of Similarly-Responding Lesions in Metastatic Prostate Cancer

    SciTech Connect

    Lin, C; Harmon, S; Perk, T; Jeraj, R

    2014-06-15

    Purpose: In patients with multiple lesions, resistance to cancer treatments and subsequent disease recurrence may be due to heterogeneity of response across lesions. This study aims to identify subpopulations of similarly-responding metastatic prostate cancer lesions in bone using quantitative PET metrics. Methods: Seven metastatic prostate cancer patients treated with AR-directed therapy received pre-treatment and mid-treatment [F-18]NaF PET/CT scans. Images were registered using an articulated CT registration algorithm and transformations were applied to PET segmentations. Midtreatment response was calculated on PET-based texture features. Hierarchical agglomerative clustering was used to form groups of similarly-responding lesions, with the number of natural clusters (K) determined by the inconsistency coefficient. Lesion clustering was performed within each patient, and for the pooled population. The cophenetic coefficient (C) quantified how well the data was clustered. The Jaccard Index (JI) assessed similarity of cluster assignments from patient clustering and from population clustering. Results: 188 lesions in seven patients were identified for analysis (between 6 to 53 lesions per patient). Lesion response was defined as percent change relative to pre-treatment for 23 uncorrelated PET-based feature identifiers. . High response heterogeneity was found across all lesions (i.e. range ΔSUVmax =−95.98% to 775.00%). For intra-patient clustering, K ranged from 1–20. Population-based clustering resulted in 75 clusters, of 1-6 lesions each. Intra-patient clustering resulted in higher quality clusters than population clustering (mean C=0.95, range=0.89 to 1.00). For all patients, cluster assignments from population clustering showed good agreement to intra-patient clustering (mean JI=0.87, range=0.68 to 1.00). Conclusion: Subpopulations of similarly-responding lesions were identified in patients with multiple metastatic lesions. Good agreement was found between

  3. Epithelial dynamics of pancreatic branching morphogenesis

    PubMed Central

    Villasenor, Alethia; Chong, Diana C.; Henkemeyer, Mark; Cleaver, Ondine

    2010-01-01

    The mammalian pancreas is a highly branched gland, essential for both digestion and glucose homeostasis. Pancreatic branching, however, is poorly understood, both at the ultrastructural and cellular levels. In this article, we characterize the morphogenesis of pancreatic branches, from gross anatomy to the dynamics of their epithelial organization. We identify trends in pancreatic branch morphology and introduce a novel mechanism for branch formation, which involves transient epithelial stratification and partial loss of cell polarity, changes in cell shape and cell rearrangements, de novo tubulogenesis and epithelial tubule remodeling. In contrast to the classical epithelial budding and tube extension observed in other organs, a pancreatic branch takes shape as a multi-lumen tubular plexus coordinately extends and remodels into a ramifying, single-lumen ductal system. Moreover, our studies identify a role for EphB signaling in epithelial remodeling during pancreatic branching. Overall, these results illustrate distinct, step-wise cellular mechanisms by which pancreatic epithelium shapes itself to create a functional branching organ. PMID:21098570

  4. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    SciTech Connect

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  5. Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation

    PubMed Central

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2016-01-01

    Summary Cellular senescence suppresses cancer by arresting cells at risk of malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation and branching morphogenesis. Furthermore, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts – the ability to alter epithelial differentiation – that might also explain the loss of tissue function and organization that is a hallmark of aging. PMID:15657080

  6. Origins and determinants of HDL populations and their subpopulations

    SciTech Connect

    Nichols, A.V.; Gong, E.L.

    1990-06-01

    This paper describes the origins and determinants of High Density Lipoproteins (HDL) populations and their subpopulations. Our survey of compositional properties of small HDL particles indicates considerable variation in core lipid content reflecting in large part the origins of such particles. Whether small HDL particles of different core content and apolipoprotein composition differ in their metabolic properties and function in reverse cholesterol transport remains to be established. Our studies demonstrate that lipolysis-derived products can facilitate formation in vitro of small Apolipoprotein (AI) particles with properties approximating those of plasma pre-{beta} HDL. Of particular interest is our observation that small AI particles are an exclusive reassembly product in mixtures containing POPE and FFA. This observation may be relevant to the physiologic origins of PE in lipoprotein structure and its role in metabolism and secretion of nascent HDL. Lastly our observations on the reactivity of small AI particles, containing FFA, with LCAT and LDL suggest further linkages between triglyceride and HDL metabolism. 19 refs., 4 figs., 5 tabs.

  7. Cisplatin and CCNU synergism in spheroid cell subpopulations.

    PubMed Central

    Durand, R. E.

    1990-01-01

    The cytotoxicity of two antineoplastic drugs, cisplatin and CCNU, was evaluated in Chinese hamster V79 multicell spheroids using the drugs as single agents or combinations. Cells obtained from different depths within spheroids 550-750 microns in diameter showed different sensitivities to the two agents; the external cells of the spheroids were more sensitive than the internal cells to cisplatin, whereas the internal cells were most effectively killed by CCNU. Combining the two agents produced the expected 'complementary' activity, and in addition, synergism was observed between the drugs at exposure levels practical for clinical use. For the combination treatments, both the net pattern of cell killing through the spheroid and the degree of interaction between the agents (quantified using the combination index method) were a function of the dose ratio of the two drugs, and of overall treatment intensity. BCNU produced patterns of cell killing similar to CCNU, but showed little interaction with cisplatin. Our results suggest significant clinical potential in using CCNU with cisplatin, particularly since CCNU-cisplatin combinations were synergistic even in the cell subpopulations most resistant to each drug as a single agent. PMID:2257225

  8. B-1-cell subpopulations contribute differently to gut immunity.

    PubMed

    Roy, Bishnudeo; Agarwal, Shiwani; Brennecke, Anne-Margarete; Krey, Martina; Pabst, Oliver; Düber, Sandra; Weiss, Siegfried

    2013-08-01

    In mice, B-1 (B1a/B1b) cells are mainly located in the peritoneal cavity. B-1 cells are well known for their role in the early stages of Ab-mediated immune responses against pathogenic invasion as well as for the production of natural IgM antibodies. Although such B cells have been claimed to give rise to intestinal plasma cells producing IgA, a clear role of B-1 cells in IgA production in the gut-associated tissues is still not defined. Here, we employed the transgenic L2 mouse model characterized by the lack of B-2 cells and presence of B-1 cells as major B-cell subpopulation. The oligoclonality of the Ab repertoire in this mouse allowed us to take typical B1a cell VH sequences as indicators of the presence of IgM-producing B-1a cells in Peyer's patches as well as in lamina propria. However, amongst the IgAVH sequences recovered from the same tissues, none of the sequences showed B1a-cell specificity. Interestingly, all IgAVH sequences derived from the lamina propria of L2 mice displayed extensive numbers of nucleotide exchanges, indicating somatic hypermutation, and affinity maturation. This suggests that the contribution of natural unmutated IgA by B-1a cells to intestinal immunity is negligible. PMID:23677546

  9. Population genomic analysis reveals differential evolutionary histories and patterns of diversity across subgenomes and subpopulations of Brassica napus L.

    DOE PAGESBeta

    Gazave, Elodie; Tassone, Erica E.; Ilut, Daniel C.; Wingerson, Megan; Datema, Erwin; Witsenboer, Hanneke M. A.; Davis, James B.; Grant, David; Dyer, John M.; Jenks, Matthew A.; et al

    2016-04-21

    Here, the allotetraploid species Brassica napus L. is a global crop of major economic importance, providing canola oil (seed) and vegetables for human consumption and fodder and meal for livestock feed. Characterizing the genetic diversity present in the extant germplasm pool of B. napus is fundamental to better conserve, manage and utilize the genetic resources of this species. We used sequence-based genotyping to identify and genotype 30,881 SNPs in a diversity panel of 782 B. napus accessions, representing samples of winter and spring growth habits originating from 33 countries across Europe, Asia, and America. We detected strong population structure broadlymore » concordant with growth habit and geography, and identified three major genetic groups: spring (SP), winter Europe (WE), and winter Asia (WA). Subpopulation-specific polymorphism patterns suggest enriched genetic diversity within the WA group and a smaller effective breeding population for the SP group compared to WE. Interestingly, the two subgenomes of B. napus appear to have different geographic origins, with phylogenetic analysis placing WE and WA as basal clades for the other subpopulations in the C and A subgenomes, respectively. Finally, we identified 16 genomic regions where the patterns of diversity differed markedly from the genome-wide average, several of which are suggestive of genomic inversions. The results obtained in this study constitute a valuable resource for worldwide breeding efforts and the genetic dissection and prediction of complex B. napus traits.« less

  10. Population Genomic Analysis Reveals Differential Evolutionary Histories and Patterns of Diversity across Subgenomes and Subpopulations of Brassica napus L.

    PubMed

    Gazave, Elodie; Tassone, Erica E; Ilut, Daniel C; Wingerson, Megan; Datema, Erwin; Witsenboer, Hanneke M A; Davis, James B; Grant, David; Dyer, John M; Jenks, Matthew A; Brown, Jack; Gore, Michael A

    2016-01-01

    The allotetraploid species Brassica napus L. is a global crop of major economic importance, providing canola oil (seed) and vegetables for human consumption and fodder and meal for livestock feed. Characterizing the genetic diversity present in the extant germplasm pool of B. napus is fundamental to better conserve, manage and utilize the genetic resources of this species. We used sequence-based genotyping to identify and genotype 30,881 SNPs in a diversity panel of 782 B. napus accessions, representing samples of winter and spring growth habits originating from 33 countries across Europe, Asia, and America. We detected strong population structure broadly concordant with growth habit and geography, and identified three major genetic groups: spring (SP), winter Europe (WE), and winter Asia (WA). Subpopulation-specific polymorphism patterns suggest enriched genetic diversity within the WA group and a smaller effective breeding population for the SP group compared to WE. Interestingly, the two subgenomes of B. napus appear to have different geographic origins, with phylogenetic analysis placing WE and WA as basal clades for the other subpopulations in the C and A subgenomes, respectively. Finally, we identified 16 genomic regions where the patterns of diversity differed markedly from the genome-wide average, several of which are suggestive of genomic inversions. The results obtained in this study constitute a valuable resource for worldwide breeding efforts and the genetic dissection and prediction of complex B. napus traits. PMID:27148342

  11. Expressional Subpopulation of Cancers Determined by G64, a Co-regulated Module

    PubMed Central

    Min, Jae-Woong

    2015-01-01

    Studies of cancer heterogeneity have received considerable attention recently, because the presence or absence of resistant sub-clones may determine whether or not certain therapeutic treatments are effective. Previously, we have reported G64, a co-regulated gene module composed of 64 different genes, can differentiate tumor intra- or inter-subpopulations in lung adenocarcinomas (LADCs). Here, we investigated whether the G64 module genes were also expressed distinctively in different subpopulations of other cancers. RNA sequencing-based transcriptome data derived from 22 cancers, except LADC, were downloaded from The Cancer Genome Atlas (TCGA). Interestingly, the 22 cancers also expressed the G64 genes in a correlated manner, as observed previously in an LADC study. Considering that gene expression levels were continuous among different tumor samples, tumor subpopulations were investigated using extreme expressional ranges of G64—i.e., tumor subpopulation with the lowest 15% of G64 expression, tumor subpopulation with the highest 15% of G64 expression, and tumor subpopulation with intermediate expression. In each of the 22 cancers, we examined whether patient survival was different among the three different subgroups and found that G64 could differentiate tumor subpopulations in six other cancers, including sarcoma, kidney, brain, liver, and esophageal cancers. PMID:26865844

  12. Estimates of vital rates for a declining loggerhead turtle (Caretta caretta) subpopulation: implications for management

    USGS Publications Warehouse

    Lamont, Margaret M.; Fujisaki, Ikuko; Carthy, Raymond R.

    2014-01-01

    Because subpopulations can differ geographically, genetically and/or phenotypically, using data from one subpopulation to derive vital rates for another, while often unavoidable, is not optimal. We used a two-state open robust design model to analyze a 14-year dataset (1998–2011) from the St. Joseph Peninsula, Florida (USA; 29.748°, −85.400°) which is the densest loggerhead (Caretta caretta) nesting beach in the Northern Gulf of Mexico subpopulation. For these analyses, 433 individuals were marked of which only 7.2 % were observed re-nesting in the study area in subsequent years during the study period. Survival was estimated at 0.86 and is among the highest estimates for all subpopulations in the Northwest Atlantic population. The robust model estimated a nesting assemblage size that ranged from 32 to 230 individuals each year with an annual average of 110. The model estimates indicated an overall population decline of 17 %. The results presented here for this nesting group represent the first estimates for this subpopulation. These data provide managers with information specific to this subpopulation that can be used to develop recovery plans and conduct subpopulation-specific modeling exercises explicit to the challenges faced by turtles nesting in this region.

  13. TMPRSS2- Driven ERG Expression In Vivo Increases Self-Renewal and Maintains Expression in a Castration Resistant Subpopulation

    PubMed Central

    Abou-Kheir, Wassim G.; Martin, Philip L.; Tillman, Heather S.; Petrovics, Gyorgy; Awwad, Hibah O.; Ward, Yvona; Lake, Ross; Zhang, Luhua; Kelly, Kathleen

    2012-01-01

    Genomic rearrangements commonly occur in many types of cancers and often initiate or alter the progression of disease. Here we describe an in vivo mouse model that recapitulates the most frequent rearrangement in prostate cancer, the fusion of the promoter region of TMPRSS2 with the coding region of the transcription factor, ERG. A recombinant bacterial artificial chromosome including an extended TMPRSS2 promoter driving genomic ERG was constructed and used for transgenesis in mice. TMPRSS2-ERG expression was evaluated in tissue sections and FACS-fractionated prostate cell populations. In addition to the anticipated expression in luminal cells, TMPRSS2-ERG was similarly expressed in the Sca-1hi/EpCAM+ basal/progenitor fraction, where expanded numbers of clonogenic self-renewing progenitors were found, as assayed by in vitro sphere formation. These clonogenic cells increased intrinsic self renewal in subsequent generations. In addition, ERG dependent self-renewal and invasion in vitro was demonstrated in prostate cell lines derived from the model. Clinical studies have suggested that the TMPRSS2-ERG translocation occurs early in prostate cancer development. In the model described here, the presence of the TMPRSS2-ERG fusion alone was not transforming but synergized with heterozygous Pten deletion to promote PIN. Taken together, these data suggest that one function of TMPRSS2-ERG is the expansion of self-renewing cells, which may serve as targets for subsequent mutations. Primary prostate epithelial cells demonstrated increased post transcriptional turnover of ERG compared to the TMPRSS2-ERG positive VCaP cell line, originally isolated from a prostate cancer metastasis. Finally, we determined that TMPRSS2-ERG expression occurred in both castration-sensitive and resistant prostate epithelial subpopulations, suggesting the existence of androgen-independent mechanisms of TMPRSS2 expression in prostate epithelium. PMID:22860005

  14. Scattering attenuation microscopy of oral epithelial dysplasia

    NASA Astrophysics Data System (ADS)

    Tomlins, Pete H.; Adegun, Oluyori; Hagi-Pavli, Eleni; Piper, Kim; Bader, Dan; Fortune, Farida

    2010-11-01

    We present a new method for quantitative visualization of premalignant oral epithelium called scattering attenuation microscopy (SAM). Using low-coherence interferometry, SAM projects measurements of epithelial optical attenuation onto an image of the tissue surface as a color map. The measured attenuation is dominated by optical scattering that provides a metric of the severity of oral epithelial dysplasia (OED). Scattering is sensitive to the changes in size and distribution of nuclear material that are characteristic of OED, a condition recognized by the occurrence of basal-cell-like features throughout the epithelial depth. SAM measures the axial intensity change of light backscattered from epithelial tissue. Scattering measurements are obtained from sequential axial scans of a 3-D tissue volume and displayed as a 2-D SAM image. A novel segmentation method is used to confine scattering measurement to epithelial tissue. This is applied to oral biopsy samples obtained from 19 patients. Our results show that imaging of tissue scattering can be used to discriminate between different dysplastic severities and furthermore presents a powerful tool for identifying the most representative tissue site for biopsy.

  15. Genomewide Identification of Genes Under Directional Selection: Gene Transcription QST Scan in Diverging Atlantic Salmon Subpopulations

    PubMed Central

    Roberge, C.; Guderley, H.; Bernatchez, L.

    2007-01-01

    Evolutionary genomics has benefited from methods that allow identifying evolutionarily important genomic regions on a genomewide scale, including genome scans and QTL mapping. Recently, genomewide scanning by means of microarrays has permitted assessing gene transcription differences among species or populations. However, the identification of differentially transcribed genes does not in itself suffice to measure the role of selection in driving evolutionary changes in gene transcription. Here, we propose and apply a “transcriptome scan” approach to investigating the role of selection in shaping differential profiles of gene transcription among populations. We compared the genomewide transcription levels between two Atlantic salmon subpopulations that have been diverging for only six generations. Following assessment of normality and unimodality on a gene-per-gene basis, the additive genetic basis of gene transcription was estimated using the animal model. Gene transcription h2 estimates were significant for 1044 (16%) of all detected cDNA clones. In an approach analogous to that of genome scans, we used the distribution of the QST values estimated from intra- and intersubpopulation additive genetic components of the transcription profiles to identify 16 outlier genes (average QST estimate = 0.11) whose transcription levels are likely to have evolved under the influence of directional selection within six generations only. Overall, this study contributes both empirically and methodologically to the quantitative genetic exploration of gene transcription data. PMID:17720934

  16. Effects of Beryllium on Human Serum Immunoglobulin and Lymphocyte Subpopulation

    PubMed Central

    Kim, DaeSeong; Won, Yong Lim; Kang, Seong-Kyu

    2013-01-01

    To investigate the effects of short-term exposure of beryllium on the human immune system, the proportion of T-lymphocytes such as CD3+, CD4+, CD8+, CD95, and NK cells, andthe proportion of B cells and TNFα level in peripheral blood and immunoglobulins in the serum of 43 exposed workers and 34 healthy control subjects were studied. External exposure to beryllium was measured by atomic absorption spectrometer as recommended by the NIOSH analytical method 7300. T lymphocyte subpopulation analysis was carried out with flow cytometer. The working duration of exposed workers was less than 3 months and the mean ambient beryllium level was 3.4 μg/m3, 112.3 μg/m3, and 2.3 μg/m3 in molding (furnace), deforming (grinding), and sorting processes, respectively (cited from Kim et al., 2008). However, ambient beryllium level after process change was non-detectable (< 0.1 μg/m3). The number of T lymphocytes and the amount of immunoglobulins in the beryllium-exposed workers and control subjects were not significantly different, except for the total number of lymphocytes and CD95 (APO1/FAS). The total number of lymphocytes was higher in the beryllium-exposed individuals than in the healthy control subjects. Multiple logistic regression analysis showed lymphocytes to be affected by beryllium exposure (odd ratio = 7.293; p < 0.001). These results show that short-term exposure to beryllium does not induce immune dysfunction but is probably associated with lymphocytes proliferation. PMID:24278637

  17. Restricted Gene Flow among Hospital Subpopulations of Enterococcus faecium

    PubMed Central

    Willems, Rob J. L.; Top, Janetta; van Schaik, Willem; Leavis, Helen; Bonten, Marc; Sirén, Jukka; Hanage, William P.; Corander, Jukka

    2012-01-01

    ABSTRACT Enterococcus faecium has recently emerged as an important multiresistant nosocomial pathogen. Defining population structure in this species is required to provide insight into the existence, distribution, and dynamics of specific multiresistant or pathogenic lineages in particular environments, like the hospital. Here, we probe the population structure of E. faecium using Bayesian-based population genetic modeling implemented in Bayesian Analysis of Population Structure (BAPS) software. The analysis involved 1,720 isolates belonging to 519 sequence types (STs) (491 for E. faecium and 28 for Enterococcus faecalis). E. faecium isolates grouped into 13 BAPS (sub)groups, but the large majority (80%) of nosocomial isolates clustered in two subgroups (2-1 and 3-3). Phylogenetic and eBURST analysis of BAPS groups 2 and 3 confirmed the existence of three separate hospital lineages (17, 18, and 78), highlighting different evolutionary trajectories for BAPS 2-1 (lineage 78) and 3-3 (lineage 17 and lineage 18) isolates. Phylogenomic analysis of 29 E. faecium isolates showed agreement between BAPS assignment of STs and their relative positions in the phylogenetic tree. Odds ratio calculation confirmed the significant association between hospital isolates with BAPS 3-3 and lineages 17, 18, and 78. Admixture analysis showed a scarce number of recombination events between the different BAPS groups. For the E. faecium hospital population, we propose an evolutionary model in which strains with a high propensity to colonize and infect hospitalized patients arise through horizontal gene transfer. Once adapted to the distinct hospital niche, this subpopulation becomes isolated, and recombination with other populations declines. PMID:22807567

  18. Cell electrophoretic characterization of peripheral blood lymphocyte subpopulations enriched by rosette formation, from normal individuals and CLL patients.

    PubMed

    Rychly, J; Babusíková, O; Koníková, E; Anders, O

    1984-01-01

    Peripheral blood lymphocytes from healthy subjects and patients with chronic lymphatic leukemia (CLL) were isolated and their subpopulations enriched through formation of spontaneous rosettes with sheep or mouse red blood cells, respectively. Electrophoretic measurements were performed in unseparated as well as in fractionated cell populations. Normal blood lymphocytes showed two clearly distinguishable populations of different electrophoretic mobilities. After separation by SRBC rosette formation the rosette-forming cells could be identified as high mobility cells. CLL lymphocytes showed in most cases an unimodally distributed cytopherogram, the mean electrophoretic mobility being intermediate between the low and high mobility cells of control persons. After separation through mouse erythrocytes rosette formation these cells contained two cell fractions differing in their electrophoretic mobility: a fraction of slower mouse rosette-forming cells and a fraction of the non-MRFC which contained mainly cells of higher mobility that could be identified as enriched T cells. These both fractions showed unimodal distributions. This study shows that CLL lymphocyte subpopulations can be further characterized by surface charge density. PMID:6700796

  19. Bovine myocardial epithelial inclusions.

    PubMed

    Baker, D C; Schmidt, S P; Langheinrich, K A; Cannon, L; Smart, R A

    1993-01-01

    Light microscopic, histochemical, immunohistochemical, and ultrastructural methods were used to examine myocardial epithelial masses in the hearts of ten cattle. The tissues consisted of paraffin-embedded or formalin-fixed samples from eight hearts that were being inspected in slaughter houses and from two hearts from calves that died of septicemia. The ages of the cattle ranged from 4 days to 12 years; the breeds were unspecified for all but one Hereford female and the two Holstein calves; and there were three males, four females, and three steers. The masses in these cases were compared with similar appearing lesions found in other animal species. The lesions in the bovine hearts were single to multiple, well circumscribed, found in the left ventricle wall, and composed of squamous to cuboidal epithelial cells that formed tubular, ductular, and acinar structures with lumens that were void or filled with amorphous protein globules. Electron microscopic examination revealed epithelial cells that had sparse apical microvilli, tight apical intercellular junctions, perinuclear bundles of filaments, and rare cilia. Almost half of the bovine epithelial masses (4/9) had occasional diastase-resistant periodic acid-Schiff-positive granules in their cytoplasm, and few had hyaluronidase-resistant alcian blue-positive granules (2/9) or colloidal iron-positive granules (1/9). All myocardial masses had abundant collagen surrounding the tubular and acinar structures, and 2/9 had elastin fibers as well. None of the myocardial masses had Churukian-Schenk or Fontana Masson's silver staining granules in epithelial cells. Immunohistochemically, all bovine myocardial tumors stained positively for cytokeratin (8/8), and occasional masses stained positively for vimentin (3/8) or carcinoembryonic antigen (3/8). None of the masses stained positively for desmin. The myocardial epithelial tumors most likely represent endodermal rests of tissue misplaced during organogenesis. PMID:7680178

  20. Sensory neuron subpopulation-specific dysregulation of intracellular calcium in a rat model of chemotherapy-induced peripheral neuropathy.

    PubMed

    Yilmaz, E; Gold, M S

    2015-08-01

    The purpose of the present study was to test the prediction that the unique manifestation of chemotherapeutic-induced peripheral neuropathy (CIPN) would be reflected in a specific pattern of changes in the regulation of the intracellular Ca(2+) concentration ([Ca(2+)]i) in subpopulations of cutaneous neurons. To test this prediction, we characterized the pattern of changes in mechanical nociceptive threshold associated with paclitaxel administration (2mg/kg, iv, every other day for four days), as well as the impact of target of innervation and paclitaxel treatment on the regulation of [Ca(2+)]i in subpopulations of putative nociceptive and non-nociceptive neurons. Neurons innervating the glabrous and hairy skin of the hindpaw as well as the thigh were identified with retrograde tracers, and fura-2 was used to assess changes in [Ca(2+)]i. Paclitaxel was associated with a persistent decrease in mechanical nociceptive threshold in response to stimuli applied to the glabrous skin of the hindpaw, but not the hairy skin of the hindpaw or the thigh. However, in both putative nociceptive and non-nociceptive neurons, resting [Ca(2+)]i was significantly lower in neurons innervating the thigh after treatment. The magnitude of the depolarization-evoked Ca(2+) transient was also lower in putative non-nociceptive thigh neurons. More interestingly, while paclitaxel had no detectable influence on either resting or depolarization-evoked Ca(2+) transients in putative non-nociceptive neurons, in putative nociceptive neurons there was a subpopulation-specific decrease in the duration of the evoked Ca(2+) transient that was largely restricted to neurons innervating the glabrous skin. These results suggest that peripheral nerve length alone, does not account for the selective distribution of CIPN symptoms. Rather, they suggest the symptoms of CIPN reflect an interaction between the toxic actions of the therapeutic and unique properties of the neurons deleteriously impacted. PMID

  1. Epithelial plasticity in urothelial carcinoma: Current advancements and future challenges

    PubMed Central

    Garg, Minal

    2016-01-01

    Urothelial carcinoma (UC) of the bladder is characterized by high recurrence rate where a subset of these cells undergoes transition to deadly muscle invasive disease and later metastasizes. Urothelial cancer stem cells (UroCSCs), a tumor subpopulation derived from transformation of urothelial stem cells, are responsible for heterogeneous tumor formation and resistance to systemic treatment in UC of the bladder. Although the precise reason for pathophysiologic spread of tumor is not clear, transcriptome analysis of microdissected cancer cells expressing multiple progenitor/stem cell markers validates the upregulation of genes that derive epithelial-to-mesenchymal transition. Experimental studies on human bladder cancer xenografts describe the mechanistic functions and regulation of epithelial plasticity for its cancer-restraining effects. It has been further examined to be associated with the recruitment of a pool of UroCSCs into cell division in response to damages induced by adjuvant therapies. This paper also discusses the various probable therapeutic approaches to attenuate the progressive manifestation of chemoresistance by co-administration of inhibitors of epithelial plasticity and chemotherapeutic drugs by abrogating the early tumor repopulation as well as killing differentiated cancer cells. PMID:27621760

  2. Epithelial plasticity in urothelial carcinoma: Current advancements and future challenges.

    PubMed

    Garg, Minal

    2016-08-26

    Urothelial carcinoma (UC) of the bladder is characterized by high recurrence rate where a subset of these cells undergoes transition to deadly muscle invasive disease and later metastasizes. Urothelial cancer stem cells (UroCSCs), a tumor subpopulation derived from transformation of urothelial stem cells, are responsible for heterogeneous tumor formation and resistance to systemic treatment in UC of the bladder. Although the precise reason for pathophysiologic spread of tumor is not clear, transcriptome analysis of microdissected cancer cells expressing multiple progenitor/stem cell markers validates the upregulation of genes that derive epithelial-to-mesenchymal transition. Experimental studies on human bladder cancer xenografts describe the mechanistic functions and regulation of epithelial plasticity for its cancer-restraining effects. It has been further examined to be associated with the recruitment of a pool of UroCSCs into cell division in response to damages induced by adjuvant therapies. This paper also discusses the various probable therapeutic approaches to attenuate the progressive manifestation of chemoresistance by co-administration of inhibitors of epithelial plasticity and chemotherapeutic drugs by abrogating the early tumor repopulation as well as killing differentiated cancer cells. PMID:27621760

  3. COMPARISON OF CELLULAR AND NUCLEAR FLOW CYTOMETRIC TECHNIQUES FOR DISCRIMINATING APOPTOTIC SUBPOPULATIONS

    EPA Science Inventory

    We compared cellular flow cytometric methods employing carboxyfluorescein (CF), Hoechst 33342, and Hoechst 33258 with a nuclear method in their ability to discriminate apoptotic subpopulations in rat thymocyte cultures exposed to dexamethasone r tributyltin. n the nuclear techniq...

  4. Cells release subpopulations of exosomes with distinct molecular and biological properties.

    PubMed

    Willms, Eduard; Johansson, Henrik J; Mäger, Imre; Lee, Yi; Blomberg, K Emelie M; Sadik, Mariam; Alaarg, Amr; Smith, C I Edvard; Lehtiö, Janne; El Andaloussi, Samir; Wood, Matthew J A; Vader, Pieter

    2016-01-01

    Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wide range of putative biological functions have been attributed to exosomes, they are assumed to represent a homogenous population of EVs. We hypothesized the existence of subpopulations of exosomes with defined molecular compositions and biological properties. Density gradient centrifugation of isolated exosomes revealed the presence of two distinct subpopulations, differing in biophysical properties and their proteomic and RNA repertoires. Interestingly, the subpopulations mediated differential effects on the gene expression programmes in recipient cells. In conclusion, we demonstrate that cells release distinct exosome subpopulations with unique compositions that elicit differential effects on recipient cells. Further dissection of exosome heterogeneity will advance our understanding of exosomal biology in health and disease and accelerate the development of exosome-based diagnostics and therapeutics. PMID:26931825

  5. Cells release subpopulations of exosomes with distinct molecular and biological properties

    PubMed Central

    Willms, Eduard; Johansson, Henrik J.; Mäger, Imre; Lee, Yi; Blomberg, K. Emelie M.; Sadik, Mariam; Alaarg, Amr; Smith, C.I. Edvard; Lehtiö, Janne; EL Andaloussi, Samir; Wood, Matthew J.A.; Vader, Pieter

    2016-01-01

    Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wide range of putative biological functions have been attributed to exosomes, they are assumed to represent a homogenous population of EVs. We hypothesized the existence of subpopulations of exosomes with defined molecular compositions and biological properties. Density gradient centrifugation of isolated exosomes revealed the presence of two distinct subpopulations, differing in biophysical properties and their proteomic and RNA repertoires. Interestingly, the subpopulations mediated differential effects on the gene expression programmes in recipient cells. In conclusion, we demonstrate that cells release distinct exosome subpopulations with unique compositions that elicit differential effects on recipient cells. Further dissection of exosome heterogeneity will advance our understanding of exosomal biology in health and disease and accelerate the development of exosome-based diagnostics and therapeutics. PMID:26931825

  6. Functional studies on Calliphora vomitoria haemocyte subpopulations defined by lectin staining and density centrifugation.

    PubMed

    McKenzie, A N; Preston, T M

    1992-01-01

    Haemocyte subpopulations of Calliphora vomitoria have been categorized by their surface staining properties using fluorescently labelled lectins, and their mobilities in Percoll density gradients. These methods of identification were exploited to determine the roles of these cell types in cellular defence reactions. Soybean agglutinin clearly defined the cell subpopulation involved in phagocytosis, while purified thrombocytoid fragments proved to be the main haemocyte population involved in encapsulation and nodule formation. PMID:1377650

  7. Male and Female Subpopulations of Salix viminalis Present High Genetic Diversity and High Long-Term Migration Rates between Them.

    PubMed

    Zhai, Feifei; Mao, Jinmei; Liu, Junxiang; Peng, Xiangyong; Han, Lei; Sun, Zhenyuan

    2016-01-01

    Dioecy distributed in 157 flowering plant families and 959 flowering plant genera. Morphological and physiological differences between male and female plants have been studied extensively, but studies of sex-specific genetic diversity are relatively scarce in dioecious plants. In this study, 20 SSR loci were employed to examine the genetic variance of male subpopulations and female subpopulations in Salix viminalis. The results showed that all of the markers were polymorphic (Na = 14.15, He = 0.7566) and workable to reveal the genetic diversity of S. viminalis. No statistically significant difference was detected between male and female subpopulations, but the average genetic diversity of male subpopulations (Na = 7.12, He = 0.7071) and female subpopulations (Na = 7.31, He = 0.7226) were high. Under unfavorable environments (West Liao basin), the genetic diversity between male and female subpopulations was still not significantly different, but the genetic diversity of sexual subpopulations were lower. The differentiation of the ten subpopulations in S. viminalis was moderate (FST = 0.0858), which was conformed by AMOVA that most of genetic variance (94%) existed within subpopulations. Pairwise FST indicated no differentiation between sexual subpopulations, which was accompanied by high long-term migrate between them (M = 0.73~1.26). However, little recent migration was found between sexual subpopulations. Therefore, artificial crossing or/and transplantation by cutting propagation should be carried out so as to increase the migration during the process of ex situ conservation. PMID:27047511

  8. Male and Female Subpopulations of Salix viminalis Present High Genetic Diversity and High Long-Term Migration Rates between Them

    PubMed Central

    Zhai, Feifei; Mao, Jinmei; Liu, Junxiang; Peng, Xiangyong; Han, Lei; Sun, Zhenyuan

    2016-01-01

    Dioecy distributed in 157 flowering plant families and 959 flowering plant genera. Morphological and physiological differences between male and female plants have been studied extensively, but studies of sex-specific genetic diversity are relatively scarce in dioecious plants. In this study, 20 SSR loci were employed to examine the genetic variance of male subpopulations and female subpopulations in Salix viminalis. The results showed that all of the markers were polymorphic (Na = 14.15, He = 0.7566) and workable to reveal the genetic diversity of S. viminalis. No statistically significant difference was detected between male and female subpopulations, but the average genetic diversity of male subpopulations (Na = 7.12, He = 0.7071) and female subpopulations (Na = 7.31, He = 0.7226) were high. Under unfavorable environments (West Liao basin), the genetic diversity between male and female subpopulations was still not significantly different, but the genetic diversity of sexual subpopulations were lower. The differentiation of the ten subpopulations in S. viminalis was moderate (FST = 0.0858), which was conformed by AMOVA that most of genetic variance (94%) existed within subpopulations. Pairwise FST indicated no differentiation between sexual subpopulations, which was accompanied by high long-term migrate between them (M = 0.73~1.26). However, little recent migration was found between sexual subpopulations. Therefore, artificial crossing or/and transplantation by cutting propagation should be carried out so as to increase the migration during the process of ex situ conservation. PMID:27047511

  9. CXCR4+CD45− BMMNC subpopulation is superior to unfractionated BMMNCs for protection after ischemic stroke in mice

    PubMed Central

    Wang, Jianping; Liu, Xi; Lu, Hong; Jiang, Chao; Cui, Xiaobing; Yu, Lie; Fu, Xiaojie; Li, Qian; Wang, Jian

    2015-01-01

    Cell-based therapy is considered to be a promising therapeutic strategy for stroke treatment. Although unfractionated bone marrow mononuclear cells (BMMNCs) have been tried in both preclinical and clinical trials, the effective subpopulations need to be identified. In this study, we used fluorescence-activated cell sorting to harvest the CXCR4+CD45+ and CXCR4+CD45− BMMNC subpopulations from transgenic mice that express enhanced green fluorescent protein. We then allogeneically grafted unfractionated BMMNCs or a subpopulation into mice subjected to transient middle cerebral artery occlusion (tMCAO) and compared the effects on stroke outcomes. We found that CXCR4+CD45− BMMNCs, but not CXCR4+CD45+ BMMNCs, more effectively reduced infarction volume and neurologic deficits than did unfractionated BMMNCs. Brain tissue from the ischemic hemisphere of mice treated with CXCR4+CD45− BMMNCs had higher levels of vascular endothelial growth factor and lower levels of TNF-α than did tissue from mice treated with unfractionated BMMNCs. In contrast, CXCR4+CD45+ BMMNCs showed an increase in TNF-α. Additionally, CXCR4+CD45+ and CXCR4+CD45− populations exhibited more robust migration into the lesion areas and were better able to express cell-specific markers of different linages than were the unfractionated BMMNCs. Endothelial and astrocyte cell markers did not colocalize with eGFP+ cells in the brains of tMCAO mice that received CXCR4+CD45+ BMMNCs. In vitro, the CXCR4+CD45− BMMNCs expressed significantly more Oct-4 and Nanog mRNA than did the unfractionated BMMNCs. However, we did not detect gene expression of these two pluripotent markers in CXCR4+CD45+ BMMNCs. Taken together, our study shows for the first time that the CXCR4+CD45− BMMNC subpopulation is superior to unfractionated BMMNCs in ameliorating cerebral damage in a mouse model of tMCAO and could represent a new therapeutic approach for stroke treatment. PMID:25526817

  10. Epithelial hyperplasia, airways —

    Cancer.gov

    Number of respiratory epithelial cells is increased diffusely or focally. Frequently luminal protrusions are observed, sometimes forming papillae. Mucous (goblet) cell metaplastic hyperplasia is a variant, in which the respiratory epithelium of conducting airways is replaced by mucous cells either as a single or a pseudostratified layer.

  11. Circulating Biomphalaria glabrata hemocyte subpopulations possess shared schistosome glycans and receptors capable of binding larval glycoconjugates.

    PubMed

    Yoshino, Timothy P; Wu, Xiao-Jun; Gonzalez, Laura A; Hokke, Cornelis H

    2013-01-01

    Host lectin-like recognition molecules may play an important role in innate resistance in Biomphalaria glabrata snails to larval schistosome infection, thus implicating parasite-expressed glycans as putative ligands for these lectin receptors. While host lectins may utilize specific glycan structures for parasite recognition, it also has been hypothesized that the parasite may use this system to evade immune detection by mimicking naturally-expressed host glycans, resulting in reduced immunorecognition capacity. By employing immunocytochemical (ICC) and Western blot assays using schistosome glycan-specific monoclonal antibodies (mABs) we sought to identify specific glycan epitopes (glycotopes) shared in common between larval Schistosoma mansoni and B. glabrata hemocytes, the primary immune effector cells in snails. Results confirmed the presence of selected larval glycotopes on subpopulations of hemocytes by ICC and association with numerous hemocyte proteins by Western blot analyses, including a trimannosyl core N-glycan (TriMan), and two fucosylated lacdiNAc (LDN) variants, F-LDN and F-LDN-F. Snail strain differences were seen in the prevalence of constitutively expressed F-LDN on hemocytes, and in the patterns of protein immunoreactivity with these mABs. In contrast, there was little to no hemocyte reactivity with mABs for Lewis X (LeX), LDN, LDN-F or LDN-DF. When intact hemocytes were exposed to larval transformation products (LTPs), distinct cell subpopulations displayed weak (LeX, LDN-DF) to moderate (LDN, LDN-F) glycotope reactivity by ICC, including snail strain differences in the prevalence of LDN-reactive cellular subsets. Far-Western blot analyses of the hemocytes following exposure to larval transformation proteins (LTPs) also revealed multiple mAB-reactive hemocyte protein bands for LeX, LDN, LDN-F, and LDN-DF. These results demonstrate the existence of complex patterns of shared larval glycan constitutively expressed on hemocytes and their proteins

  12. CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

    SciTech Connect

    Malizia, Andrea P.; Lacey, Noreen; Walls, Dermot; Egan, Jim J.; Doran, Peter P.

    2009-07-01

    Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGF{beta}1-mediated lytic phase. EBV lytic reactivation by TGF{beta}1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM{sub 1}81552) expression, inducing activation of non-canonical Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.

  13. Allele frequency data for 15 autosomal STR loci in eight Indonesian subpopulations.

    PubMed

    Venables, Samantha J; Daniel, Runa; Sarre, Stephen D; Soedarsono, Nurtami; Sudoyo, Herawati; Suryadi, Helena; van Oorschot, Roland A H; Walsh, Simon J; Widodo, Putut T; McNevin, Dennis

    2016-01-01

    Evolutionary and cultural history can affect the genetic characteristics of a population and influences the frequency of different variants at a particular genetic marker (allele frequency). These characteristics directly influence the strength of forensic DNA evidence and make the availability of suitable allele frequency information for every discrete country or jurisdiction highly relevant. Population sub-structure within Indonesia has not been well characterised but should be expected given the complex geographical, linguistic and cultural architecture of the Indonesian population. Here we use forensic short tandem repeat (STR) markers to identify a number of distinct genetic subpopulations within Indonesia and calculate appropriate population sub-structure correction factors. This data represents the most comprehensive investigation of population sub-structure within Indonesia to date using these markers. The results demonstrate that significant sub-structure is present within the Indonesian population and must be accounted for using island specific allele frequencies and corresponding sub-structure correction factors in the calculation of forensic DNA match statistics. PMID:26517173

  14. Functional features of hemocyte subpopulations of the invasive mollusk species Dreissena polymorpha.

    PubMed

    Evariste, Lauris; Auffret, Michel; Audonnet, Sandra; Geffard, Alain; David, Elise; Brousseau, Pauline; Fournier, Michel; Betoulle, Stéphane

    2016-09-01

    Dreissena polymorpha is a mussel species that invaded many lotic and lentic inland waters in Western Europe and North America. Its positive or negative interactions with biotic and abiotic components of ecosystems are numerous, making this bivalve the subject of numerous studies in ecology, ecophysiology and ecotoxicology. In these contexts, the functional characterization of the zebra mussel hemocytes is of particular interest, as hemocytes are central cells involved in vital functions (immunity, growth, reproduction) of molluscan physiology. Dreissena polymorpha circulating hemocytes populations were characterized by a combination of structural and functional analysis. Assessments were performed during two contrasted physiological periods for mussels (gametogenesis and spawning). Three hemocyte types were identified as hyalinocytes and blast-like cells for agranular hemocytes and one granulocyte population. Flow cytometry analysis of hemocytes functionalities indicated that blast-like cells had low oxidative and mitochondrial activities and low lysosomal content. Hyalinocytes and granulocytes are fully equipped to perform innate immune response. Hyalinocytes exhibit higher oxidative activity than granulocytes. Such observation is not common since numerous studies show that granulocytes are usually cells that have the highest cellular activities. This result demonstrates the significant functional variability of hemocyte subpopulations. Moreover, our findings reveal that spawning period of Dreissena polymorpha was associated with an increase of hyalinocyte percentage in relation to low levels of biological activities in hemocytes. This reduction in hemocyte activity would reflect the important physiological changes associated with the spawning period of this invasive species known for its high reproductive potential. PMID:27374433

  15. Distinct Proteasome Subpopulations in the Alveolar Space of Patients with the Acute Respiratory Distress Syndrome

    PubMed Central

    Sixt, S. U.; Alami, R.; Hakenbeck, J.; Adamzik, M.; Kloß, A.; Costabel, U.; Jungblut, P. R.; Dahlmann, B.; Peters, J.

    2012-01-01

    There is increasing evidence that proteasomes have a biological role in the extracellular alveolar space, but inflammation could change their composition. We tested whether immunoproteasome protein-containing subpopulations are present in the alveolar space of patients with lung inflammation evoking the acute respiratory distress syndrome (ARDS). Bronchoalveolar lavage (BAL) supernatants and cell pellet lysate from ARDS patients (n = 28) and healthy subjects (n = 10) were analyzed for the presence of immunoproteasome proteins (LMP2 and LMP7) and proteasome subtypes by western blot, chromatographic purification, and 2D-dimensional gelelectrophoresis. In all ARDS patients but not in healthy subjects LMP7 and LMP2 were observed in BAL supernatants. Proteasomes purified from pooled ARDS BAL supernatant showed an altered enzyme activity ratio. Chromatography revealed a distinct pattern with 7 proteasome subtype peaks in BAL supernatant of ARDS patients that differed from healthy subjects. Total proteasome concentration in BAL supernatant was increased in ARDS (971 ng/mL ± 1116 versus 59 ± 25; P < 0.001), and all fluorogenic substrates were hydrolyzed, albeit to a lesser extent, with inhibition by epoxomicin (P = 0.0001). Thus, we identified for the first time immunoproteasome proteins and a distinct proteasomal subtype pattern in the alveolar space of ARDS patients, presumably in response to inflammation. PMID:22363101

  16. Effect of the amyloidogenic L75P apolipoprotein A-I variant on HDL subpopulations

    PubMed Central

    Gomaraschi, Monica; Obici, Laura; Simonelli, Sara; Gregorini, Gina; Negrinelli, Alessandro; Merlini, Giampaolo; Franceschini, Guido; Calabresi, Laura

    2011-01-01

    Background Hereditary amyloidosis due to mutations of apolipoprotein A-I (apoA-I) is a rare disease characterized by the deposition of amyloid fibrils constituted by the N-terminal fragment of apoA-I in several organs. L75P is a variant of apoA-I associated with systemic amyloidosis predominantly involving the liver, kidneys, and testis, identified in a large number of unrelated subjects. Objective of the present paper was to evaluate the impact of the L75P apoA-I variant on HDL subpopulations and cholesterol esterification in carriers. Methods and results Plasma samples were collected from 30 carriers of the amyloidogenic L75P apoA-I (Carriers) and from 15 non affected relatives (Controls). Carriers displayed significantly reduced plasma levels of HDL-cholesterol, apoA-I, and apoA-II compared to Controls. Plasma levels of LpA-I, but not LpA-I:A-II, were significantly reduced in Carriers. HDL subclass distribution was not affected by the presence of the variant. The unesterified to total cholesterol ratio was higher, and cholesterol esterification rate and LCAT activity were lower in Carriers than in Controls. Conclusions The L75P apoA-I variant is associated with hypoalphalipoproteinemia, a selective reduction of LpA-I particles, and a partial defect in cholesterol esterification. PMID:21458433

  17. Claudins: Gatekeepers of lung epithelial function.

    PubMed

    Schlingmann, Barbara; Molina, Samuel A; Koval, Michael

    2015-06-01

    The lung must maintain a proper barrier between airspaces and fluid filled tissues in order to maintain lung fluid balance. Central to maintaining lung fluid balance are epithelial cells which create a barrier to water and solutes. The barrier function of these cells is mainly provided by tight junction proteins known as claudins. Epithelial barrier function varies depending on the different needs within the segments of the respiratory tree. In the lower airways, fluid is required to maintain mucociliary clearance, whereas in the terminal alveolar airspaces a thin layer of surfactant enriched fluid lowers surface tension to prevent airspace collapse and is critical for gas exchange. As the epithelial cells within the segments of the respiratory tree differ, the composition of claudins found in these epithelial cells is also different. Among these differences is claudin-18 which is uniquely expressed by the alveolar epithelial cells. Other claudins, notably claudin-4 and claudin-7, are more ubiquitously expressed throughout the respiratory epithelium. Claudin-5 is expressed by both pulmonary epithelial and endothelial cells. Based on in vitro and in vivo model systems and histologic analysis of lungs from human patients, roles for specific claudins in maintaining barrier function and protecting the lung from the effects of acute injury and disease are being identified. One surprising finding is that claudin-18 and claudin-4 control lung cell phenotype and inflammation beyond simply maintaining a selective paracellular permeability barrier. This suggests claudins have more nuanced roles for the control of airway and alveolar physiology in the healthy and diseased lung. PMID:25951797

  18. Comparison of Disability Rates Among Older Adults in Aggregated and Separate Asian American/Pacific Islander Subpopulations

    PubMed Central

    Brennenstuhl, Sarah; Hurd, Marion

    2011-01-01

    Objectives. We assessed the prevalence and adjusted odds of 4 types of disability among 7 groups of older Asian American/Pacific Islander (AAPI) subpopulations, both separately and aggregated, compared with non-Hispanic Whites. Methods. Data were from the nationally representative 2006 American Community Survey, which included institutionalized and community-dwelling Hawaiian/Pacific Islander (n = 524), Vietnamese (n = 2357), Korean (n = 2082), Japanese (n = 3230), Filipino (n = 5109), Asian Indian (n = 2942), Chinese (n = 6034), and non-Hispanic White (n = 641 177) individuals aged 55 years and older. The weighted prevalence, population estimates, and odds ratios of 4 types of disability (functional limitations, limitations in activities of daily living, cognitive problems, and blindness or deafness) were reported for each group. Results. Disability rates in older adults varied more among AAPI subpopulations than between non-Hispanic Whites and the aggregated Asian group. Asian older adults had, on average, better disability outcomes than did non-Hispanic Whites. Conclusions. This study provides the strongest evidence to date that exclusion of institutionalized older adults minimizes disparities in disabilities between Asians and Whites. The aggregation of Asians into one group obscures substantial subgroup variability and fails to identify the most vulnerable groups (e.g., Hawaiian/Pacific Islanders and Vietnamese). PMID:20299647

  19. Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion

    PubMed Central

    Schweizer, Nadine; Pupe, Stéfano; Arvidsson, Emma; Nordenankar, Karin; Smith-Anttila, Casey J. A.; Mahmoudi, Souha; Andrén, Anna; Dumas, Sylvie; Rajagopalan, Aparna; Lévesque, Daniel; Leão, Richardson N.; Wallén-Mackenzie, Åsa

    2014-01-01

    The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knockout mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects. PMID:24821804

  20. A Novel Approach to Selectively Target Neuronal Subpopulations Reveals Genetic Pathways That Regulate Tangential Migration in the Vertebrate Hindbrain

    PubMed Central

    Benzing, Karsten; Flunkert, Stefanie; Schedl, Andreas; Engelkamp, Dieter

    2011-01-01

    Vertebrate genes often play functionally distinct roles in different subsets of cells; however, tools to study the cell-specific function of gene products are poorly developed. Therefore, we have established a novel mouse model that enables the visualization and manipulation of defined subpopulations of neurons. To demonstrate the power of our system, we dissected genetic cascades in which Pax6 is central to control tangentially migrating neurons of the mouse brainstem. Several Pax6 downstream genes were identified and their function was analyzed by over-expression and knock-down experiments. One of these, Pou4f2, induces a prolonged midline arrest of growth cones to influence the proportion of ipsilaterally versus contralaterally settling neurons. These results demonstrate that our approach serves as a versatile tool to study the function of genes involved in cell migration, axonal pathfinding, and patterning processes. Our model will also serve as a general tool to specifically over-express any gene in a defined subpopulation of neurons and should easily be adapted to a wide range of applications. PMID:21698138

  1. Heteroresistance to colistin in Klebsiella pneumoniae is triggered by small colony variants sub-populations within biofilms.

    PubMed

    Silva, Ana; Sousa, Ana Margarida; Alves, Diana; Lourenço, Anália; Pereira, Maria Olívia

    2016-07-01

    The emergence of Klebsiella pneumoniae multidrug-resistant strains paves the way to the re-introduction of colistin as a salvage therapy. However, recent planktonic studies have reported several cases of heteroresistance to this antimicrobial agent. The aim of this present work was to gain better understanding about the response of K. pneumoniae biofilms to colistin antibiotherapy and inspect the occurrence of heteroresistance in biofilm-derived cells. Biofilm formation and its susceptibility to colistin were evaluated through the determination of biofilm-cells viability. The profiling of planktonic and biofilm cell populations was conducted to assess the occurrence of heteroresistance. Colony morphology was further characterized in order to inspect the potential role of colistin in K. pneumoniae phenotypic differentiation. Results show that K. pneumoniae was susceptible to colistin in its planktonic form, but biofilms presented enhanced resistance. Population analysis profiles pointed out that K. pneumoniae manifest heteroresistance to colistin only when grown in biofilm arrangements, and it was possible to identify a resistant sub-population presenting a small colony morphology (diameter around 5 mm). To the best of our knowledge, this is the first report linking heteroresistance to biofilm formation and a morphological distinctive sub-population. Moreover, this is the first evidence that biofilm formation can trigger the emergence of heteroresistance in an apparently susceptible strain. PMID:27140200

  2. Monoclonal antibody identification of subpopulations of cerebral cortical neurons affected in Alzheimer disease.

    PubMed Central

    Miller, C A; Rudnicka, M; Hinton, D R; Blanks, J C; Kozlowski, M

    1987-01-01

    Neuronal degeneration is one of the hallmarks of Alzheimer disease (AD). Given the paucity of molecular markers available for the identification of neuronal subtypes, the specificity of neuronal loss within the cerebral cortex has been difficult to evaluate. With a panel of four monoclonal antibodies (mAbs) applied to central nervous system tissues from AD patients, we have immunocytochemically identified a population of vulnerable cortical neurons; a subpopulation of pyramidal neurons is recognized by mABs 3F12 and 44.1 in the hippocampus and neocortex, and clusters of multipolar neurons in the entorhinal cortex reactive with mAb 44.1 show selective degeneration. Closely adjacent stellate-like neurons in these regions, identified by mAB 6A2, show striking preservation in AD. The neurons recognized by mAbs 3F12 and 44.1, to the best of our knowledge, do not comprise a single known neurotransmitter system. mAb 3A4 identifies a phosphorylated antigen that is undetectable in normal brain but accumulates early in the course of AD in somas of vulnerable neurons. Antigen 3A4 is distinct from material reactive with thioflavin S or antibody generated against paired helical filaments. Initially, antigen 3A4 is localized to neurons in the entorhinal cortex and subiculum, later in the association neocortex, and, ultimately in cases of long duration, in primary sensory cortical regions. mAb 3F12 recognizes multiple bands on immunoblots of homogenates of normal and AD cortical tissues, whereas mAb 3A4 does not bind to immunoblots containing neurofilament proteins or brain homogenates from AD patients. Ultrastructurally, antigen 3A4 is localized to paired helical filaments. Using these mAbs, further molecular characterization of the affected cortical neurons is now possible. Images PMID:3120196

  3. Rates of upgrade to malignancy for 271 cases of flat epithelial atypia (FEA) diagnosed by breast core biopsy.

    PubMed

    Peres, Alexandre; Barranger, Emmanuel; Becette, Véronique; Boudinet, Alain; Guinebretiere, Jean-Marc; Cherel, Pascal

    2012-06-01

    Flat epithelial atypia (FEA) is a borderline lesion that might represent an early stage in the development of certain low-grade carcinomas in situ and invasive cancers. There are no guidelines on its management. Our objectives were to determine the upgrade to malignancy rate and identify a subpopulation of patients that might undergo just mammographic surveillance. We retrospectively reviewed the data for 271 FEA cases among 5,555 breast core biopsies obtained over a 7-year period (January 2003-2010). We collated clinical data (age, history of cancer, menopausal status), radiological data (lesion type, size, Bi-Rads category), technical data (number of biopsies, needle gauge, excision quality) and histological data and sought correlations between these factors and upgrade rate. The 271 FEA comprised 128 cases of pure FEA, 135 cases of FEA + atypical ductal hyperplasia, and 8 cases of FEA + atypical lobular hyperplasia. Overall, 184 patients underwent surgery and 46 mammographic surveillance. Surgery detected 34 cases of malignancy (23 CIS, 7 invasive cancers, and 4 mixed cases) giving a 15% upgrade rate. Quality of excision was the only factor associated with under-diagnosis. The presence of FEA at biopsy warrants surgery. PMID:22042365

  4. Angiomyolipoma With Epithelial Cysts.

    PubMed

    LeRoy, Michael A; Rao, Priya

    2016-06-01

    Angiomyolipoma with epithelial cysts is a rare mesenchymal tumor of the kidney that enters in the differential diagnosis of adult cystic renal neoplasms. These tumors demonstrate a slight female predominance and can present either incidentally or with symptoms, commonly flank pain and hematuria. Unlike conventional angiomyolipoma, this variant is characterized grossly by both solid and cystic areas, and histologically by the presence of single or multiple cysts lined by epithelial cells, a subepithelial "cambium-like" layer of small stromal cells with a prominent capillary vasculature, and a thick exterior wall composed of poorly formed fascicles of smooth muscle and thick-walled dysplastic blood vessels. Tumors show a distinct immunohistochemical profile and are often reactive for melanocytic markers (HMB-45 and Melan-A), as well as estrogen receptor and progesterone receptor. These tumors have an indolent clinical course, with no reports of progression or metastasis in reported cases thus far. PMID:27232352

  5. Depth sensitive oblique polarized reflectance spectroscopy of oral epithelial tissue

    NASA Astrophysics Data System (ADS)

    Jimenez, Maria K.; Lam, Sylvia; Poh, Catherine; Sokolov, Konstantin

    2014-05-01

    Identifying depth-dependent alterations associated with epithelial cancerous lesions can be challenging in the oral cavity where variable epithelial thicknesses and troublesome keratin growths are prominent. Spectroscopic methods with enhanced depth resolution would immensely aid in isolating optical properties associated with malignant transformation. Combining multiple beveled fibers, oblique collection geometry, and polarization gating, oblique polarized reflectance spectroscopy (OPRS) achieves depth sensitive detection. We report promising results from a clinical trial of patients with oral lesions suspected of dysplasia or carcinoma demonstrating the potential of OPRS for the analysis of morphological and architectural changes in the context of multilayer, epithelial oral tissue.

  6. Are cementoblasts a subpopulation of osteoblasts or a unique phenotype?

    PubMed

    Bosshardt, D D

    2005-05-01

    Experimental studies have shown a great potential for periodontal regeneration. The limitations of periodontal regeneration largely depend on the regenerative potential at the root surface. Cellular intrinsic fiber cementum (CIFC), so-called bone-like tissue, may form instead of the desired acellular extrinsic fiber cementum (AEFC), and the interfacial tissue bonding may be weak. The periodontal ligament harbors progenitor cells that can differentiate into periodontal ligament fibroblasts, osteoblasts, and cementoblasts, but their precise location is unknown. It is also not known whether osteoblasts and cementoblasts arise from a common precursor cell line, or whether distinct precursor cell lines exist. Thus, there is limited knowledge about how cell diversity evolves in the space between the developing root and the alveolar bone. This review supports the hypothesis that AEFC is a unique tissue, while CIFC and bone share some similarities. Morphologically, functionally, and biochemically, however, CIFC is distinctly different from any bone type. There are several lines of evidence to propose that cementoblasts that produce both AEFC and CIFC are unique phenotypes that are unrelated to osteoblasts. Cementum attachment protein appears to be cementum-specific, and the expression of two proteoglycans, fibromodulin and lumican, appears to be stronger in CIFC than in bone. A theory is presented that may help explain how cell diversity evolves in the periodontal ligament. It proposes that Hertwig's epithelial root sheath and cells derived from it play an essential role in the development and maintenance of the periodontium. The role of enamel matrix proteins in cementoblast and osteoblast differentiation and their potential use for tissue engineering are discussed. PMID:15840773

  7. Autoantibodies to tumor-associated antigens in epithelial ovarian carcinoma.

    PubMed

    Piura, Benjamin; Piura, Ettie

    2009-01-01

    This review will focus on recent knowledge related to circulating autoantibodies (AAbs) to tumor-associated antigens (TAAs) in epithelial ovarian carcinoma. So far, the following TAAs have been identified to elicit circulating AAbs in epithelial ovarian carcinoma: p53, homeobox proteins (HOXA7, HOXB7), heat shock proteins (HSP-27, HSP-90), cathepsin D, cancer-testis antigens (NY-ESO-1/LAGE-1), MUC1, GIPC-1, IL-8, Ep-CAM, and S100A7. Since AAbs to TAAs have been identified in the circulation of patients with early-stage cancer, it has been speculated that the assessment of a panel of AAbs specific for epithelial ovarian carcinoma TAAs might hold great potential as a novel tool for early diagnosis of epithelial ovarian carcinoma. PMID:20145720

  8. Catfish consumption as a contributor to elevated PCB levels in a non-Hispanic black subpopulation.

    PubMed

    Weintraub, Max; Birnbaum, Linda S

    2008-07-01

    The human body burden of polychlorinated biphenyls (PCBs) sharply declined after production was banned in the US in 1979. For the 10% of the US population that remains most exposed to PCBs, fish consumption is the primary source. National Health and Nutrition Examination Survey (NHANES) data indicates that the highest remaining PCB levels exist in a non-Hispanic black subpopulation. Our review suggests that catfish consumption may be a significant PCB source for the one million non-Hispanic black anglers who fish for catfish. In comparison to non-Hispanic white anglers, non-Hispanic black anglers consume more catfish, are more likely to eat the whole fish rather than just the fillets that contain less PCBs, and are more likely to fish in watersheds with high PCB contamination. Efforts to diminish potential racial disparities in PCB exposure are challenged by geographic, economic, cultural, and educational barriers. In response, we propose that a fish consumption survey be performed that identifies the extent of subsistence fishing by non-Hispanic black anglers for catfish in watersheds with PCB contamination, the type and quantity of catfish subsistence fishing provides, and what actions would help moderate PCB exposure due to subsistence fishing for catfish in such areas. Understanding the contamination and consumption factors that contribute to higher PCB body burdens will help identify and offer solutions to racial disparities in exposure to PCBs due to subsistence fishing while providing a model to prevent similar disparities in exposure to toxics ranging from mercury to polybrominated diphenyl ethers. PMID:18407261

  9. Escape from Lethal Bacterial Competition through Coupled Activation of Antibiotic Resistance and a Mobilized Subpopulation

    PubMed Central

    Stubbendieck, Reed M.; Straight, Paul D.

    2015-01-01

    Bacteria have diverse mechanisms for competition that include biosynthesis of extracellular enzymes and antibiotic metabolites, as well as changes in community physiology, such as biofilm formation or motility. Considered collectively, networks of competitive functions for any organism determine success or failure in competition. How bacteria integrate different mechanisms to optimize competitive fitness is not well studied. Here we study a model competitive interaction between two soil bacteria: Bacillus subtilis and Streptomyces sp. Mg1 (S. Mg1). On an agar surface, colonies of B. subtilis suffer cellular lysis and progressive degradation caused by S. Mg1 cultured at a distance. We identify the lytic and degradative activity (LDA) as linearmycins, which are produced by S. Mg1 and are sufficient to cause lysis of B. subtilis. We obtained B. subtilis mutants spontaneously resistant to LDA (LDAR) that have visibly distinctive morphology and spread across the agar surface. Every LDAR mutant identified had a missense mutation in yfiJK, which encodes a previously uncharacterized two-component signaling system. We confirmed that gain-of-function alleles in yfiJK cause a combination of LDAR, changes in colony morphology, and motility. Downstream of yfiJK are the yfiLMN genes, which encode an ATP-binding cassette transporter. We show that yfiLMN genes are necessary for LDA resistance. The developmental phenotypes of LDAR mutants are genetically separable from LDA resistance, suggesting that the two competitive functions are distinct, but regulated by a single two-component system. Our findings suggest that a subpopulation of B. subtilis activate an array of defensive responses to counter lytic stress imposed by competition. Coordinated regulation of development and antibiotic resistance is a streamlined mechanism to promote competitive fitness of bacteria. PMID:26647299

  10. Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies

    PubMed Central

    Thapa, Dharendra; Shepherd, Danielle L.

    2014-01-01

    Cardiac tissue contains discrete pools of mitochondria that are characterized by their subcellular spatial arrangement. Subsarcolemmal mitochondria (SSM) exist below the cell membrane, interfibrillar mitochondria (IFM) reside in rows between the myofibrils, and perinuclear mitochondria are situated at the nuclear poles. Microstructural imaging of heart tissue coupled with the development of differential isolation techniques designed to sequentially separate spatially distinct mitochondrial subpopulations have revealed differences in morphological features including shape, absolute size, and internal cristae arrangement. These findings have been complemented by functional studies indicating differences in biochemical parameters and, potentially, functional roles for the ATP generated, based upon subcellular location. Consequently, mitochondrial subpopulations appear to be influenced differently during cardiac pathologies including ischemia/reperfusion, heart failure, aging, exercise, and diabetes mellitus. These influences may be the result of specific structural and functional disparities between mitochondrial subpopulations such that the stress elicited by a given cardiac insult differentially impacts subcellular locales and the mitochondria contained within. The goal of this review is to highlight some of the inherent structural and functional differences that exist between spatially distinct cardiac mitochondrial subpopulations as well as provide an overview of the differential impact of various cardiac pathologies on spatially distinct mitochondrial subpopulations. As an outcome, we will instill a basis for incorporating subcellular spatial location when evaluating the impact of cardiac pathologies on the mitochondrion. Incorporation of subcellular spatial location may offer the greatest potential for delineating the influence of cardiac pathology on this critical organelle. PMID:24778166

  11. Characterization of Discrete Subpopulations of Progenitor Cells in Traumatic Human Extremity Wounds

    PubMed Central

    Woodard, Geoffrey E.; Ji, Youngmi; Christopherson, Gregory T.; Wolcott, Karen M.; Hall, David J.; Jackson, Wesley M.; Nesti, Leon J.

    2014-01-01

    Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics. PMID:25490403

  12. Subpopulations of MCF7 cells separate by Percoll gradient centrifugation: a model to analyze the heterogeneity of human breast cancer

    SciTech Connect

    Resnicoff, M.; Medrano, E.E.; Podhajcer, O.L.; Bravo, A.I.; Bover, L.; Mordoh, J.

    1987-10-01

    Exponentially growing MCF7 human breast cancer cells were separated in Percoll gradients into six different fractions of increasing density (A to F). These fractions could be subcultured and were found to contain different cellular subpopulations as defined by the following criteria: ability to generate other cellular subpopulations; growth rate; DNA synthesis; and expression of estrogen receptors, ras oncogene-encoded protein p21, and carcinoembryonic antigen. One of the minor fractions (E), which contained about 5% of the total cell number, appeared to contain the stem cells, on the basis of the following criteria: (i) its ability to reproduce the other cellular subpopulations, (ii) its high rate of growth and DNA synthesis, and (iii) the inability of the other subpopulations to generate it. The most differentiated subpopulation appeared to be the densest one (F), since it was the slowest growing and appeared to be the end point of the other subpopulations.

  13. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    PubMed Central

    Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M.; Pluth, Janice M.; Cucinotta, Francis A.

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1. PMID:22844446

  14. Intracellular calcium regulation among subpopulations of rat dorsal root ganglion neurons

    PubMed Central

    Lu, Shao-Gang; Zhang, Xiulin; Gold, Michael S

    2006-01-01

    Primary afferent neurons are functionally heterogeneous. To determine whether this functional heterogeneity reflects, in part, heterogeneity in the regulation of the concentration of intracellular Ca2+ ([Ca2+]i), the magnitude and decay of evoked Ca2+ transients were assessed in subpopulations of dorsal root ganglion (DRG) neurons with voltage clamp and fura-2 ratiometric imaging. To determine whether differences in evoked Ca2+ transients among subpopulations of DRG neurons reflected differences in the contribution of Ca2+ regulatory mechanisms, pharmacological techniques were employed to assess the contribution of influx, efflux, release and uptake pathways. Subpopulations of DRG neurons were defined by cell body size, binding of the plant lectin IB4 and responsiveness to the algogenic compound capsaicin (CAP). Ca2+ transients were evoked with 30 mm K+ or voltage steps to 0 mV. There were marked differences between subpopulations of neurons with respect to both the magnitude and decay of the Ca2+ transient, with the largest and most slowly decaying Ca2+ transients in small-diameter, IB4-positive, CAP-responsive neurons. The smallest and most rapidly decaying transients were in large-diameter, IB4-negative and CAP-unresponsive DRG neurons. These differences were not due to a differential distribution of voltage-gated Ca2+ currents. However, these differences did appear to reflect a differential contribution of other influx, efflux, release and uptake mechanisms between subpopulations of neurons. These results suggest that electrical activity in subpopulations of DRG neurons will have a differential influence on Ca2+-regulated phenomena such as spike adaptation, transmitter release and gene transcription. Significantly more activity should be required in large-diameter non-nociceptive afferents than in small-diameter nociceptive afferents to have a comparable influence on these processes. PMID:16945973

  15. Differential Impact of Tobacco Control Policies on Youth Sub-Populations

    PubMed Central

    Tauras, John A.; Huang, Jidong; Chaloupka, Frank J.

    2013-01-01

    Background: While previous studies have demonstrated the efficacy of tobacco control interventions in reducing tobacco use among youth overall, there have been very few studies that examine the potential differential impact of tobacco control policies on various youth subgroups, defined by socio-economic status (SES), race/ethnicity, and gender. Objective: We examined the relationship between state-level cigarette prices and smoke-free air laws and youth smoking prevalence and intensity for various youth sub-populations in the United States. Methods: We estimated a 2-part model of cigarette demand using data from the 1991 through 2010 nationally representative surveys of 8th-, 10th-, and 12th-grade students as part of the Monitoring the Future project. Findings: We found that real cigarette prices are strong determinants of youth smoking. Blacks, females, Hispanics, and low-SES subpopulations are found to have a larger price response with respect to smoking prevalence than the full sample. Smoke-free air laws are found to have a negative effect on smoking prevalence for the full sample and for the male, white, and high-SES sub-populations. Conclusions: This research concludes that higher cigarette prices will reduce smoking prevalence rates of Blacks, Hispanics, females, and low-SES subpopulations faster than the overall youth population and other youth sub-populations. Moreover, this research concludes that smoke-free air laws will reduce smoking prevalence for the overall youth population with the largest reductions in high SES and male subpopulations. PMID:24036487

  16. Recurrent simple tandem repeat mutations during human Y-chromosome radiation in Caucasian subpopulations.

    PubMed

    Ciminelli, B M; Pompei, F; Malaspina, P; Hammer, M; Persichetti, F; Pignatti, P F; Palena, A; Anagnou, N; Guanti, G; Jodice, C

    1995-12-01

    The haplotypes at four polymorphic loci of the Y chromosome were determined in 245 Caucasian males from 12 subpopulations. The data show that haplotype radiation occurred among Caucasians. Haplotype radiation was accompanied by recurrent mutations at STR loci that caused partial randomization of haplotype structure. The present distribution of alleles at short tandem repeats (STRs) can be explained by a mutation pattern similar to those described for autosomal STRs. The degree of variation among groups of subpopulations was assayed by using the Analysis of Molecular Variance. The results confirm a faster divergence of the Y chromosome as compared to the rest of the genome. PMID:8587142

  17. Epithelial hyperplasia, alveoli —

    Cancer.gov

    Solitary or multiple foci of increased cellularity distal to terminal bronchioles. The background of broncho-alveolar architecture remains detectable, and epithelial cells are usually single layered. Round to oval hypertrophic type II pneumocytes with abundant eosinophilic cytoplasm line alveolar walls. In bronchiolar subvariant, also called bronchiolization of alveoli, alveolar walls are lined by cuboidal to columnar cells with features of bronchiolar differentiation, such as formation of cilia, Clara cell resemblance, and presence of mucous granules. Foci of consolidation may indicate early stages of adenoma formation. Macrophages may be present in the alveolar lumens.

  18. Annexin II is associated with mRNAs which may constitute a distinct subpopulation.

    PubMed Central

    Vedeler, A; Hollås, H

    2000-01-01

    Protein-mRNA interactions affect mRNA transport, anchorage, stability and translatability in the cytoplasm. During the purification of three subpopulations of polysomes, it was observed that a 36-kDa protein, identified as annexin II, is associated with only one specific population of polysomes, namely cytoskeleton-associated polysomes. This association appears to be calcium-dependent since it was sensitive to EGTA and could be reconstituted in vitro. UV irradiation resulted in partial, EGTA-resistant cross-linking of annexin II to the polysomes. Binding of (32)P-labelled total RNA to proteins isolated from the cytoskeleton-bound polysomes on a NorthWestern blot resulted in a radioactive band having the same mobility as annexin II and, most importantly, purified native annexin II immobilized on nitrocellulose specifically binds mRNA. The mRNA population isolated from cytoskeleton-bound polysomes binds to annexin II with the highest affinity as compared with those isolated from free or membrane-bound polysomes. Interestingly, the annexin II complex, isolated from porcine small intestinal microvilli was a far better substrate for mRNA binding than the complex derived from transformed Krebs II ascites cells. When cytoskeleton-associated polysomes were split into 60 S and 40 S ribosomal subunits, and a peak containing mRNA complexes, annexin II fractionated with the mRNAs. Finally, using affinity purification of mRNA on poly(A)(+)-coupled magnetic beads, annexin II was only detected in association with messenger ribonucleoproteins (mRNPs) present in the cytoskeletal fraction (non-polysomal mRNPs). These results, derived from both in vitro experiments and cell fractionation, suggest that annexin II binds directly to the RNA moiety of mRNP complexes containing a specific population of mRNAs. PMID:10839987

  19. Identification and Characterization of CD133pos Subpopulation Cells From a Human Laryngeal Cancer Cell Line

    PubMed Central

    Qiu, Hai-ou; Wang, Huifang; Che, Na; Li, Dong; Mao, Yong; Zeng, Qiao; Ge, Rongming

    2016-01-01

    Background Recent research indicates that CD133 are expressed in several kinds of stem cells, among which, its high expression in laryngeal carcinoma has caused wide concern. To further explore efficaciously targeting drugs to laryngeal carcinoma stem cells (CSCs), we transplanted a solid tumor from CSCs into abdominal subcutaneous tissue of nude mice, and then compared the biological characteristics of laryngeal solid tumors with or without cisplatin intervention. Material/Methods In this study, the expression of CD133 was detected in the Hep-2 cell line by flow cytometry. By applying magnetic cell sorting (MACS) technology, we reported the results of purifying CD133-positive cells from a Hep-2 cell line. Cell proliferation, colony formation, and tumor-forming ability were examined in vitro and in vivo to identify the marker of CSCs in Hep-2 cell line. Results Upon flow cytometry analysis, CD133 was expressed constantly on 40.12±1.32% in Hep-2 cell line. Cell proliferation and colony formation ability were higher in CD133-positive cells compared to CD133-negative cells, and the in vivo tumorigenesis experiment showed the same results as in vitro assay. The 2 subpopulations cells were both sensitive to DDP, among which, the effect of DPP on proliferation ability and tumor-forming ability of CD133-positive cells was obviously greater than that of CD133-negative cells. Conclusions Above all, our study revealed that CD133-positive cells have properties of higher proliferation, colony formation, and tumorigenesis in Hep-2 cell line, indicating that CD133 could be a marker to characterize laryngeal cancer stem cells. PMID:27049928

  20. O-glycosylation of protein subpopulations in alcohol-extracted rice proteins.

    PubMed

    Kilcoyne, Michelle; Shah, Miti; Gerlach, Jared Q; Bhavanandan, Veer; Nagaraj, Vinay; Smith, Amy D; Fujiyama, Kazuhito; Sommer, Ulf; Costello, Catherine E; Olszewski, Neil; Joshi, Lokesh

    2009-02-15

    Mucin-type O-glycosylation has been well characterized in mammalian systems but not in plants. In this study, the purified alcohol-soluble, non-reduced protein (prolamin) fraction from rice seed was investigated for the occurrence of O-linked oligosaccharides. As storage prolamins are unlikely to be O-glycosylated, any O-glycosylation found was likely to belong to co-extracted proteins, whether because of association with the protein body or solubility. SDS-PAGE and MS analyses revealed 14 and 16kDa protein families in fractions that bound to the lectins peanut agglutinin (PNA), Vicia villosa lectin (VVL) and Jacalin, indicative of the presence of O-linked saccharides. Enzymatic cleavage, fluorescent labeling and high-performance liquid chromatography (HPLC) analysis demonstrated a peak consistent with Gal-beta-(1-->3)-GalNAc, with similar MS/MS fragmentation. Additionally, upon chemical analysis, a GlcNAc-containing O-linked carbohydrate moiety was discovered. Protein blotting with anti-O-GlcNAc antibody (clone CTD110.6) was positive in a subpopulation of the 14kDa alcohol-soluble protein fraction, but a hot capping experiment was negative. Therefore, the GlcNAc residue in this case is unlikely to be terminal. Additionally, a positive reaction with CTD110.6mAb cannot be taken as absolute proof of O-GlcNAc modification and further confirmatory experiments should be employed. We hypothesize that O-glycosylation may contribute to protein functionality or regulation. Further investigation is required to identify the specific proteins with these modifications. This 'reverse' approach could lead to the identification of proteins involved in mRNA targeting, signaling, translation, anchoring or maintenance of translational quiescence and may be applied to germinating rice seed extracts for further elucidation of protein function and regulation. PMID:18639953

  1. Comparative study of various subpopulations of cytotoxic cells in blood and ascites from patients with ovarian carcinoma

    PubMed Central

    Lukesova, Sarka; Vroblova, Vladimira; Tosner, Jindrich; Kopecky, Jindrich; Sedlakova, Iva; Čermáková, Eva; Vokurkova, Doris

    2015-01-01

    Aim of the study A number of observations have indicated that the immune system plays a significant role in patients with epithelial ovarian cancer (EOC). In cases of EOC, the prognostic significance of tumour infiltrating lymphocytes has not been clearly explained yet. The aim is to determine the phenotype and activation molecules of cytotoxic T cell and NK cell subpopulations and to compare their representation in malignant ascites and peripheral blood in patients with ovarian cancer. Material and methods Cytotoxic cells taken from blood samples of the cubital vein and malignant ascites were obtained from 53 patients with EOC. Their surface and activation characteristics were determined by means of a flow cytometer. Immunophenotype multiparametric analysis of peripheral blood lymphocytes (PBLs) and tumour infiltrating lymphocytes (TILs) was carried out. Results CD3+ T lymphocytes were the main population of TILs (75.9%) and PBLs (70.9%). The number of activating T cells was significantly higher in TILs: CD3+/69+ 6.7% vs. 0.8% (p < 0.001). The representation of (CD3–/16+56+) NK cells in TILs was significantly higher: 11.0% vs. 5.6% (p = 0.041); likewise CD56bright and CD–56bright from CD56+ cells were higher in TILs (both p < 0.001). The activation receptor NKG2D was present in 45.1% of TILs vs. 32.3% of PBLs (p = 0.034), but we did not find a significant difference in the numbers of CD56+/NKG2D+ in TILs and PBLs. Conclusions These results prove that the characteristics and intensity of anti-tumour responses are different in compared compartments (ascites/PBLs). The knowledge of phenotype and functions of effector cells is the basic precondition for understanding the anti-tumour immune response. PMID:26557777

  2. Efficacy of several candidate protein biomarkers in the differentiation of vaginal from buccal epithelial cells.

    PubMed

    Simons, Joanne L; Vintiner, Sue K

    2012-11-01

    Currently, there is no accurate method to differentiate vaginal epithelial cells from buccal epithelial cells in biological samples typically encountered in forensic casework. This study tested the expression of a selection of candidate proteins in buccal and vaginal epithelial cells. We investigated six candidate biomarkers, such as loricrin, vimentin, stratifin, cytokeratin 4, cytokeratin 13, small proline-rich protein 2, and involucrin, using Western blot analysis on whole protein extracts and immunohistochemistry (IHC) on intact cells in an attempt to identify cell-specific markers that would differentiate these cells by microscopy. Involucrin, loricrin, and stratifin showed differential expression during Western blot analysis and were carried through to IHC. Although proteins unique to vaginal epithelial cells and buccal epithelial cells were not identified from among the proteins tested, the increased expression levels of two proteins, loricrin and stratifin in vaginal cells, when compared to buccal cells, do provide encouraging results in the search for epithelial cell-specific markers. PMID:22612601

  3. Hydraulic fracture during epithelial stretching

    NASA Astrophysics Data System (ADS)

    Casares, Laura; Vincent, Romaric; Zalvidea, Dobryna; Campillo, Noelia; Navajas, Daniel; Arroyo, Marino; Trepat, Xavier

    2015-03-01

    The origin of fracture in epithelial cell sheets subject to stretch is commonly attributed to excess tension in the cells’ cytoskeleton, in the plasma membrane, or in cell-cell contacts. Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension. We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres. After pressure equilibration, cracks heal readily through actomyosin-dependent mechanisms. The observed phenomenology is captured by the theory of poroelasticity, which predicts the size and healing dynamics of epithelial cracks as a function of the stiffness, geometry and composition of the hydrogel substrate. Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics.

  4. Hydraulic fracture during epithelial stretching.

    PubMed

    Casares, Laura; Vincent, Romaric; Zalvidea, Dobryna; Campillo, Noelia; Navajas, Daniel; Arroyo, Marino; Trepat, Xavier

    2015-03-01

    The origin of fracture in epithelial cell sheets subject to stretch is commonly attributed to excess tension in the cells' cytoskeleton, in the plasma membrane, or in cell-cell contacts. Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension. We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres. After pressure equilibration, cracks heal readily through actomyosin-dependent mechanisms. The observed phenomenology is captured by the theory of poroelasticity, which predicts the size and healing dynamics of epithelial cracks as a function of the stiffness, geometry and composition of the hydrogel substrate. Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics. PMID:25664452

  5. Hydraulic fracture during epithelial stretching

    PubMed Central

    Casares, Laura; Vincent, Romaric; Zalvidea, Dobryna; Campillo, Noelia; Navajas, Daniel; Arroyo, Marino; Trepat, Xavier

    2015-01-01

    The origin of fracture in epithelial cell sheets subject to stretch is commonly attributed to excess tension in the cells’ cytoskeleton, in the plasma membrane, or in cell-cell contacts. Here we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension. We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression maneuvers. After pressure equilibration cracks heal readily through actomyosin-dependent mechanisms. The observed phenomenology is captured by the theory of poroelasticity, which predicts the size and healing dynamics of epithelial cracks as a function of the stiffness, geometry and composition of the hydrogel substrate. Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics. PMID:25664452

  6. V3 interneuron subpopulations in the mouse spinal cord undergo distinctive postnatal maturation processes.

    PubMed

    Borowska, J; Jones, C T; Deska-Gauthier, D; Zhang, Y

    2015-06-01

    Mice develop weight-bearing locomotion within the first 2-3 weeks of birth, a period during which motoneurons (MNs) and interneurons (INs) that control locomotor activities undergo rapid maturation. In this study, we investigate the maturation of two subpopulations of V3 INs in the mouse spinal cord during this period. To do this, we conducted whole-cell patch-clamp recordings of tdTomato fluorescent protein-expressing spinal V3 INs from Sim1(Cre/+);tdTom mice at post-natal day (P) 0, P4, P9 and P14 and compared their properties to those at P21. Combining electrophysiology with computational analyses, we show that dorsal and ventral V3 subpopulations are physiologically distinct at birth, but the electrophysiological properties of V3 INs change significantly during the first three post-natal weeks. We further reveal that there are multiple developmental phases of both V3 subpopulations during the maturation process. The different developmental trajectories of physiological properties also coincide with changes in an animal's locomotor behavior. These properties likely reflect the differential functions of V3 subpopulations in maturing spinal locomotor circuits. PMID:25800308

  7. Correlation between high density lipoprotein and monocyte subpopulations among stable coronary atherosclerotic heart disease patients

    PubMed Central

    Yang, Rong-Hai; Liu, Ying-Feng; Wang, Xue-Jun; Liang, Jian-Guang; Liu, Jia-Chao

    2015-01-01

    High density lipoprotein (HDL) is a structurally and functionally heterogeneous molecular particle whose function is unclear in atherosclerosis at present. Studies show that small HDL functional imbalance may exist in Coronary Atherosclerotic Heart Disease (CAD) patients. Monocyte is considered to play an important role in atherosclerosis, in accordance with the expression of superficial CD14 and CD16, it can be divided into three subpopulations. The purpose of this study was to explore the relation between HDL and monocyte subpopulations among CAD patients. We report 90 cases of stable CAD patients and define the monocyte subpopulations as classical monocyte (CD14++CD16-; CM), intermediate monocyte (CD14+CD16+; IM), and non-classical monocyte (CD14+CD16++; NCM); HDL group is measured by polyacrylamide gel electrophoresis. The results indicated that the small HDL in blood serum has a correlation with proinflammatory NCM in circulation but a negative correction with CM and no relationship with diabetes, saccharify hemoglobin, hypertension, smoking history and taking dose of statins drugs and severity of disease. In conclusion, this study primarily confirms that micromolecule HDL level correlates with the increase of non-classical monocyte subpopulations and decrease of classical monocyte quantity. Thus demonstrates the proinflammatory correlation between micromolecule HDL and internal immunity in the development of stable atherosclerosis. PMID:26629252

  8. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth

    PubMed Central

    Roesch, Alexander; Fukunaga-Kalabis, Mizuho; Schmidt, Elizabeth C.; Zabierowski, Susan E.; Brafford, Patricia A.; Vultur, Adina; Basu, Devraj; Gimotty, Phyllis; Vogt, Thomas; Herlyn, Meenhard

    2010-01-01

    Summary Melanomas are highly heterogeneous tumors, but the biological significance of their different subpopulations is not clear. Using the H3K4 demethylase JARID1B (KDM5B/PLU-1/RBP2-H1) as a biomarker, we have characterized a small subpopulation of slow-cycling melanoma cells that cycle with doubling times of >4 weeks within the rapidly proliferating main population. Isolated JARID1B-positive melanoma cells give rise to a highly proliferative progeny. Knock-down of JARID1B leads to an initial acceleration of tumor growth followed by exhaustion which suggests that the JARID1B-positive subpopulation is essential for continuous tumor growth. Expression of JARID1B is dynamically regulated and does not follow a hierarchical cancer stem cell model because JARID1B-negative cells can become positive and even single melanoma cells irrespective of selection are tumorigenic. These results suggest a new understanding of melanoma heterogeneity with tumor maintenance as a dynamic process mediated by a temporarily distinct subpopulation. PMID:20478252

  9. Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

    SciTech Connect

    Rubin, D.B.; Drab, E.A.; Bauer, K.D. )

    1989-10-01

    Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: (1) cell volume; (2) cell cycle position; and (3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings (1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and (2) demonstrate EC subpopulations in culture.

  10. A non-invasive method for in situ quantification of subpopulation behaviour in mixed cell culture.

    PubMed

    MacArthur, Ben D; Tare, Rahul S; Please, Colin P; Prescott, Philip; Oreffo, Richard O C

    2006-02-22

    Ongoing advances in quantitative molecular- and cellular-biology highlight the need for correspondingly quantitative methods in tissue-biology, in which the presence and activity of specific cell-subpopulations can be assessed in situ. However, many experimental techniques disturb the natural tissue balance, making it difficult to draw realistic conclusions concerning in situ cell behaviour. In this study, we present a widely applicable and minimally invasive method which combines fluorescence cell labelling, retrospective image analysis and mathematical data processing to detect the presence and activity of cell subpopulations, using adhesion patterns in STRO-1 immunoselected human mesenchymal populations and the homogeneous osteoblast-like MG63 continuous cell line as an illustration. Adhesion is considered on tissue culture plastic and fibronectin surfaces, using cell area as a readily obtainable and individual cell specific measure of spreading. The underlying statistical distributions of cell areas are investigated and mappings between distributions are examined using a combination of graphical and non-parametric statistical methods. We show that activity can be quantified in subpopulations as small as 1% by cell number, and outline behaviour of significant subpopulations in both STRO-1+/- fractions. This method has considerable potential to understand in situ cell behaviour and thus has wide applicability, for example in developmental biology and tissue engineering. PMID:16849218

  11. CD14+CD16+ and CD14+CD163+ monocyte subpopulations in kidney allograft transplantation

    PubMed Central

    2014-01-01

    Background Monocytes represent a heterogeneous population of cells subdivided according to the expression level of membrane antigens. A pro-inflammatory (intermediate/nonclassical) subpopulation of monocytes is defined by expression of CD16. CD163 seems to be characteristically preferentially expressed by immunosuppressive monocytes. The aim of our study was to evaluate the distribution of monocyte subpopulations in 71 patients with kidney allograft transplantation. Results The phenotype was evaluated by flow cytometry in defined time points. The proportions of peripheral CD14+CD16+ monocytes were downregulated immediately after the kidney transplantation and basiliximab treatment partially attenuated this trend. The transient downregulation of the CD14+CD16+ subpopulation was adjusted to basal values in two months. The proportions of CD14+CD163+ monocytes were transiently upregulated early after the kidney transplantation and remained higher during the first month in most patients. In ATG treated patients, the expansion of CD14+CD163+ monocytes was delayed but their upregulation lasted longer. In vitro data showed the direct effect of ATG and methylprednisolone on expression of CD16 and CD163 molecules while basiliximab did not affect the phenotype of cultured monocytes. Conclusions We assume from our data that kidney allograft transplantation is associated with modulation of monocyte subpopulations (CD14+CD16+ and CD14+CD163+) partially affected by an immunosuppressive regime used. PMID:24499053

  12. Meta-STEPP: subpopulation treatment effect pattern plot for individual patient data meta-analysis.

    PubMed

    Wang, Xin Victoria; Cole, Bernard; Bonetti, Marco; Gelber, Richard D

    2016-09-20

    We have developed a method, called Meta-STEPP (subpopulation treatment effect pattern plot for meta-analysis), to explore treatment effect heterogeneity across covariate values in the meta-analysis setting for time-to-event data when the covariate of interest is continuous. Meta-STEPP forms overlapping subpopulations from individual patient data containing similar numbers of events with increasing covariate values, estimates subpopulation treatment effects using standard fixed-effects meta-analysis methodology, displays the estimated subpopulation treatment effect as a function of the covariate values, and provides a statistical test to detect possibly complex treatment-covariate interactions. Simulation studies show that this test has adequate type-I error rate recovery as well as power when reasonable window sizes are chosen. When applied to eight breast cancer trials, Meta-STEPP suggests that chemotherapy is less effective for tumors with high estrogen receptor expression compared with those with low expression. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27073066

  13. State-Dependent Propagation of Neuronal Sub-Population in Spontaneous Synchronized Bursts

    PubMed Central

    Yada, Yuichiro; Kanzaki, Ryohei; Takahashi, Hirokazu

    2016-01-01

    Repeating stable spatiotemporal patterns emerge in synchronized spontaneous activity in neuronal networks. The repertoire of such patterns can serve as memory, or a reservoir of information, in a neuronal network; moreover, the variety of patterns may represent the network memory capacity. However, a neuronal substrate for producing a repertoire of patterns in synchronization remains elusive. We herein hypothesize that state-dependent propagation of a neuronal sub-population is the key mechanism. By combining high-resolution measurement with a 4096-channel complementary metal-oxide semiconductor (CMOS) microelectrode array (MEA) and dimensionality reduction with non-negative matrix factorization (NMF), we investigated synchronized bursts of dissociated rat cortical neurons at approximately 3 weeks in vitro. We found that bursts had a repertoire of repeating spatiotemporal patterns, and different patterns shared a partially similar sequence of sub-population, supporting the idea of sequential structure of neuronal sub-populations during synchronized activity. We additionally found that similar spatiotemporal patterns tended to appear successively and periodically, suggesting a state-dependent fluctuation of propagation, which has been overlooked in existing literature. Thus, such a state-dependent property within the sequential sub-population structure is a plausible neural substrate for performing a repertoire of stable patterns during synchronized activity. PMID:27065820

  14. Plasma levels of HDL subpopulations and remnant lipoproteins predict the extent of angiographically defined disease in post-menopausal women

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The association of coronary heart disease (CHD) with subpopulations of triglyceride (TG)-rich lipoproteins and high-density lipoproteins (HDL) is established in men, but has not been well characterized in women. Plasma HDL subpopulation concentrations, quantified by 2-dimensional gel electrophoresis...

  15. Lymphocyte subpopulations in peripheral blood of healthy persons. Characterization by surface markers and lack of selection during purification

    PubMed Central

    Holm, G.; Pettersson, D.; Mellstedt, H.; Hedfors, E.; Bloth, B.

    1975-01-01

    Lymphocytes were isolated at 99% purity from peripheral blood of healthy persons by defibrination, gelatine sedimentation, treatment with carbonyl iron powder and centrifugation on Ficoll–Isopaque. Subpopulations were identified by three surface markers: cells forming rosettes with sheep red blood cells (SRBC) (E-binding lymphocytes) as a measure of T lymphocytes; lymphocytes with surface immunoglobulin identified by indirect immunofluorescence (B lymphocytes); lymphocytes with receptors for C3 observed by the rosette method using SRBC treated with rabbit antiserum and human complement (EAC-binding lymphocytes). The yield of lymphocytes after purification varied from 15 to 65%. No selection of lymphocytes was observed either by counting immunoglobulin-bearing and EAC-binding lymphocytes in whole blood and in purified cells from the same sample, or by statistical analysis of lymphocytes in subpopulations as a function of the yields from twenty-six experiments. In the absence of selection during purification the total numbers of T and B lymphocytes could be calculated from the percentages and the total numbers of lymphocytes. Our normal values are close to those reported using other non-selective methods of purification. When lymphocytes were simultaneously stained for immunoglobulin and rosetted with EAC, cells bearing either or both markers were found. In total, 27–35% cells were identified by these markers. Since about 70% of the cells were E-binding, practically all lymphocytes could be identified. A small overlap between E-binding and immunoglobulin-bearing/EAC-binding lymphocytes may occur. Either the IgM or the IgG-containing fractions obtained after fractionation of rabbit anti-SRBC serum on Sephadex G-200 could be used for sensitization of SRBC with complement. Formation of rosettes was not prevented by pretreating the lymphocytes with aggregated IgG, while rosettes formed with EA prepared by high concentrations of IgG antibody (Fc-binding lymphocytes

  16. Induced pluripotency of human prostatic epithelial cells.

    PubMed

    Zhao, Hongjuan; Sun, Ning; Young, Sarah R; Nolley, Rosalie; Santos, Jennifer; Wu, Joseph C; Peehl, Donna M

    2013-01-01

    Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that

  17. The forkhead transcription factors, Foxp1 and Foxp2, identify different subpopulations of projection neurons in the mouse cerebral cortex.

    PubMed

    Hisaoka, T; Nakamura, Y; Senba, E; Morikawa, Y

    2010-03-17

    Foxp1 and Foxp2, which belong to the forkhead transcription factor family, are expressed in the developing and adult mouse brain, including the striatum, thalamus, and cerebral cortex. Recent reports suggest that FOXP1 and FOXP2 are involved in the development of speech and language in humans. Although both Foxp1 and Foxp2 are expressed in the neural circuits that mediate speech and language, including the corticostriatal circuit, the functions of Foxp1 and Foxp2 in the cerebral cortex remain unclear. To gain insight into the functions of Foxp1 and Foxp2 in the cerebral cortex, we characterized Foxp1- and Foxp2-expressing cells in postnatal and adult mice using immunohistochemistry. In adult mice, Foxp1 was expressed in neurons of layers III-VIa in the neocortex, whereas the expression of Foxp2 was restricted to dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa (DARPP-32)(+) neurons of layer VI. In addition, Foxp2 was weakly expressed in the neurons of layer V of the motor cortex and hindlimb and forelimb regions of the primary somatosensory cortex. Both Foxp1 and Foxp2 were expressed in the ionotropic glutamate receptor (GluR) 2/3(+) neurons, and colocalized with none of GluR1, gamma-aminobutyric acid, calbindin, and parvalbumin, indicating that expression of Foxp1 and Foxp2 is restricted to projection neurons. During the postnatal stages, Foxp1 was predominantly expressed in Satb2(+)/Ctip2(-) corticocortical projection neurons of layers III-V and in Tbr1(+) corticothalamic projection neurons of layer VIa. Although Foxp2 was also expressed in Tbr1(+) corticothalamic projection neurons of layer VI, no colocalization of Foxp1 with Foxp2 was observed from postnatal day (P) 0 to P7. These findings suggest that Foxp1 and Foxp2 may be involved in the development of different cortical projection neurons during the early postnatal stages in addition to the establishment and maintenance of different cortical circuits from the late postnatal stage to adulthood. PMID:20040367

  18. Diversity of epithelial stem cell types in adult lung.

    PubMed

    Li, Feng; He, Jinxi; Wei, Jun; Cho, William C; Liu, Xiaoming

    2015-01-01

    Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. PMID:25810726

  19. Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Sullivan, David A.; Liu, Yang; Kam, Wendy R.; Ding, Juan; Green, Karin M.; Shaffer, Scott A.; Hatton, Mark P.; Liu, Shaohui

    2014-01-01

    Purpose. We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Methods. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Results. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Conclusions. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland. PMID:24867579

  20. Diversity of Epithelial Stem Cell Types in Adult Lung

    PubMed Central

    Li, Feng; He, Jinxi; Wei, Jun; Cho, William C.; Liu, Xiaoming

    2015-01-01

    Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. PMID:25810726

  1. Awareness assessment in Turkish subpopulation with chronic oral mucosal diseases

    PubMed Central

    Okumus, Ozlem; Kalkan, Sevda; Keser, Gaye; Pekiner, Filiz Namdar

    2015-01-01

    Objectives: The aim of this study was to evaluate the awareness of group Turkish patients with chronic oral mucosal diseases by chronic oral mucosal diseases questionnaires (COMDQ). Materials and Methods: Eighty patients with chronic oral mucosal diseases were participated in the study. A detailed medical history of each patient was taken, and all the COMDQ questions, which were translated from English version, were filled out. The data were analyzed with the IBM Statistical Package for Social Sciences Statistics 22.0. Results: The mean ages of patients were 48.91 ± 13.36 years. Of the total 80 cases of chronic oral mucosal diseases identified 52 (65%) were female and 28 (35%) male. The standardized mean scores for COMDQ were 1.72 ± 1.11 for “pain and functional limitation,” 1.09 ± 0.94 for “medication and treatment,” 2.31 ± 1.06 for “social and emotional,” and 2.27 ± 0.83 for “patient support,” respectively. Conclusions: The results of this study indicate that the Turkish version of the COMDQ has the profitable psychometric peculiarity and comfortable to patients with chronic oral mucosal diseases in Turkey. PMID:26929697

  2. Radiation-Induced Reprogramming of Pre-Senescent Mammary Epithelial Cells Enriches Putative CD44+/CD24−/low Stem Cell Phenotype

    PubMed Central

    Gao, Xuefeng; Sishc, Brock J.; Nelson, Christopher B.; Hahnfeldt, Philip; Bailey, Susan M.; Hlatky, Lynn

    2016-01-01

    The enrichment of putative CD44+/CD24−/low breast stem cell populations following exposure to ionizing radiation (IR) has been ascribed to their inherent radioresistance and an elevated frequency of symmetric division during repopulation. However, recent studies demonstrating radiation-induced phenotypic reprogramming (the transition of non-CD44+/CD24−/low cells into the CD44+/CD24−/low phenotype) as a potential mechanism of CD44+/CD24−/low cell enrichment have raised the question of whether a higher survival and increased self-renewal of existing CD44+/CD24−/low cells or induced reprogramming is an additional mode of enrichment. To investigate this question, we combined a cellular automata model with in vitro experimental data using both MCF-10A non-tumorigenic human mammary epithelial cells and MCF-7 breast cancer cells, with the goal of identifying the mechanistic basis of CD44+/CD24−/low stem cell enrichment in the context of radiation-induced cellular senescence. Quantitative modeling revealed that incomplete phenotypic reprogramming of pre-senescent non-stem cells (reprogramming whereby the CD44+/CD24−/low phenotype is conveyed, along with the short-term proliferation capacity of the original cell) could be an additional mode of enriching the CD44+/CD24−/low subpopulation. Furthermore, stem cell enrichment in MCF-7 cells occurs both at lower doses and earlier time points, and has longer persistence, than that observed in MCF-10A cells, suggesting that phenotypic plasticity appears to be less regulated in breast cancer cells. Taken together, these results suggest that reprogramming of pre-senescent non-stem cells may play a significant role in both cancer and non-tumorigenic mammary epithelial populations following exposure to IR, a finding with important implications for both radiation therapy and radiation carcinogenesis. PMID:27379202

  3. Ovarian yolk sac tumors in older women arising from epithelial ovarian tumors or with no detectable epithelial component.

    PubMed

    Roth, Lawrence M; Talerman, Aleksander; Levy, Tally; Sukmanov, Oleg; Czernobilsky, Bernard

    2011-09-01

    Yolk sac tumor (YST) occurs rarely in older women, either in association with a variety of ovarian epithelial tumors or, considerably less often, without an identifiable epithelial precursor. The patients often have elevated serum levels of α-fetoprotein that roughly correlate with the amount of the YST component. In postmenopausal women with an ovarian mass and elevated serum levels of α-fetoprotein, a tumor of this type should be suspected. Endometrioid carcinoma is the most common putative precursor, and the tumor is often associated with an endometriotic cyst; however, malignant Müllerian mixed tumor and mucinous neoplasms have also been reported as precursors. We report 4 cases of YST in postmenopausal women. Of the 3 cases with an identified epithelial component, 1 was serous carcinoma, another was clear cell adenocarcinoma, and the third was an admixture of endometrioid and clear cell adenocarcinoma arising from an endometriotic cyst. Although a precursor epithelial ovarian neoplasm, typically a malignancy (somatic carcinoma), is usually identified, no precursor neoplasm was observed in 1 of our cases and in 5 cases from the literature. We believe that YSTs in older women, whether or not an epithelial component is detected histologically, constitute a single entity that is distinct from YSTs in younger patients and should be treated aggressively. Neoplasms with a YST component in older women are less responsive to the chemotherapy currently used for ovarian germ cell tumors; therefore, adjuvant therapy should include platinum-based chemotherapy designed to treat both epithelial ovarian cancer and germ cell tumors. Of the 24 reported cases, including our own, 17 died of neoplasms within 25 months and another was living with disease at 2 months. However, 2 more recent patients treated aggressively with platinum-based chemotherapy designed to treat both epithelial and germ cell tumor components with stage 1 disease are living and have been disease free >1

  4. Epithelial Nitration by a Peroxidase/NOX5 System Mediates Mosquito Antiplasmodial Immunity

    PubMed Central

    de Almeida Oliveira, Giselle; Lieberman, Joshua; Barillas-Mury, Carolina

    2012-01-01

    Plasmodium ookinetes traverse midgut epithelial cells before they encounter the complement system in the mosquito hemolymph. We identified a heme peroxidase (HPX2) and NADPH oxidase 5 (NOX5) as critical mediators of midgut epithelial nitration and antiplasmodial immunity that enhance nitric oxide toxicity in Anopheles gambiae. We show that the two immune mechanisms that target ookinetes—epithelial nitration and thioester-containing protein 1 (TEP1)-mediated lysis—work sequentially and propose that epithelial nitration works as an opsonization-like system that promotes activation of the mosquito complement cascade. PMID:22282475

  5. Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity.

    PubMed

    Oliveira, Giselle de Almeida; Lieberman, Joshua; Barillas-Mury, Carolina

    2012-02-17

    Plasmodium ookinetes traverse midgut epithelial cells before they encounter the complement system in the mosquito hemolymph. We identified a heme peroxidase (HPX2) and NADPH oxidase 5 (NOX5) as critical mediators of midgut epithelial nitration and antiplasmodial immunity that enhance nitric oxide toxicity in Anopheles gambiae. We show that the two immune mechanisms that target ookinetes-epithelial nitration and thioester-containing protein 1 (TEP1)-mediated lysis-work sequentially, and we propose that epithelial nitration works as an opsonization-like system that promotes activation of the mosquito complement cascade. PMID:22282475

  6. Cigarette Smoke Induces Human Epidermal Receptor 2-Dependent Changes in Epithelial Permeability.

    PubMed

    Mishra, Rangnath; Foster, Daniel; Vasu, Vihas T; Thaikoottathil, Jyoti V; Kosmider, Beata; Chu, Hong Wei; Bowler, Russell P; Finigan, James H

    2016-06-01

    The airway epithelium constitutes a protective barrier against inhaled insults, such as viruses, bacteria, and toxic fumes, including cigarette smoke (CS). Maintenance of bronchial epithelial integrity is central for airway health, and defective epithelial barrier function contributes to the pathogenesis of CS-mediated diseases, such as chronic obstructive pulmonary disease. Although CS has been shown to increase epithelial permeability, current understanding of the mechanisms involved in CS-induced epithelial barrier disruption remains incomplete. We have previously identified that the receptor tyrosine kinase human epidermal receptor (HER) 2 growth factor is activated by the ligand neuregulin-1 and increases epithelial permeability in models of inflammatory acute lung injury. We hypothesized that CS activates HER2 and that CS-mediated changes in barrier function would be HER2 dependent in airway epithelial cells. We determined that HER2 was activated in whole lung, as well as isolated epithelial cells, from smokers, and that acute CS exposure resulted in HER2 activation in cultured bronchial epithelial cells. Mechanistic studies determined that CS-mediated HER2 activation is independent of neuregulin-1 but required upstream activation of the epidermal growth factor receptor. HER2 was required for CS-induced epithelial permeability as knockdown of HER2 blocked increases in permeability after CS. CS caused an increase in IL-6 production by epithelial cells that was dependent on HER2-mediated extracellular signal-regulated kinases (Erk) activation. Finally, blockade of IL-6 attenuated CS-induced epithelial permeability. Our data indicate that CS activates pulmonary epithelial HER2 and that HER2 is a central mediator of CS-induced epithelial barrier dysfunction. PMID:26600084

  7. Volumetric imaging of oral epithelial neoplasia by MPM-SHGM: epithelial connective tissue interface (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Pal, Rahul; Yang, Jinping; Qiu, Suimin; Resto, Vicente; McCammon, Susan; Vargas, Gracie

    2016-03-01

    The majority of oral cancers are comprised of oral squamous cell carcinoma in which neoplastic epithelial cells invade across the epithelial connective tissue interface (ECTI). Invasion is preceded by a multi-component process including epithelial hyperproliferation, loss of cell polarity, and remodeling of the extracellular matrix. Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) show promise for revealing indicators of neoplasia. In particular, volumetric imaging by these methods can reveal aspects of the 3D microstructure that are not possible by other methods and which could both further our understanding of neoplastic transformation and be explored for development of diagnostic approaches in this disease having only 55% 5-year survival rate. MPAM-SHG were applied to reveal the 3D structure of the critical ECTI interface that plays an integral part toward invasion. Epithelial dysplasia was induced in an established hamster model. MPAM-SHGM was applied to lesion sites, using 780 nm excitation (450-600nm emission) for autofluroescence of cellular and extracellular components; 840 nm using 420 nm bandpass filter for SHG. The ECTI surface was identified as the interface at which SHG signal began following the epithelium and was modeled as a 3D surface using Matlab. ECTI surface area and cell features at sites of epithelial expansion where ECTI was altered were measured; Imaged sites were biopsied and processed for histology. ROC analysis using ECTI image metrics indicated the ability to delineate normal from neoplasia with high sensitivity and specificity and it is noteworthy that inflammation did not significantly alter diagnostic potential of MPAM-SHGM .

  8. Direction- and distance-dependent interareal connectivity of pyramidal cell subpopulations in the rat frontal cortex

    PubMed Central

    Ueta, Yoshifumi; Hirai, Yasuharu; Otsuka, Takeshi; Kawaguchi, Yasuo

    2013-01-01

    The frontal cortex plays an important role in the initiation and execution of movements via widespread projections to various cortical and subcortical areas. Layer 2/3 (L2/3) pyramidal cells in the frontal cortex send axons mainly to other ipsilateral/contralateral cortical areas. Subpopulations of layer 5 (L5) pyramidal cells that selectively project to the pontine nuclei or to the contralateral cortex [commissural (COM) cells] also target diverse and sometimes overlapping ipsilateral cortical areas. However, little is known about target area-dependent participation in ipsilateral corticocortical (iCC) connections by subclasses of L2/3 and L5 projection neurons. To better understand the functional hierarchy between cortical areas, we compared iCC connectivity between the secondary motor cortex (M2) and adjacent areas, such as the orbitofrontal and primary motor cortices, and distant non-frontal areas, such as the perirhinal and posterior parietal cortices. We particularly assessed the laminar distribution of iCC cells and fibers, and identified the subtypes of pyramidal cells participating in those projections. For connections between M2 and frontal areas, L2/3 and L5 cells in both areas contributed to reciprocal projections, which can be viewed as “bottom-up” or “top-down” on the basis of their differential targeting of cortical lamina. In connections between M2 and non-frontal areas, neurons participating in bottom-up and top-down projections were segregated into the different layers: bottom-up projections arose primarily from L2/3 cells, while top-down projections were dominated by L5 COM cells. These findings suggest that selective participation in iCC connections by pyramidal cell subtypes lead to directional connectivity between M2 and other cortical areas. Based on these findings, we propose a provisional unified framework of interareal hierarchy within the frontal cortex, and discuss the interaction of local circuits with long-range interareal

  9. Genetic differences among Anopheles vestitipennis subpopulations collected using different methods in Chiapas state, southern México.

    PubMed

    Arredondo-Jiménez, J I; Gimnig, J; Rodríguez, M H; Washino, R K

    1996-09-01

    Biting activity and population genetic studies of the malaria vector Anopheles vestitipennis were conducted in southern México. Three subpopulations were collected from 2 villages; 2 subpopulations were from the same village, one on human bait and one with an animal-baited trap; the third was collected from a cattle corral in the 2nd village (280 km away SSE). The anthropophilic subpopulation had steady activity with 61% of bites occurring before midnight, significantly different from those of the 2 zoophilic subpopulations, which had 78-82% of bites before midnight and 2 biting peaks, one at 1900-2100 h and the other at 0400-0500 h. Isozyme analysis (13 enzymes) of these subpopulations indicated that differences between the 2 sympatric subpopulations (D = 0.07), collected using 2 different methods, were greater than that between the 2 allopatric ones (D = 0.03). These studies suggest the existence of 2 genetically different subpopulations of An. vestitipennis with specific host preferences. PMID:8887216

  10. Transcriptional Landscape of Glomerular Parietal Epithelial Cells

    PubMed Central

    Gharib, Sina A.; Pippin, Jeffrey W.; Ohse, Takamoto; Pickering, Scott G.; Krofft, Ronald D.; Shankland, Stuart J.

    2014-01-01

    Very little is known about the function of glomerular parietal epithelial cells (PECs). In this study, we performed genome-wide expression analysis on PEC-enriched capsulated vs. PEC-deprived decapsulated rat glomeruli to determine the transcriptional state of PECs under normal conditions. We identified hundreds of differentially expressed genes that mapped to distinct biologic modules including development, tight junction, ion transport, and metabolic processes. Since developmental programs were highly enriched in PECs, we characterized several of their candidate members at the protein level. Collectively, our findings confirm that PECs are multifaceted cells and help define their diverse functional repertoire. PMID:25127402

  11. Theory of epithelial elasticity

    NASA Astrophysics Data System (ADS)

    Krajnc, Matej; Ziherl, Primož

    2015-11-01

    We propose an elastic theory of epithelial monolayers based on a two-dimensional discrete model of dropletlike cells characterized by differential surface tensions of their apical, basal, and lateral sides. We show that the effective tissue bending modulus depends on the apicobasal differential tension and changes sign at the transition from the flat to the fold morphology. We discuss three mechanisms that stabilize the finite-wavelength fold structures: Physical constraint on cell geometry, hard-core interaction between non-neighboring cells, and bending elasticity of the basement membrane. We show that the thickness of the monolayer changes along the waveform and thus needs to be considered as a variable rather than a parameter. Next we show that the coupling between the curvature and the thickness is governed by the apicobasal polarity and that the amplitude of thickness modulation along the waveform is proportional to the apicobasal differential tension. This suggests that intracellular stresses can be measured indirectly by observing easily measurable morphometric parameters. We also study the mechanics of three-dimensional structures with cylindrical symmetry.

  12. Experimental allergic encephalomyelitis. Early T cells and TG cells are separate subpopulations.

    PubMed

    Traugott, U; Raine, C S

    1982-10-01

    To investigate whether previously described fluctuations in circulating early T cell levels reflected a change in suppressor activity, early T cell and TG cell values were determined in guinea pigs with acute and chronic experimental allergic encephalomyelitis (EAE) and normals. It was found that while both subpopulations showed similar changes in EAE, particularly decreases during periods of clinical worsening, changes in the two subpopulations were not always synchronous. Also, similar levels of TG cells could be found both in isolated early and isolated late (total) T cells. These two lines of evidence indicate that early T cells and TG cells are heterologous populations and that early T cells do not reflect suppressor activity, findings in accord with a similar, recent study on multiple sclerosis. PMID:6216327

  13. T lymphocytes in rat germinal centres belong to an ER3+ subpopulation of CD4+ cells.

    PubMed Central

    Vonderheide, R H; Hunt, S V

    1990-01-01

    Two-colour immunofluorescence histochemistry showed directly that greater than 90% of CD4+ germinal centre T cells in rat spleen or lymph node examined 7 days after immunization bear the antigen recognized by the monoclonal antibody (mAb) ER3. By contrast, only 30-40% of all thoracic duct or lymph node CD4+ cells were ER3+, as determined by two-colour flow cytometry. CD8+ cells were ER3+, but nearly all B cells were ER3-. Thus, germinal centre T cells belong to a subpopulation of CD4+ cells. Because only 25-30% of CD4+ cells that lack higher molecular weight forms of CD45 (i.e. mAb MRC OX32 cells, equivalent to MRC OX22 cells) express ER3, the CD4+ subpopulations defined by ER3 are neither identical nor complementary to the subsets defined by restricted expression of CD45 epitopes. Images Figure 1 PMID:1970805

  14. Characterization of different CTC subpopulations in non-small cell lung cancer

    PubMed Central

    Hanssen, Annkathrin; Wagner, Jenny; Gorges, Tobias M.; Taenzer, Aline; Uzunoglu, Faik G.; Driemel, Christiane; Stoecklein, Nikolas H.; Knoefel, Wolfram T.; Angenendt, Sebastian; Hauch, Siegfried; Atanackovic, Djordje; Loges, Sonja; Riethdorf, Sabine; Pantel, Klaus; Wikman, Harriet

    2016-01-01

    Circulating tumour cells (CTCs) serve as valuable biomarkers. However, EpCAM positive CTCs are less frequently detected in NSCLC patients compared to other epithelial tumours. First, EpCAM protein expression was analysed in primary and metastatic lung cancer tissue. In both groups 21% of the samples were EpCAM negative. Second, the CellSearch system identified 15% of patients (n = 48) as CTC positive whereas a multiplex RT-PCR for PIK3CA, AKT2, TWIST, and ALDH1 following EGFR, HER2 and EpCAM based enrichment detected CTCs in 29% of the patients. Interestingly, 86% of CTC positive patients were found to express ALDH1. Only 11% of the patients were CTC-positive by both techniques. CTC positivity was associated with patient disease state when assessed by the multiplex RT-PCR assay (p = 0.015). Patients harbouring tumours with an altered EGFR genotype were more frequently CTC-positive compared to patients with EGFR wildtype tumours. In subsets of patients, CTCs were found to express genes involved in resistance to therapy such as HER3 and MET. In conclusion, using multiple targets for CTC capture and identification increases the sensitivity of CTC detection in NSCLC patients, which can be explained by the presence of different CTC subtypes with distinct molecular features. PMID:27302574

  15. Characterization of different CTC subpopulations in non-small cell lung cancer.

    PubMed

    Hanssen, Annkathrin; Wagner, Jenny; Gorges, Tobias M; Taenzer, Aline; Uzunoglu, Faik G; Driemel, Christiane; Stoecklein, Nikolas H; Knoefel, Wolfram T; Angenendt, Sebastian; Hauch, Siegfried; Atanackovic, Djordje; Loges, Sonja; Riethdorf, Sabine; Pantel, Klaus; Wikman, Harriet

    2016-01-01

    Circulating tumour cells (CTCs) serve as valuable biomarkers. However, EpCAM positive CTCs are less frequently detected in NSCLC patients compared to other epithelial tumours. First, EpCAM protein expression was analysed in primary and metastatic lung cancer tissue. In both groups 21% of the samples were EpCAM negative. Second, the CellSearch system identified 15% of patients (n = 48) as CTC positive whereas a multiplex RT-PCR for PIK3CA, AKT2, TWIST, and ALDH1 following EGFR, HER2 and EpCAM based enrichment detected CTCs in 29% of the patients. Interestingly, 86% of CTC positive patients were found to express ALDH1. Only 11% of the patients were CTC-positive by both techniques. CTC positivity was associated with patient disease state when assessed by the multiplex RT-PCR assay (p = 0.015). Patients harbouring tumours with an altered EGFR genotype were more frequently CTC-positive compared to patients with EGFR wildtype tumours. In subsets of patients, CTCs were found to express genes involved in resistance to therapy such as HER3 and MET. In conclusion, using multiple targets for CTC capture and identification increases the sensitivity of CTC detection in NSCLC patients, which can be explained by the presence of different CTC subtypes with distinct molecular features. PMID:27302574

  16. Circulating Melanoma Cell Subpopulations: Their Heterogeneity and Differential Responses to Treatment

    PubMed Central

    Gray, Elin S; Reid, Anna L; Bowyer, Samantha; Calapre, Leslie; Siew, Kelvin; Pearce, Robert; Cowell, Lester; Frank, Markus H; Millward, Michael; Ziman, Mel

    2015-01-01

    Metastatic melanoma is a highly heterogeneous tumor; thus, methods to analyze tumor-derived cells circulating in blood should address this diversity. Taking this into account, we analyzed, using multiparametric flow cytometry, the co-expression of the melanoma markers melanoma cell adhesion molecule and melanoma-associated chondroitin sulphate proteoglycan and the tumor-initiating markers ATP-binding cassette sub-family B member 5 (ABCB5), CD271, and receptor activator of NF-κβ (RANK) in individual circulating tumor cells (CTCs) from 40 late-stage (III–IV) and 16 early-stage (I–II) melanoma patients. CTCs were heterogeneous within and between patients, with limited co-expression between the five markers analyzed. Analysis of patient matched blood and metastatic tumors revealed that ABCB5 and RANK subpopulations are more common among CTCs than in the solid tumors, suggesting a preferential selection for these cells in circulation. Pairwise comparison of CTC subpopulations longitudinally before and 6–13 weeks after treatment initiation showed that the percentage of RANK+ CTCs significantly increased in the patients undergoing targeted therapy (N=16, P<0.01). Moreover, the presence of ⩾5 RANK+ CTCs in the blood of patients undergoing targeted therapies was prognostic of shorter progression-free survival (hazards ratio 8.73, 95% confidence interval 1.82–41.75, P<0.01). Taken together, our results provide evidence of the heterogeneity among CTC subpopulations in melanoma and the differential response of these subpopulations to targeted therapy. PMID:25830652

  17. Characterization of circulating lymphocyte subpopulations in canine leishmaniasis throughout treatment with antimonials and allopurinol.

    PubMed

    Miranda, Sonia; Martorell, Susanna; Costa, Manuela; Ferrer, Lluis; Ramis, Antonio

    2007-03-31

    Canine leishmaniasis (CL) is a systemic parasitic disease with a wide variability of response to specific therapy: the majority of patients apparently improve with treatment, some of them respond but later relapse, and few of them do not respond at all. It has been demonstrated that the immune response plays a key role in the development and outcome of Leishmania infection in the dog and in the response to the treatment, although this response is not well understood. Some authors have suggested that ill dogs show a reduction in the percentage of circulating CD4+ lymphocytes and in the CD4+/CD8+ ratio, both of which normalize after treatment and clinical recovery. The present paper discusses the variation of the different lymphocyte subpopulations (CD3, CD4, CD8, CD21) of peripheral blood mononuclear cells (PBMC) in 28 dogs diagnosed with CL and submitted to conventional treatment with meglumine antimoniate (Glucantime) for 1 month and with allopurinol (Zyloric) for 1 year, in order to evaluate the usefulness of these parameters as indicators of the immunological condition of the ill animals and of the prognosis of their evolution during the treatment. It is concluded that circulating lymphocyte subpopulations are similar in dogs with leishmaniasis and in healthy dogs and that there is no correlation between the clinical status or response to therapy and the values of the counts of the different lymphocyte subpopulations. Therefore, the percentage of different lymphocyte subpopulations cannot be used as a parameter to predict the evolution of an individual patient in a clinical context. PMID:17110042

  18. Comparison of the chemotactic responsiveness of two fibrosarcoma subpopulations of differing malignancy.

    PubMed Central

    Orr, F. W.; Varani, J.; Delikatny, J.; Jain, N.; Ward, P. A.

    1981-01-01

    There are several points of similarity between the processes of cancer metastasis and inflammation. In both, cells circulate in the vasculature, arrest, and cross vessel walls, thereby entering the extravascular tissues. In vitro, leukocytes and some, but not all, tumor cells exhibit chemotaxis. Since the chemotactic response of leukocytes effect their transvascular migration, we propose that chemotactic responsiveness contributes to the ability of circulating tumor cells to localize in extravascular tissues. This study was done to seek a relationship between chemotactic responsiveness of tumor cells and their behavior in vivo. Two subpopulations of cells were isolated from a methylcholanthrene-induced fibrosarcoma. The two cell lines were compared with regard to their biologic behavior in vivo and their chemotactic responsiveness in vitro. In vivo one subpopulation was highly malignant. An injection of 2.0 x 10(5) cells into the footpad of syngeneic mice led to the development of primary tumors in 87% of the animals and lung metastases in 61% of the animals with primary tumors. This line demonstrated chemotaxis to a factor that behaved similarly in gel filtration and showed immunologic reactivity similar to that of a previously described tumor cell chemotactic factor derived from the fifth component of complement. In contrast, an injection of the same number of cells from the second subpopulation of fibrosarcoma cells led to the development of primary tumors in only 12% of syngeneic mice, and lung metastases did not occur. Neither this subpopulation nor normal embryonic fibroblasts demonstrated chemotactic responsiveness. We postulate that the ability of tumor cells to respond to specific chemotactic stimuli may be one of the many unique properties which distinguish malignant from benign tumor cells. This is the first report documenting the chemotactic responsiveness of non-ascites tumors and fibrosarcomas. PMID:7468766

  19. Maternal cigarette smoking and its effect on neonatal lymphocyte subpopulations and replication

    PubMed Central

    2013-01-01

    Background Significant immunomodulatory effects have been described as result of cigarette smoking in adults and pregnant women. However, the effect of cigarette smoking during pregnancy on the lymphocyte subpopulations in newborns has been discussed, controversially. Methods In a prospective birth cohort, we analyzed the peripheral lymphocyte subpopulations of smoking (SM) and non-smoking mothers (NSM) and their newborns and the replicative history of neonatal, mostly naive CD4 + CD45RA + T cells by measurements of T-cell-receptor-excision-circles (TRECs), relative telomere lengths (RTL) and the serum cytokine concentrations. Results SM had higher lymphocyte counts than NSM. Comparing SM and NSM and SM newborns with NSM newborns, no significant differences in proportions of lymphocyte subpopulations were seen. Regardless of their smoking habits, mothers had significantly lower naive T cells and higher memory and effector T cells than newborns. NSM had significantly lower percentages of CD4 + CD25++ T cells compared to their newborns, which was not significant in SM. There were no differences regarding cytokine concentrations in newborns of SM and NSM. However, NSM had significantly higher Interleukin-7 concentrations than their newborns. Regardless of smoking habits of mothers, newborns had significantly longer telomeres and higher TRECs than their mothers. Newborns of SM had significantly longer telomeres than newborns of NSM. Conclusions Apart from higher lymphocyte counts in SM, our results did not reveal differences between lymphocyte subpopulations of SM and NSM and their newborns, respectively. Our finding of significantly longer RTL in newborns of SM may reflect potential harm on lymphocytes, such as cytogenetic damage induced by smoking. PMID:23597118

  20. Characterization of radiation resistant hypoxic cell subpopulations in KHT sarcomas. (II). Cell sorting.

    PubMed Central

    Siemann, D. W.; Keng, P. C.

    1988-01-01

    Hypoxic cells in KHT sarcomas were characterized using fluorescence activated cell sorting based on the diffusion properties of the fluorochrome Hoechst 33342. Tumour-bearing female C3H/HeJ mice were injected i.v. with 10 micrograms g-1 Hoechst 33342 and the cells derived from the tumours sorted on the basis of their staining intensities. For each sorted fraction the DNA histogram was evaluated using FCM analysis. The results indicated that the bright and dim cells were not equally distributed about the cell cycle. For example, a greater proportion of S phase cells were in the bright subpopulations whereas the dim subpopulations contained an increased proportion of cells in G1. When the tumours were irradiated with a single dose of radiation prior to cell sorting, the dim cells survived preferentially. Dose response curves for the 20% most dim and 20% most bright cells, sorted on the basis of fluorescence intensity, then were determined. The survival curves of the dim and bright cells were found to have slopes similar to those of KHT cells irradiated in situ in dead animals or in vitro under fully oxic conditions, respectively. In addition, when KHT sarcoma-bearing mice were given a 2.5 mmol kg-1 dose of misonidazole (MISO) prior to irradiation and cell sorting, the dim subpopulation was sensitized whereas the bright subpopulation was not. These findings suggest that (i) compared to well-oxygenated areas, hypoxic regions of KHT tumours contain a smaller percentage of cells actively proliferating and (ii) Hoechst 33342 sorting may allow the detailed in situ evaluation of agents acting directly against hypoxic cells in solid tumours. PMID:3179180

  1. Heritability of Susceptibility to Ionizing Radiation-Induced Apoptosis of Human Lymphocyte Subpopulations

    SciTech Connect

    Schmitz, Annette; Dechamps, Nathalie; Goldin, Lynn; Thomas, Gilles

    2007-07-15

    Purpose: To evaluate the heritability of intrinsic radiosensitivity, the induction of apoptosis in lymphocyte subpopulations was determined on samples from related individuals belonging to large kindred families. Methods and Materials: Quiescent lymphocytes from 334 healthy individuals were gamma-irradiated in vitro. Apoptosis was determined 18 h after irradiation by eight-color flow cytometry. Radiosensitivity was quantified from dose-effect curves. Intrafamilial correlations and heritability were computed for 199 father-mother-offspring trios using the programs SOLAR (Sequential Oligogenic Linkage Analysis Routines) and SAGE (Statistical Analysis for Genetic Epidemiology). Segregation analyses were conducted using SAGE. Results: Marked differential susceptibility of naive and memory T lymphocytes was demonstrated. Also, although age and gender were significant covariates, their effects only accounted for a minor part of the inter-individual variation. Parent-offspring and sib-sib correlations were significant for the radiosensitivity of B cells, T4, and T8 and of effector memory T4 and T8 subpopulations. In the T4-effector memory subpopulation, the phenotype showed correlations most consistent with dominant or additive genetic effects, and the results of the segregation analysis were consistent with the contribution of a bi-allelic dominant locus. Conclusions: Heritability was demonstrated for the susceptibility to ionizing radiation-induced apoptosis of lymphocyte populations, and the segregation of the T4-effector memory radiosensitivity phenotype was consistent with a simple mendelian transmission model involving one major gene.

  2. Metabolite Profiling and Stable Isotope Tracing in Sorted Subpopulations of Mammalian Cells.

    PubMed

    Roci, Irena; Gallart-Ayala, Hector; Schmidt, Angelika; Watrous, Jeramie; Jain, Mohit; Wheelock, Craig E; Nilsson, Roland

    2016-03-01

    Biological samples such as tissues, blood, or tumors are often complex and harbor heterogeneous populations of cells. Separating out specific cell types or subpopulations from such complex mixtures to study their metabolic phenotypes is challenging because experimental procedures for separation may disturb the metabolic state of cells. To address this issue, we developed a method for analysis of cell subpopulations using stable isotope tracing and fluorescence-activated cell sorting followed by liquid chromatography-high-resolution mass spectrometry. To ensure a faithful representation of cellular metabolism after cell sorting, we benchmarked sorted extraction against direct extraction. While peak areas differed markedly with lower signal for amino acids but higher signal for nucleotides, mass isotopomer distributions from sorted cells were generally in good agreement with those obtained from direct extractions, indicating that they reflect the true metabolic state of cells prior to sorting. In proof-of-principle studies, our method revealed metabolic phenotypes specific to T cell subtypes, and also metabolic features of cells in the committed phase of the cell division cycle. Our approach enables studies of a wide range of adherent and suspension cell subpopulations, which we anticipate will be of broad importance in cell biology and biomedicine. PMID:26855138

  3. Activation of a distinct subpopulation of pulmonary macrophages following exposure to biological response modifiers.

    PubMed

    Drath, D B; Do, C; Burd, T; Hong, L L

    1994-03-01

    A distinct subpopulation of tissue-associated pulmonary macrophages (TAPM) displayed tumoricidal activity towards syngeneic and xenogeneic targets following in vitro incubation with N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP). This subpopulation, as well as, the predominant population of freely lavagable alveolar macrophages destroyed allogeneic targets following a similar incubation with either 6-0-stearoyl MDP (S-MDP) or recombinant interferon-gamma (IFN-gamma). IFN-gamma-induced in vivo tumoricidal activation of both populations of pulmonary macrophage was most effective when delivered either intravenously or via osmotic minipump infusion and least effective when administered by direct intratracheal instillation. The separate populations also displayed in vivo activation in response to liposome-encapsulated i.v. administered S-MDP. Under comparable conditions, IFN-alpha was not nearly as effective. Metabolic activation of TAPM, assessed by the release of increased levels of superoxide free radicals during phagocytosis, occurred following 24 hr exposure to S-MDP or lipopolysaccharide. Incorporation of these agents into multilamellar vesicle liposomes further augmented the release of superoxide observed at 24 hrs. Our results collectively demonstrated that a subpopulation of lung macrophage, a tissue-associated pulmonary macrophage, may be activated to a tumoricidal state and to release pronounced levels of oxygen free radicals following either in vitro or in situ treatment with several biological response modifiers. PMID:8194852

  4. Pulmonary and thoracic macrophage subpopulations and clearance of particles from the lung.

    PubMed Central

    Lehnert, B E

    1992-01-01

    Pulmonary macrophages consist of several subpopulations that can be defined by their anatomical locations as well as by other criteria. In addition to the well-known alveolar macrophages that reside on the alveolar surface, pulmonary macrophages also occur in the conducting airways, in various pulmonary interstitial regions, and, in some mammalian species, in the lung's intravascular compartment. Other thoracic macrophages of relevance to pulmonary defense and some lung disease processes are the pleural macrophages resident in the pleural space and macrophages present in regional lymph nodes that receive lymphatic drainage from the lung. Of the above subpopulations of pulmonary and thoracic macrophages, the alveolar macrophages have received the most experimental attention in the context of the pulmonary clearance and retention of deposited particles. Accordingly, less information is currently available regarding the roles other pulmonary and thoracic populations of macrophages may play in the removal of particles from the lower respiratory tract and associated tissue compartments. This report provides an overview of the various subpopulations of pulmonary and thoracic macrophages, as defined by their anatomical locations. The known and postulated roles of macrophages in the pulmonary clearance and retention of particles are reviewed, with particular emphasis on macrophage-associated processes involved in the pulmonary clearance of relatively insoluble particles. Images FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 5. FIGURE 8. FIGURE 12. FIGURE 14. FIGURE 15. FIGURE 16. FIGURE 17. FIGURE 18. FIGURE 19. A FIGURE 19. B FIGURE 21. FIGURE 22. PMID:1396454

  5. The subpopulation of microglia expressing functional muscarinic acetylcholine receptors expands in stroke and Alzheimer's disease.

    PubMed

    Pannell, Maria; Meier, Maria Almut; Szulzewsky, Frank; Matyash, Vitali; Endres, Matthias; Kronenberg, Golo; Prinz, Vincent; Waiczies, Sonia; Wolf, Susanne A; Kettenmann, Helmut

    2016-03-01

    Microglia undergo a process of activation in pathology which is controlled by many factors including neurotransmitters. We found that a subpopulation (11 %) of freshly isolated adult microglia respond to the muscarinic acetylcholine receptor agonist carbachol with a Ca(2+) increase and a subpopulation of similar size (16 %) was observed by FACS analysis using an antibody against the M3 receptor subtype. The carbachol-sensitive population increased in microglia/brain macrophages isolated from tissue of mouse models for stroke (60 %) and Alzheimer's disease (25 %), but not for glioma and multiple sclerosis. Microglia cultured from adult and neonatal brain contained a carbachol-sensitive subpopulation (8 and 9 %), which was increased by treatment with interferon-γ to around 60 %. This increase was sensitive to blockers of protein synthesis and correlated with an upregulation of the M3 receptor subtype and with an increased expression of MHC-I and MHC-II. Carbachol was a chemoattractant for microglia and decreased their phagocytic activity. PMID:25523105

  6. ODE constrained mixture modelling: a method for unraveling subpopulation structures and dynamics.

    PubMed

    Hasenauer, Jan; Hasenauer, Christine; Hucho, Tim; Theis, Fabian J

    2014-07-01

    Functional cell-to-cell variability is ubiquitous in multicellular organisms as well as bacterial populations. Even genetically identical cells of the same cell type can respond differently to identical stimuli. Methods have been developed to analyse heterogeneous populations, e.g., mixture models and stochastic population models. The available methods are, however, either incapable of simultaneously analysing different experimental conditions or are computationally demanding and difficult to apply. Furthermore, they do not account for biological information available in the literature. To overcome disadvantages of existing methods, we combine mixture models and ordinary differential equation (ODE) models. The ODE models provide a mechanistic description of the underlying processes while mixture models provide an easy way to capture variability. In a simulation study, we show that the class of ODE constrained mixture models can unravel the subpopulation structure and determine the sources of cell-to-cell variability. In addition, the method provides reliable estimates for kinetic rates and subpopulation characteristics. We use ODE constrained mixture modelling to study NGF-induced Erk1/2 phosphorylation in primary sensory neurones, a process relevant in inflammatory and neuropathic pain. We propose a mechanistic pathway model for this process and reconstructed static and dynamical subpopulation characteristics across experimental conditions. We validate the model predictions experimentally, which verifies the capabilities of ODE constrained mixture models. These results illustrate that ODE constrained mixture models can reveal novel mechanistic insights and possess a high sensitivity. PMID:24992156

  7. Deconvolution of Ensemble Chromatin Interaction Data Reveals the Latent Mixing Structures in Cell Subpopulations.

    PubMed

    Sefer, Emre; Duggal, Geet; Kingsford, Carl

    2016-06-01

    Chromosome conformation capture (3C) experiments provide a window into the spatial packing of a genome in three dimensions within the cell. This structure has been shown to be correlated with gene regulation, cancer mutations, and other genomic functions. However, 3C provides mixed measurements on a population of typically millions of cells, each with a different genome structure due to the fluidity of the genome and differing cell states. Here, we present several algorithms to deconvolve these measured 3C matrices into estimations of the contact matrices for each subpopulation of cells and relative densities of each subpopulation. We formulate the problem as that of choosing matrices and densities that minimize the Frobenius distance between the observed 3C matrix and the weighted sum of the estimated subpopulation matrices. Results on HeLa 5C and mouse and bacteria Hi-C data demonstrate the methods' effectiveness. We also show that domain boundaries from deconvolved matrices are often more enriched or depleted for regulatory chromatin markers when compared to boundaries from convolved matrices. PMID:27267775

  8. Probing diversity within subpopulations of locomotor-related V0 interneurons.

    PubMed

    Griener, Anna; Zhang, Wei; Kao, Henry; Wagner, Christine; Gosgnach, Simon

    2015-11-01

    The V0 interneuronal population is derived from Dbx1 expressing progenitors. Initial studies on these interneurons in the mouse spinal cord demonstrated that they project commissural axons and are involved in coordinating left-right alternation during locomotion. Subsequent work has indicated that the V0 population can be divided into genetically distinct ventral (V0V) and dorsal (V0D) subpopulations, and experimental evidence suggests that each is responsible for left-right alternation at different locomotor speeds. In this study, we perform a series of experiments to probe the location and connectivity of these subpopulations in neonatal mice and demonstrate that they are more diverse than previously predicted. While the distribution of either subpopulation remains consistent along the extent of the lumbar spinal cord, a cluster of V0D cells lateral to the central canal receive substantial input from primary afferents. Retrograde tracing and activity dependent labeling experiments demonstrate that a group of V0 interneurons located in this same region preferentially project axons towards contralateral motoneurons via an oligosynaptic pathway, and are active during fictive locomotion. Our results suggest that this subset of V0 interneurons may be primarily responsible for coordination of left-right alternation during locomotion. Furthermore these experiments indicate that while genetic identity is one determinant of the function of a neuron during locomotion, the specific position in which the cell is located may also play a key role. PMID:25649879

  9. Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis.

    PubMed

    Wang, Youji; Hu, Menghong; Chiang, M W L; Shin, P K S; Cheung, S G

    2012-03-01

    The green-lipped mussel Perna viridis is distributed widely in the estuarine and coastal areas of the Indo-Pacific region and extensively cultured as an inexpensive protein source. Morphology and immunological activities of hemocytes of P. viridis were investigated using flow cytometry and light and electron microscopy. Three major types of hemocytes were identified in the hemolymph, including dense-granulocyte, semi-granulocyte (small and large size) and hyalinocyte. Other hemocytes, which occurred in low numbers, included granulocytes with different electron-dense/lucent granules and hemoblast-like cells. Based on flow cytometry, two subpopulations were identified. Granulocytes were larger cells, and the more abundant, containing numerous granules in the cytoplasm, and hyalinocytes were the smaller and less abundant with the fewest granules. Flow cytometry revealed that the granulocytes were more active in cell phagocytosis, contained the higher lysosomal content, and showed higher esterase activity and reactive oxygen species (ROS) generation compared with hyalinocytes. Immune functions assessed by the flow cytometry indicated that the granulocytes were the main hemocytes involved in the cellular defence in P. viridis. PMID:21982876

  10. Flow-cytometric evaluation of lymphocyte subpopulations in synchronously developing Schistosoma mansoni egg and Sephadex bead pulmonary granulomas.

    PubMed Central

    Remick, D. G.; Chensue, S. W.; Hiserodt, J. C.; Higashi, G. I.; Kunkel, S. L.

    1988-01-01

    Synchronous models of T-cell-mediated and foreign body granulomas were induced in mice by intravenous embolization of Schistosoma mansoni eggs and Sephadex beads, respectively. The authors then performed flow-cytometric analysis of lymphocytes from dispersed granulomas, spleens, and peripheral blood at 4, 8, 16, and 32 days corresponding to the induction, growth, and maintenance, and resolution of these lesions. Lymphocytes were identified on the basis of light scatter characteristics, and the nature of the cells was confirmed by cell sorting and electron-microscopic examination. Lymphocyte subpopulations were characterized with antibodies to lymphocyte surface markers, specifically Ig, Thy 1.2, Lyt 1, Lyt 2, and L3T4. Natural killer cells were identified with anti-asialo GM1. Egg-induced granulomas had more lymphocytes of all phenotypes at all time points. Surprisingly, there was a significant number of cells staining positive for asialo GM1. On Day 16 after embolization there was a greater percentage of helper T cells, as defined by positive staining with L3T4, in the egg model, compared with the bead model. There was no obvious shift of lymphocytes from either the blood or spleen into the granuloma. These data confirm the importance of T cells in the direct participation of granulomatous inflammation, and the large numbers of asialo GM1-positive cells suggest a role for natural killer cells. Images Figure 4 PMID:2451888

  11. Genomewide identification of genes under directional selection: gene transcription Q(ST) scan in diverging Atlantic salmon subpopulations.

    PubMed

    Roberge, C; Guderley, H; Bernatchez, L

    2007-10-01

    Evolutionary genomics has benefited from methods that allow identifying evolutionarily important genomic regions on a genomewide scale, including genome scans and QTL mapping. Recently, genomewide scanning by means of microarrays has permitted assessing gene transcription differences among species or populations. However, the identification of differentially transcribed genes does not in itself suffice to measure the role of selection in driving evolutionary changes in gene transcription. Here, we propose and apply a "transcriptome scan" approach to investigating the role of selection in shaping differential profiles of gene transcription among populations. We compared the genomewide transcription levels between two Atlantic salmon subpopulations that have been diverging for only six generations. Following assessment of normality and unimodality on a gene-per-gene basis, the additive genetic basis of gene transcription was estimated using the animal model. Gene transcription h(2) estimates were significant for 1044 (16%) of all detected cDNA clones. In an approach analogous to that of genome scans, we used the distribution of the Q(ST) values estimated from intra- and intersubpopulation additive genetic components of the transcription profiles to identify 16 outlier genes (average Q(ST) estimate = 0.11) whose transcription levels are likely to have evolved under the influence of directional selection within six generations only. Overall, this study contributes both empirically and methodologically to the quantitative genetic exploration of gene transcription data. PMID:17720934

  12. EDAC: Epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals.

    PubMed

    Kajita, Mihoko; Fujita, Yasuyuki

    2015-07-01

    During embryonic development or under certain pathological conditions, viable but suboptimal cells are often eliminated from the cellular society through a process termed cell competition. Cell competition was originally identified in Drosophila where cells with different properties compete for survival; 'loser' cells are eliminated from tissues and consequently 'winner' cells become dominant. Recent studies have shown that cell competition also occurs in mammals. While apoptotic cell death is the major fate for losers in Drosophila, outcompeted cells show more variable phenotypes in mammals, such as cell death-independent apical extrusion and cellular senescence. Molecular mechanisms underlying these processes have been recently revealed. Especially, in epithelial tissues, normal cells sense and actively eliminate the neighbouring transformed cells via cytoskeletal proteins by the process named epithelial defence against cancer (EDAC). Here, we introduce this newly emerging research field: cell competition in mammals. PMID:25991731

  13. Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells

    PubMed Central

    Liu, Yang; Jiang, Bi-Jie; Zhao, Run-Zhen; Ji, Hong-Long

    2016-01-01

    Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na+ channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium. PMID:27570489

  14. Porphyromonas gingivalis Fimbriae Bind to Cytokeratin of Epithelial Cells

    PubMed Central

    Sojar, Hakimuddin T.; Sharma, Ashu; Genco, Robert J.

    2002-01-01

    The adherence of Porphyromonas gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis-induced periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of P. gingivalis fimbriae. Using an overlay assay with purified P. gingivalis fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which P. gingivalis fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that P. gingivalis fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified P. gingivalis fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial

  15. Phenotypical and functional characterization of alveolar macrophage subpopulations in the lungs of NO2-exposed rats

    PubMed Central

    Garn, Holger; Siese, Anette; Stumpf, Sabine; Wensing, Anka; Renz, Harald; Gemsa, Diethard

    2006-01-01

    Background Alveolar macrophages (AM) are known to play an important role in the regulation of inflammatory reactions in the lung, e.g. during the development of chronic lung diseases. Exposure of rats to NO2 has recently been shown to induce a shift in the activation type of AM that is characterized by reduced TNF-α and increased IL-10 production. So far it is unclear, whether a functional shift in the already present AM population or the occurrence of a new, phenotypically different AM population is responsible for these observations. Methods AM from rat and mice were analyzed by flow cytometry for surface marker expression and in vivo staining with PKH26 was applied to characterize newly recruited macrophages. Following magnetic bead separation, AM subpopulations were further analyzed for cytokine, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP) mRNA expression using quantitative RT-PCR. Following in vitro stimulation, cytokines were quantitated in the culture supernatants by ELISA. Results In untreated rats the majority of AM showed a low expression of the surface antigen ED7 (CD11b) and a high ED9 (CD172) expression (ED7-/ED9high). In contrast, NO2 exposure induced the occurrence of a subpopulation characterized by the marker combination ED7+/ED9low. Comparable changes were observed in mice and by in vivo labeling of resident AM using the dye PKH26 we could demonstrate that CD11b positive cells mainly comprise newly recruited AM. Subsequent functional analyses of separated AM subpopulations of the rat revealed that ED7+ cells showed an increased expression and production of the antiinflammatory cytokine IL-10 whereas TNF-α production was lower compared to ED7- AM. However, iNOS and IL-12 expression were also increased in the ED7+ subpopulation. In addition, these cells showed a significantly higher mRNA expression for the matrix metalloproteinases MMP-7, -8, -9, and -12. Conclusion NO2 exposure induces the infiltration of an AM subpopulation

  16. Intestinal epithelial dysplasia (tufting enteropathy).

    PubMed

    Goulet, Olivier; Salomon, Julie; Ruemmele, Frank; de Serres, Natacha Patey-Mariaud; Brousse, Nicole

    2007-01-01

    Intestinal epithelial dysplasia (IED), also known as tufting enteropathy, is a congenital enteropathy presenting with early-onset severe intractable diarrhea causing sometimes irreversible intestinal failure. To date, no epidemiological data are available, however, the prevalence can be estimated at around 1/50,000-100,000 live births in Western Europe. The prevalence seems higher in areas with high degree of consanguinity and in patients of Arabic origin. Infants develop within the first days after birth a watery diarrhea persistent in spite of bowel rest and parenteral nutrition. Some infants are reported to have associated choanal rectal or esophageal atresia. IED is thought to be related to abnormal enterocytes development and/or differentiation. Nonspecific punctuated keratitis was reported in more than 60% of patients. Histology shows various degree of villous atrophy, with low or without mononuclear cell infiltration of the lamina propria but specific histological abnormalities involving the epithelium with disorganization of surface enterocytes with focal crowding, resembling tufts. Several associated specific features were reported, including abnormal deposition of laminin and heparan sulfate proteoglycan (HSPG) in the basement membrane, increased expression of desmoglein and ultrastructural changes in the desmosomes, and abnormal distribution of alpha2beta1 integrin adhesion molecules. One model of transgenic mice in which the gene encoding the transcription factor Elf3 is disrupted have morphologic features resembling IED. Parental consanguinity and/or affected siblings suggest an autosomal recessive transmission but the causative gene(s) have not been yet identified making prenatal diagnosis unavailable. Some infants have a milder phenotype than others but in most patients, the severity of the intestinal malabsorption even with enteral feeding make them totally dependent on daily long-term parenteral nutrition with a subsequent risk of complications

  17. LIM Homeobox Domain 2 Is Required for Corneal Epithelial Homeostasis

    PubMed Central

    Sartaj, Rachel; Chee, Ru‐ik; Yang, Jing; Wan, Pengxia; Liu, Aihong; Guaiquil, Victor; Fuchs, Elaine

    2016-01-01

    Abstract The cornea requires constant epithelial renewal to maintain clarity for appropriate vision. A subset of stem cells residing at the limbus is primarily responsible for maintaining corneal epithelium homeostasis. Trauma and disease may lead to stem cell deficiency and therapeutic targeting to replenish the stemness capacity has been stalled by the lack of reliable corneal epithelial stem cell markers. Here we identified the location of Lhx2 in mice (mLhx2) cornea and conjunctival tissue using an Lhx2eGFP reporter model and in human tissues (hLHX2). Lhx2 localized to the basal cells of central cornea, the conjunctiva and the entire limbal epithelium in humans and mice. To ascribe a functional role we generated Lhx2 conditional knockout (cKO) mice and the phenotypic effects in corneas were analyzed by slit lamp microscopy, in cell‐based assays and in a model of corneal epithelium debridement. Immunodetection on corneal sections were used to visualize conjunctivalization, a sign of limbal barrier failure. Lhx2cKO mice produced reduced body hair and spontaneous epithelial defects in the cornea that included neovascularization, perforation with formation of scar tissue and opacification. Cell based assays showed that Lhx2cKO derived corneal epithelial cells have a significantly lower capacity to form colonies over time and delayed wound‐healing recovery when compared to wildtype cells. Repeated corneal epithelial wounding resulted in decreased re‐epithelialization and multiple cornea lesions in Lhx2cKO mice compared to normal recovery seen in wildtype mice. We conclude that Lhx2 is required for maintenance of the corneal epithelial cell compartment and the limbal barrier. Stem Cells 2016;34:493–503 PMID:26661907

  18. LIM Homeobox Domain 2 Is Required for Corneal Epithelial Homeostasis.

    PubMed

    Sartaj, Rachel; Chee, Ru-ik; Yang, Jing; Wan, Pengxia; Liu, Aihong; Guaiquil, Victor; Fuchs, Elaine; Rosenblatt, Mark I

    2016-02-01

    The cornea requires constant epithelial renewal to maintain clarity for appropriate vision. A subset of stem cells residing at the limbus is primarily responsible for maintaining corneal epithelium homeostasis. Trauma and disease may lead to stem cell deficiency and therapeutic targeting to replenish the stemness capacity has been stalled by the lack of reliable corneal epithelial stem cell markers. Here we identified the location of Lhx2 in mice (mLhx2) cornea and conjunctival tissue using an Lhx2eGFP reporter model and in human tissues (hLHX2). Lhx2 localized to the basal cells of central cornea, the conjunctiva and the entire limbal epithelium in humans and mice. To ascribe a functional role we generated Lhx2 conditional knockout (cKO) mice and the phenotypic effects in corneas were analyzed by slit lamp microscopy, in cell-based assays and in a model of corneal epithelium debridement. Immunodetection on corneal sections were used to visualize conjunctivalization, a sign of limbal barrier failure. Lhx2cKO mice produced reduced body hair and spontaneous epithelial defects in the cornea that included neovascularization, perforation with formation of scar tissue and opacification. Cell based assays showed that Lhx2cKO derived corneal epithelial cells have a significantly lower capacity to form colonies over time and delayed wound-healing recovery when compared to wildtype cells. Repeated corneal epithelial wounding resulted in decreased re-epithelialization and multiple cornea lesions in Lhx2cKO mice compared to normal recovery seen in wildtype mice. We conclude that Lhx2 is required for maintenance of the corneal epithelial cell compartment and the limbal barrier. PMID:26661907

  19. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    SciTech Connect

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  20. The Epithelial Cell in Lung Health and Emphysema Pathogenesis

    PubMed Central

    Mercer, Becky A.; Lemaître, Vincent; Powell, Charles A.; D’Armiento, Jeanine

    2009-01-01

    Cigarette smoking is the primary cause of the irreversible lung disease emphysema. Historically, inflammatory cells such as macrophages and neutrophils have been studied for their role in emphysema pathology. However, recent studies indicate that the lung epithelium is an active participant in emphysema pathogenesis and plays a critical role in the lung’s response to cigarette smoke. Tobacco smoke increases protease production and alters cytokine expression in isolated epithelial cells, suggesting that these cells respond potently even in the absence of a complete inflammatory program. Tobacco smoke also acts as an immunosuppressant, reducing the defense function of airway epithelial cells and enhancing colonization of the lower airways. Thus, the paradigm that emphysema is strictly an inflammatory-cell based disease is shifting to consider the involvement of resident epithelial cells. Here we review the role of epithelial cells in lung development and emphysema. To better understand tobacco-epithelial interactions we performed microarray analyses of RNA from human airway epithelial cells exposed to smoke extract for 24 hours. These studies identified differential regulation of 425 genes involved in diverse biological processes, such as apoptosis, immune function, cell cycle, signal transduction, proliferation, and antioxidants. Some of these genes, including VEGF, glutathione peroxidase, IL-13 receptor, and cytochrome P450, have been previously reported to be altered in the lungs of smokers. Others, such as pirin, cathepsin L, STAT1, and BMP2, are shown here for the first time to have a potential role in smoke-associated injury. These data broaden our understanding of the importance of epithelial cells in lung health and cigarette smoke-induced emphysema. PMID:19662102

  1. Normal cycling patterns of hematopoietic stem cell subpopulations: an assay using long-term in vivo BrdU infusion

    SciTech Connect

    Pietrzyk, M.E.; Priestley, G.V.; Wolf, N.S.

    1985-12-01

    It was found in a long-term bromodeoxyuridine (BrdU) infusion study that two or more different subpopulations of bone marrow stem cells exist in mice. One of these subpopulations appears to be noncycling and forms approximately 10% of eight-day CFU-S. Another one, a subpopulation of slowly cycling bone marrow cells, is represented as 14-day CFU-S. The 14-day CFU-S have a regular increment in the percentage of the subpopulation entering the cycle over time, with a cell generation half-time of 21 days. The cycling status in these experiments was ascertained by in vivo continuous long-term BrdU infusion. An improved method is presented for long-term BrdU infusion with UV killing of cycled cells.

  2. Optical Coherence Tomography Compared With Colposcopy for Assessment of Vaginal Epithelial Damage: A Randomized Controlled Trial

    PubMed Central

    Vincent, Kathleen L.; Stanberry, Lawrence R.; Moench, Thomas R.; Breitkopf, Carmen Radecki; Loza, Melissa L.; Wei, Jingna; Grady, James; Paull, Jeremy; Motamedi, Massoud; Rosenthal, Susan L.

    2011-01-01

    Objective Colposcopy has been used to detect epithelial damage with vaginal microbicides. In animal models, optical coherence tomography (OCT) provided increased sensitivity over colposcopy in detecting epithelial injury. This randomized double-blinded clinical study compared OCT to colposcopy for the evaluation of epithelial injury in women using placebo or nonoxynol-9. Methods Thirty women aged 18–45 were randomized to use hydroxyethyl cellulose placebo or nonoxynol-9 vaginal gel twice daily for 5.5 days. Imaging with colposcopy and OCT was performed prior to product use, after the last dose, and 1 week later. Colposcopy was graded using standard criteria. OCT images were scored for epithelial integrity based on a published scoring system and measured for epithelial thickness. Results Colposcopy findings and OCT scores and epithelial thicknesses were similar between treatment groups at baseline. After treatment, there were significant differences between the nonoxynol-9 (1.37) and control group (1.15) OCT scores (p<0.001, indicating epithelial injury, and there was epithelial thinning in the nonoxynol-9 group (237μm) compared to the control group (292μm) (p=0.008). There were no significant posttreatment colposcopic differences in epithelial disruption between treatment groups, with only increased erythema noted after nonoxynol-9 use (p=0.02). Conclusion OCT detected epithelial disruption and thinning not identified by colposcopy. Vaginal epithelial thickness, a measure previously available only through biopsy, decreased after nonoxynol-9 use, a finding that may contribute to increased susceptibility to HIV after frequent use. OCT shows promise for the noninvasive clinical assessment of vaginal epithelial damage. Clinical Trial Registration UMIN Clinical Trials Registry, www.umin.ac.jp/ctr/index.htm, R000006186. PMID:22105265

  3. Plasmolipin—a new player in endocytosis and epithelial development

    PubMed Central

    Le Guelte, Armelle; Macara, Ian G

    2015-01-01

    Polarized vesicle sorting is essential not only for epithelial cell function but also for cell polarization and tissue morphogenesis. Endocytosis is a key determinant of the surface abundance of plasma membrane proteins and is highly regulated. In an important recent paper, Rodríguez-Fraticelli et al (4) identify a new player in apical endocytosis—a previously uncharacterized protein called Plasmolipin. They report not only its mechanism of action through binding to an epsin, but also highlight an essential role in regulating Notch signaling, which controls epithelial differentiation. PMID:25825384

  4. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  5. Interferons Mediate Terminal Differentiation of Human Cortical Thymic Epithelial Cells

    PubMed Central

    Vidalain, Pierre-Olivier; Laine, David; Zaffran, Yona; Azocar, Olga; Servet-Delprat, Christine; Wild, T. Fabian; Rabourdin-Combe, Chantal; Valentin, Hélène

    2002-01-01

    In the thymus, epithelial cells comprise a heterogeneous population required for the generation of functional T lymphocytes, suggesting that thymic epithelium disruption by viruses may compromise T-cell lymphopoiesis in this organ. In a previous report, we demonstrated that in vitro, measles virus induced differentiation of cortical thymic epithelial cells as characterized by (i) cell growth arrest, (ii) morphological and phenotypic changes, and (iii) apoptotis as a final step of this process. In the present report, we have analyzed the mechanisms involved. First, measles virus-induced differentiation of thymic epithelial cells is shown to be strictly dependent on beta interferon (IFN-β) secretion. In addition, transfection with double-stranded RNA, a common intermediate of replication for a broad spectrum of viruses, is reported to similarly mediate thymic epithelial cell differentiation through IFN-β induction. Finally, we demonstrated that recombinant IFN-α, IFN-β, or IFN-γ was sufficient to induce differentiation and apoptosis of uninfected thymic epithelial cells. These observations suggested that interferon secretion by either infected cells or activated leukocytes, such as plasmacytoid dendritic cells or lymphocytes, may induce thymic epithelium disruption in a pathological context. Thus, we have identified a new mechanism that may contribute to thymic atrophy and altered T-cell lymphopoiesis associated with many infections. PMID:12050353

  6. Epithelial impedance analysis in experimentally induced colon cancer.

    PubMed Central

    Davies, R J; Joseph, R; Kaplan, D; Juncosa, R D; Pempinello, C; Asbun, H; Sedwitz, M M

    1987-01-01

    Epithelial impedance analysis was used to measure the alterations in resistance of the large bowel in a murine model of large bowel cancer. The technique was able to resolve the epithelial resistance from the total resistance of the bowel wall. A progressive decrease in resistance of the bowel epithelium occurs during carcinogenesis induced with dimethyhydrazine. About a 21% decrease in epithelial resistance from 22.0 +/- 1.3 omega.cm-2 to 17.5 +/- 1.1 omega cm-2 (p less than 0.025) was observed after 20 wk of carcinogen administration. The sensitivity of the technique in detecting altered epithelial resistance in premalignant bowel mucosa was improved by examining the impedance profile in a sodium-free Ringer's solution where the epithelium of control colons had a resistance of 24.4 +/- 1.8 omega.cm-2 compared with 19.0 +/- 1.1 omega.cm-2 (p less than 0.02) in colons from animals treated for only 4 wk with the carcinogen. Epithelial impedance analysis would seem to be a sensitive technique capable of identifying changes in the electrical properties or the large bowel early in disease states. PMID:3427187

  7. Porphyromonas gingivalis invades oral epithelial cells in vitro.

    PubMed

    Sandros, J; Papapanou, P; Dahlén, G

    1993-05-01

    The aim of the present study was to analyze the adhesive and invasive potential of a number of P. gingivalis strains, in an in vitro system utilizing cultures of human oral epithelial cells (KB cell line, ATCC CCL 17). P. gingivalis strains W50 and FDC 381 (laboratory strains) and OMGS 1738, 1743 and 1439 (clinical isolates) as well as E. coli strain HB 101 (non-adhering, non-invasive control) were used. Adherence was assessed by means of scintillation counting and light microscopy, after incubation of radiolabelled bacteria with epithelial cells. In the invasion assay, monolayers were infected with the P. gingivalis and E. coli strains and further incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). Invasion was evaluated by (i) assessing presence of bacteria surviving the antibiotic treatment, and (ii) electron microscopy. All P. gingivalis strains adhered to and entered into the oral epithelial cells. After 3 hours of incubation, bacteria were frequently identified intracellularly by means of electron microscopy. The cellular membranes, encapsulating the microorganisms in early stages of the invasive process, appeared later to disintegrate. The presence of coated pits on the epithelial cell surfaces suggested that internalization of P. gingivalis was associated with receptor-mediated endocytosis (RME). Formation of outer membrane vesicles (blebs) by intracellular bacteria indicated that internalized P. gingivalis was able to retain its viability. E. coli strain HB 101 neither adhered to nor invaded epithelial cells. PMID:8388449

  8. Lingual Epithelial Stem Cells and Organoid Culture of Them

    PubMed Central

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-01

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine. PMID:26828484

  9. Neurofilament protein is differentially distributed in subpopulations of corticocortical projection neurons in the macaque monkey visual pathways

    NASA Technical Reports Server (NTRS)

    Hof, P. R.; Ungerleider, L. G.; Webster, M. J.; Gattass, R.; Adams, M. M.; Sailstad, C. A.; Morrison, J. H.; Bloom, F. E. (Principal Investigator)

    1996-01-01

    Previous studies of the primate cerebral cortex have shown that neurofilament protein is present in pyramidal neuron subpopulations displaying specific regional and laminar distribution patterns. In order to characterize further the neurochemical phenotype of the neurons furnishing feedforward and feedback pathways in the visual cortex of the macaque monkey, we performed an analysis of the distribution of neurofilament protein in corticocortical projection neurons in areas V1, V2, V3, V3A, V4, and MT. Injections of the retrogradely transported dyes Fast Blue and Diamidino Yellow were placed within areas V4 and MT, or in areas V1 and V2, in 14 adult rhesus monkeys, and the brains of these animals were processed for immunohistochemistry with an antibody to nonphosphorylated epitopes of the medium and heavy molecular weight subunits of the neurofilament protein. Overall, there was a higher proportion of neurons projecting from areas V1, V2, V3, and V3A to area MT that were neurofilament protein-immunoreactive (57-100%), than to area V4 (25-36%). In contrast, feedback projections from areas MT, V4, and V3 exhibited a more consistent proportion of neurofilament protein-containing neurons (70-80%), regardless of their target areas (V1 or V2). In addition, the vast majority of feedback neurons projecting to areas V1 and V2 were located in layers V and VI in areas V4 and MT, while they were observed in both supragranular and infragranular layers in area V3. The laminar distribution of feedforward projecting neurons was heterogeneous. In area V1, Meynert and layer IVB cells were found to project to area MT, while neurons projecting to area V4 were particularly dense in layer III within the foveal representation. In area V2, almost all neurons projecting to areas MT or V4 were located in layer III, whereas they were found in both layers II-III and V-VI in areas V3 and V3A. These results suggest that neurofilament protein identifies particular subpopulations of

  10. NME genes in epithelial morphogenesis

    PubMed Central

    2012-01-01

    The NME family of genes encodes highly conserved multifunctional proteins that have been shown to participate in nucleic acid metabolism, energy homeostasis, cell signaling, and cancer progression. Some family members, particularly isoforms 1 and 2, have attracted extensive interests because of their potential anti-metastasis activity. Unfortunately, there have been few consensus mechanistic explanations for this critical function because of the numerous molecular functions ascribed to these proteins, including nucleoside diphosphate kinase, protein kinase, nuclease, transcription factor, growth factor, among others. In addition, different studies showed contradictory prognostic correlations between NME expression levels and tumor progression in clinical samples. Thus, analyses using pliable in vivo systems have become critical for unraveling at least some aspects of the complex functions of this family of genes. Recent works using the Drosophila genetic system have suggested a role for NME in regulating epithelial cell motility and morphogenesis, which has also been demonstrated in mammalian epithelial cell culture. This function is mediated by promoting internalization of growth factor receptors in motile epithelial cells, and the adherens junction components such as E-cadherin and β-catenin in epithelia that form the tissue linings. Interestingly, NME genes in epithelial cells appear to function in a defined range of expression levels. Either down-regulation or over-expression can perturb epithelial integrity, resulting in different aspects of epithelial abnormality. Such biphasic functions provide a plausible explanation for the documented anti-metastatic activity and the suspected oncogenic function. This review summarizes these recent findings and discusses their implications. PMID:21336542

  11. AMP-18 Targets p21 to Maintain Epithelial Homeostasis

    PubMed Central

    Chen, Peili; Li, Yan Chun; Toback, F. Gary

    2015-01-01

    Dysregulated homeostasis of epithelial cells resulting in disruption of mucosal barrier function is an important pathogenic mechanism in inflammatory bowel diseases (IBD). We have characterized a novel gastric protein, Antrum Mucosal Protein (AMP)-18, that has pleiotropic properties; it is mitogenic, anti-apoptotic and can stimulate formation of tight junctions. A 21-mer synthetic peptide derived from AMP-18 exhibits the same biological functions as the full-length protein and is an effective therapeutic agent in mouse models of IBD. In this study we set out to characterize therapeutic mechanisms and identify molecular targets by which AMP-18 maintains and restores disrupted epithelial homeostasis in cultured intestinal epithelial cells and a mouse model of IBD. Tumor necrosis factor (TNF)-α, a pro-inflammatory cytokine known to mediate gastrointestinal (GI) mucosal injury in IBD, was used to induce intestinal epithelial cell injury, and study the effects of AMP-18 on apoptosis and the cell cycle. An apoptosis array used to search for targets of AMP-18 in cells exposed to TNF-α identified the cyclin-dependent kinase inhibitor p21WAF1/CIP1. Treatment with AMP-18 blunted increases in p21 expression and apoptosis, while reversing disturbed cell cycle kinetics induced by TNF-α. AMP-18 appears to act through PI3K/AKT pathways to increase p21 phosphorylation, thereby reducing its nuclear accumulation to overcome the antiproliferative effects of TNF-α. In vitamin D receptor-deficient mice with TNBS-induced IBD, the observed increase in p21 expression in colonic epithelial cells was suppressed by treatment with AMP peptide. The results indicate that AMP-18 can maintain and/or restore the homeostatic balance between proliferation and apoptosis in intestinal epithelial cells to protect and repair mucosal barrier homeostasis and function, suggesting a therapeutic role in IBD. PMID:25919700

  12. Regulation of intestinal epithelial cells transcriptome by enteric glial cells: impact on intestinal epithelial barrier functions

    PubMed Central

    2009-01-01

    Background Emerging evidences suggest that enteric glial cells (EGC), a major constituent of the enteric nervous system (ENS), are key regulators of intestinal epithelial barrier (IEB) functions. Indeed EGC inhibit intestinal epithelial cells (IEC) proliferation and increase IEB paracellular permeability. However, the role of EGC on other important barrier functions and the signalling pathways involved in their effects are currently unknown. To achieve this goal, we aimed at identifying the impact of EGC upon IEC transcriptome by performing microarray studies. Results EGC induced significant changes in gene expression profiling of proliferating IEC after 24 hours of co-culture. 116 genes were identified as differentially expressed (70 up-regulated and 46 down-regulated) in IEC cultured with EGC compared to IEC cultured alone. By performing functional analysis of the 116 identified genes using Ingenuity Pathway Analysis, we showed that EGC induced a significant regulation of genes favoring both cell-to-cell and cell-to-matrix adhesion as well as cell differentiation. Consistently, functional studies showed that EGC induced a significant increase in cell adhesion. EGC also regulated genes involved in cell motility towards an enhancement of cell motility. In addition, EGC profoundly modulated expression of genes involved in cell proliferation and cell survival, although no clear functional trend could be identified. Finally, important genes involved in lipid and protein metabolism of epithelial cells were shown to be differentially regulated by EGC. Conclusion This study reinforces the emerging concept that EGC have major protective effects upon the IEB. EGC have a profound impact upon IEC transcriptome and induce a shift in IEC phenotype towards increased cell adhesion and cell differentiation. This concept needs to be further validated under both physiological and pathophysiological conditions. PMID:19883504

  13. Subpopulations of GFP-Marked Mouse Pancreatic β-Cells Differ in Size, Granularity, and Insulin Secretion

    PubMed Central

    Katsuta, Hitoshi; Aguayo-Mazzucato, Cristina; Katsuta, Rimiko; Akashi, Tomoyuki; Hollister-Lock, Jennifer; Sharma, Arun J.; Bonner-Weir, Susan

    2012-01-01

    There is growing information about the heterogeneity of pancreatic β-cells and how it relates to insulin secretion. This study used the approach of flow cytometry to sort and analyze β-cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter. Three populations of β-cells with differing GFP brightness could be identified, which were classified as GFP-low, GFP-medium, and GFP-bright. The GFP-medium population comprised about 70% of the total. The GFP-low population had less insulin secretion as determined by the reverse hemolytic plaque assay and reduced insulin gene expression. Additionally, all three subpopulations of β-cells were found in mice of varying ages (embryonic d 15.5 and postnatal wk 1–9). The three populations from the youngest had larger cells (forward scatter) and less granularity (side scatter) than those from the adults. This approach opens up new ways to advance knowledge about β-cell heterogeneity. PMID:22919061

  14. The fine structure of normal lymphocyte subpopulations--a study with monoclonal antibodies and the immunogold technique.

    PubMed Central

    Matutes, E; Catovsky, D

    1982-01-01

    The ultrastructural characteristics of normal lymphocyte subpopulations, identified by monoclonal antibodies and visualized by a colloidal gold labelled anti-mouse IgG were analysed. Our study demonstrates: (1) the major T lymphocyte subsets (OKT4+ and OKT8+) have distinct ultrastructural morphology. The majority of OKT4+ cells have a high nuclear/cytoplasmic ratio (N/C) and few cytoplasmic organelles whilst most OKT8+ cells have a low N/C ratio and numerous organelles, namely a well developed Golgi apparatus, lysosomal granules and parallel tubular arrays (PTA); (2) a unique subtype with irregular nuclear outline that resembles Sézary cells was seen in 5-10% of OKT4+ lymphocytes; (3) OKM1, a reagent that reacts with monocytes and granulocytes, is positive in a small lymphocyte subset which appears to be negative with the OKT reagents and is morphologically identical to OKT8+ cells; (4) 'hand-mirror' cells were only seen labelled with OKT8 and OKM1; (5) B lymphocytes labelled with FMC4 (anti-IA) could be distinguished from OKT3+ lymphocytes by having numerous profiles of endoplasmic reticulum (ER) and ribosomes; these were particularly prominent in lymphoplasmacytoid cells. Morphological similarities between normal T lymphocyte subsets and T neoplasias of the same membrane phenotype suggest that these disorders arise from specific T cell types present in normal peripheral blood or from common precursors. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:6185261

  15. Ion Channels in Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Palmer, Lawrence G.

    Ion channels in epithelial cells serve to move ions, and in some cases fluid, between compartments of the body. This function of the transfer of material is fundamentally different from that of the transfer of information, which is the main job of most channels in excitable cells. Nevertheless the basic construction of the channels is similar in many respects in the two tissue types. This chapter reviews the nature of channels in epithelia and discusses how their functions have evolved to accomplish the basic tasks for which they are responsible. I will focus on three channel types: epithelial Na+ channels, inward-rectifier K+ channels, and CFTR Cl- channels.

  16. Alix-mediated assembly of the actomyosin–tight junction polarity complex preserves epithelial polarity and epithelial barrier

    PubMed Central

    Campos, Yvan; Qiu, Xiaohui; Gomero, Elida; Wakefield, Randall; Horner, Linda; Brutkowski, Wojciech; Han, Young-Goo; Solecki, David; Frase, Sharon; Bongiovanni, Antonella; d'Azzo, Alessandra

    2016-01-01

    Maintenance of epithelial cell polarity and epithelial barrier relies on the spatial organization of the actin cytoskeleton and proper positioning/assembly of intercellular junctions. However, how these processes are regulated is poorly understood. Here we reveal a key role for the multifunctional protein Alix in both processes. In a knockout mouse model of Alix, we identified overt structural changes in the epithelium of the choroid plexus and in the ependyma, such as asymmetrical cell shape and size, misplacement and abnormal beating of cilia, blebbing of the microvilli. These defects culminate in excessive cell extrusion, enlargement of the lateral ventricles and hydrocephalus. Mechanistically, we find that by interacting with F-actin, the Par complex and ZO-1, Alix ensures the formation and maintenance of the apically restricted actomyosin–tight junction complex. We propose that in this capacity Alix plays a role in the establishment of apical–basal polarity and in the maintenance of the epithelial barrier. PMID:27336173

  17. Alix-mediated assembly of the actomyosin-tight junction polarity complex preserves epithelial polarity and epithelial barrier.

    PubMed

    Campos, Yvan; Qiu, Xiaohui; Gomero, Elida; Wakefield, Randall; Horner, Linda; Brutkowski, Wojciech; Han, Young-Goo; Solecki, David; Frase, Sharon; Bongiovanni, Antonella; d'Azzo, Alessandra

    2016-01-01

    Maintenance of epithelial cell polarity and epithelial barrier relies on the spatial organization of the actin cytoskeleton and proper positioning/assembly of intercellular junctions. However, how these processes are regulated is poorly understood. Here we reveal a key role for the multifunctional protein Alix in both processes. In a knockout mouse model of Alix, we identified overt structural changes in the epithelium of the choroid plexus and in the ependyma, such as asymmetrical cell shape and size, misplacement and abnormal beating of cilia, blebbing of the microvilli. These defects culminate in excessive cell extrusion, enlargement of the lateral ventricles and hydrocephalus. Mechanistically, we find that by interacting with F-actin, the Par complex and ZO-1, Alix ensures the formation and maintenance of the apically restricted actomyosin-tight junction complex. We propose that in this capacity Alix plays a role in the establishment of apical-basal polarity and in the maintenance of the epithelial barrier. PMID:27336173

  18. Downregulation of miR-205 in migrating epithelial tongue facilitates skin wound re-epithelialization by derepressing ITGA5.

    PubMed

    Wang, Tao; Zhao, Na; Long, Shuang; Ge, Lan; Wang, Aiping; Sun, Huiqin; Ran, Xinze; Zou, Zhongmin; Wang, Junping; Su, Yongping

    2016-08-01

    Keratinocyte migration is essential for re-epithelialization during skin wound healing, but the molecular mechanisms regulating this cellular response remain to be completely clarified. Here we show that keratinocyte-specific miR-205 is significantly downregulated in the leading edge of the migrating epithelial tongue after skin injury in mice. In HaCaT keratinocytes, miR-205 could be downregulated by TGF-β1 stimulation. And similar to the effect of TGF-β1, miR-205 knockdown could promote keratinocyte migration in wound scratch model in vitro. Furthermore, topical inhibition of miR-205 by administrating Pluronic gel containing antagomir-205 could accelerate re-epithelialization in mouse skin wound model in vivo. Moreover, we identified integrin alpha 5 (ITGA5) as one key functional miR-205 target in the re-epithelialization process and epidermal downregulation of miR-205 may desilence ITGA5 to promote keratinocyte migration. And knockdown of ITGA5 would abolish the pro-migratory effects of miR-205 inhibition in vitro. What's more, we found dysregulation of miR-205 and its target ITGA5 in epidermis of clinical chronic wound samples with persistence of high level miR-205 and absence of ITGA5. Our findings indicate that downregulation of miR-205 in the leading migrating keratinocytes is critical for re-epithelialization and miR-205 may be a potential therapeutic target for chronic wounds. PMID:27169579

  19. Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8.

    PubMed

    Shiheido, Hirokazu; Kitagori, Koji; Sasaki, Chiyomi; Kobayashi, Shio; Aoyama, Takane; Urata, Kozue; Oku, Takuma; Hirayama, Yoshitaka; Yoshitomi, Hiroyuki; Hikida, Masaki; Yoshifuji, Hajime; Mimori, Tsuneyo; Watanabe, Takeshi; Shimizu, Jun

    2014-06-01

    BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3(+) T cells). BEND3(+) T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4(+) and CD8(+) T cells, and were also observed in cord blood. The stimulation of BEND3(+) T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3(+) T cells were higher than those by BEND3(-) T cells. The proportion of BEND3(+) T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3(+) T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3(+) T cells may be of importance and useful in understanding human T cell immunology. PMID:25400923

  20. Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8

    PubMed Central

    Shiheido, Hirokazu; Kitagori, Koji; Sasaki, Chiyomi; Kobayashi, Shio; Aoyama, Takane; Urata, Kozue; Oku, Takuma; Hirayama, Yoshitaka; Yoshitomi, Hiroyuki; Hikida, Masaki; Yoshifuji, Hajime; Mimori, Tsuneyo; Watanabe, Takeshi; Shimizu, Jun

    2014-01-01

    BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3+ T cells). BEND3+ T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4+ and CD8+ T cells, and were also observed in cord blood. The stimulation of BEND3+ T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3+ T cells were higher than those by BEND3− T cells. The proportion of BEND3+ T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3+ T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3+ T cells may be of importance and useful in understanding human T cell immunology. PMID:25400923

  1. IL-15Rα deficiency in skeletal muscle alters respiratory function and the proteome of mitochondrial subpopulations independent of changes to the mitochondrial genome.

    PubMed

    O'Connell, Grant C; Nichols, Cody; Guo, Ge; Croston, Tara L; Thapa, Dharendra; Hollander, John M; Pistilli, Emidio E

    2015-11-01

    Interleukin-15 receptor alpha knockout (IL15RαKO) mice exhibit a greater skeletal muscle mitochondrial density with an altered mitochondrial morphology. However, the mechanism and functional impact of these changes have not been determined. In this study, we characterized the functional, proteomic, and genomic alterations in mitochondrial subpopulations isolated from the skeletal muscles of IL15RαKO mice and B6129 background control mice. State 3 respiration was greater in interfibrillar mitochondria and whole muscle ATP levels were greater in IL15RαKO mice supporting the increases in respiration rate. However, the state 3/state 4 ratio was lower, suggesting some degree of respiratory uncoupling. Proteomic analyses identified several markers independently in mitochondrial subpopulations that are associated with these functional alterations. Next Generation Sequencing of mtDNA revealed a high degree of similarity between the mitochondrial genomes of IL15RαKO mice and controls in terms of copy number, consensus coding and the presence of minor alleles, suggesting that the functional and proteomic alterations we observed occurred independent of alterations to the mitochondrial genome. These data provide additional evidence to implicate IL-15Rα as a regulator of skeletal muscle phenotypes through effects on the mitochondrion, and suggest these effects are driven by alterations to the mitochondrial proteome. PMID:26458787

  2. Peripheral blood lymphocyte subpopulations and immunoglobulin concentrations in healthy foals and foals with Rhodococcus equi pneumonia.

    PubMed

    Flaminio, M J; Rush, B R; Shuman, W

    1999-01-01

    Infectious diseases are common in foals aged 1-5 months. The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4+) or cytotoxic (CD8+) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses. Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion. Flow cytometric analysis of lymphocyte subpopulations suggested age-related changes in the cell-mediated immune system in horses. Absolute circulating CD4+ and CD8+ T lymphocytes and B cells increased linearly up to 3 months of age. Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes. Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age. The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count. Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age. These data suggest age-dependent cell-mediated and humoral development in young foals. PMID:10357110

  3. Distinct functional roles of cardiac mitochondrial subpopulations revealed by a 3D simulation model.

    PubMed

    Hatano, Asuka; Okada, Jun-Ichi; Washio, Takumi; Hisada, Toshiaki; Sugiura, Seiryo

    2015-06-01

    Experimental characterization of two cardiac mitochondrial subpopulations, namely, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), has been hampered by technical difficulties, and an alternative approach is eagerly awaited. We previously developed a three-dimensional computational cardiomyocyte model that integrates electrophysiology, metabolism, and mechanics with subcellular structure. In this study, we further developed our model to include intracellular oxygen diffusion, and determined whether mitochondrial localization or intrinsic properties cause functional variations. For this purpose, we created two models: one with equal SSM and IFM properties and one with IFM having higher activity levels. Using these two models to compare the SSM and IFM responses of [Ca(2+)], tricarboxylic acid cycle activity, [NADH], and mitochondrial inner membrane potential to abrupt changes in pacing frequency (0.25-2 Hz), we found that the reported functional differences between these subpopulations appear to be mostly related to local [Ca(2+)] heterogeneity, and variations in intrinsic properties only serve to augment these differences. We also examined the effect of hypoxia on mitochondrial function. Under normoxic conditions, intracellular oxygen is much higher throughout the cell than the half-saturation concentration for oxidative phosphorylation. However, under limited oxygen supply, oxygen is mostly exhausted in SSM, leaving the core region in an anoxic condition. Reflecting this heterogeneous oxygen environment, the inner membrane potential continues to decrease in IFM, whereas it is maintained to nearly normal levels in SSM, thereby ensuring ATP supply to this region. Our simulation results provide clues to understanding the origin of functional variations in two cardiac mitochondrial subpopulations and their differential roles in maintaining cardiomyocyte function as a whole. PMID:26039174

  4. Incorporating population variability and susceptible subpopulations into dosimetry for high-throughput toxicity testing.

    PubMed

    Wetmore, Barbara A; Allen, Brittany; Clewell, Harvey J; Parker, Timothy; Wambaugh, John F; Almond, Lisa M; Sochaski, Mark A; Thomas, Russell S

    2014-11-01

    Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assessment context. Previously, we employed in vitro hepatic metabolic clearance and plasma protein binding data with in vitro in vivo extrapolation (IVIVE) modeling to estimate oral equivalent doses, or daily oral chemical doses required to achieve steady-state blood concentrations (Css) equivalent to media concentrations having a defined effect in an in vitro HTS assay. In this study, hepatic clearance rates of selected ToxCast chemicals were measured in vitro for 13 cytochrome P450 and five uridine 5'-diphospho-glucuronysyltransferase isozymes using recombinantly expressed enzymes. The isozyme-specific clearance rates were then incorporated into an IVIVE model that captures known differences in isozyme expression across several life stages and ethnic populations. Comparison of the median Css for a healthy population against the median or the upper 95th percentile for more sensitive populations revealed differences of 1.3- to 4.3-fold or 3.1- to 13.1-fold, respectively. Such values may be used to derive chemical-specific human toxicokinetic adjustment factors. The IVIVE model was also used to estimate subpopulation-specific oral equivalent doses that were directly compared with subpopulation-specific exposure estimates. This study successfully combines isozyme and physiologic differences to quantitate subpopulation pharmacokinetic variability. Incorporation of these values with dosimetry and in vitro bioactivities provides a viable approach that could be employed within a high-throughput risk assessment framework. PMID:25145659

  5. Sex differences in the regulation of spatially distinct cardiac mitochondrial subpopulations.

    PubMed

    Ribeiro, Rogério Faustino; Ronconi, Karoline Sousa; Morra, Elis Aguiar; Do Val Lima, Patrícia Ribeiro; Porto, Marcella Leite; Vassallo, Dalton Valentim; Figueiredo, Suely Gomes; Stefanon, Ivanita

    2016-08-01

    Spatially distinct mitochondrial subpopulation may mediate myocardial pathology through permeability transition pore opening (MPTP). The goal of this study was to assess sex differences on the two spatially distinct mitochondrial subpopulations: subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IFM) based on morphology, membrane potential, mitochondrial function, oxidative phosphorylation, and MPTP. Aged matched Wistar rats were used to study SSM and IFM. Mitochondrial size was larger in SSM than in IFM in both genders. However, SSM internal complexity, yield, and membrane potential were higher in male than in female. The maximal rate of mitochondrial respiration, states 3 and 4, using glutamate + malate as substrate, were higher in IFM and SSM in the male group compared to female. The respiratory control ratio (RCR-state3/state 4), was not different in both SSM and IFM with glutamate + malate. The ADP:O ratio was found higher in IFM and SSM from female compared to males. When pyruvate was used, state 3 was found unchanged in both IFM and SSM, state 4 was also greater in male IFM compared to female. The RCR increased in the SSM while IFM remained the same. State 4 was higher in male SSM while in the IFM remained the same. The IFM presented a higher Ca(2+) retention capacity compared with SSM, however, there was a greater sensitivity to Ca(2+)-induced MPTP in SSM and IFM in the male group compared to female. In conclusion, our data show that spatially distinct mitochondrial subpopulations have sex-based differences in oxidative phosphorylation, morphology, and calcium retention capacity. PMID:27370644

  6. Variability in subpopulation formation propagates into biocatalytic variability of engineered Pseudomonas putida strains

    PubMed Central

    Lindmeyer, Martin; Jahn, Michael; Vorpahl, Carsten; Müller, Susann; Schmid, Andreas; Bühler, Bruno

    2015-01-01

    Pivotal challenges in industrial biotechnology are the identification and overcoming of cell-to-cell heterogeneity in microbial processes. While the development of subpopulations of isogenic cells in bioprocesses is well described (intra-population variability), a possible variability between genetically identical cultures growing under macroscopically identical conditions (clonal variability) is not. A high such clonal variability has been found for the recombinant expression of the styrene monooxygenase genes styAB from Pseudomonas taiwanensis VLB120 in solvent-tolerant Pseudomonas putida DOT-T1E using the alk-regulatory system from P. putida GPo1. In this study, the oxygenase subunit StyA fused to eGFP was used as readout tool to characterize the population structure in P. putida DOT-T1E regarding recombinant protein content. Flow cytometric analyses revealed that in individual cultures, at least two subpopulations with highly differing recombinant StyA-eGFP protein contents appeared (intra-population variability). Interestingly, subpopulation sizes varied from culture-to-culture correlating with the specific styrene epoxidation activity of cells derived from respective cultures (clonal variability). In addition, flow cytometric cell sorting coupled to plasmid copy number (PCN) determination revealed that detected clonal variations cannot be correlated to the PCN, but depend on the combination of the regulatory system and the host strain employed. This is, to the best of our knowledge, the first work reporting that intra-population variability (with differing protein contents in the presented case study) causes clonal variability of genetically identical cultures. Respective impacts on bioprocess reliability and performance and strategies to overcome respective reliability issues are discussed. PMID:26483771

  7. Thermostability of subpopulations of H2N3 influenza virus isolates from mallard ducks.

    PubMed

    Negovetich, Nicholas J; Webster, Robert G

    2010-09-01

    Maintenance of avian influenza virus in waterfowl populations requires that virions remain infectious while in the environment. Temperature has been shown to negatively correlate with persistence time, which is the duration for which virions are infectious. However, thermostability can vary between isolates regardless of subtype, and it is not known whether this variation occurs when host and geographic location of isolation are controlled. In this study, we analyzed the thermostabilities of 7 H2N3 viruses isolated from mallard ducks in Alberta, Canada. Virus samples were incubated at 37 degrees C and 55 degrees C, and infectivity titers were calculated at different time points. Based on the rate of infectivity inactivation at 37 degrees C, isolates could be grouped into either a thermosensitive or thermostable fraction for both egg- and MDCK-grown virus populations. Titers decreased more rapidly for isolates incubated at 55 degrees C, and this loss of infectivity occurred in a nonlinear, 2-step process, which is in contrast with the consensus on thermostability. This suggests that stock samples contain a mixture of subpopulations with different thermostabilities. The rate of decrease for the sensitive fraction was approximately 14 times higher than that for the stable fraction. The presence of subpopulations is further supported by selection experiments and plaque purification, both of which result in homogenous populations that exhibit linear decreases of infectivity titer. Therefore, variation of thermostability of influenza virus isolates begins at the level of the population. The presence of subpopulations with high thermostability suggests that avian viruses can persist in water longer than previously estimated, thus increasing the probability of transmission to susceptible hosts. PMID:20610728

  8. Distinct Functional Roles of Cardiac Mitochondrial Subpopulations Revealed by a 3D Simulation Model

    PubMed Central

    Hatano, Asuka; Okada, Jun-ichi; Washio, Takumi; Hisada, Toshiaki; Sugiura, Seiryo

    2015-01-01

    Experimental characterization of two cardiac mitochondrial subpopulations, namely, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), has been hampered by technical difficulties, and an alternative approach is eagerly awaited. We previously developed a three-dimensional computational cardiomyocyte model that integrates electrophysiology, metabolism, and mechanics with subcellular structure. In this study, we further developed our model to include intracellular oxygen diffusion, and determined whether mitochondrial localization or intrinsic properties cause functional variations. For this purpose, we created two models: one with equal SSM and IFM properties and one with IFM having higher activity levels. Using these two models to compare the SSM and IFM responses of [Ca2+], tricarboxylic acid cycle activity, [NADH], and mitochondrial inner membrane potential to abrupt changes in pacing frequency (0.25–2 Hz), we found that the reported functional differences between these subpopulations appear to be mostly related to local [Ca2+] heterogeneity, and variations in intrinsic properties only serve to augment these differences. We also examined the effect of hypoxia on mitochondrial function. Under normoxic conditions, intracellular oxygen is much higher throughout the cell than the half-saturation concentration for oxidative phosphorylation. However, under limited oxygen supply, oxygen is mostly exhausted in SSM, leaving the core region in an anoxic condition. Reflecting this heterogeneous oxygen environment, the inner membrane potential continues to decrease in IFM, whereas it is maintained to nearly normal levels in SSM, thereby ensuring ATP supply to this region. Our simulation results provide clues to understanding the origin of functional variations in two cardiac mitochondrial subpopulations and their differential roles in maintaining cardiomyocyte function as a whole. PMID:26039174

  9. Association between Influenza A Virus Infection and Pigs Subpopulations in Endemically Infected Breeding Herds

    PubMed Central

    Diaz, Andres; Perez, Andres; Sreevatsan, Srinand; Davies, Peter; Culhane, Marie; Torremorell, Montserrat

    2015-01-01

    Influenza A viruses (IAVs) are distributed worldwide in birds, pigs and humans, and cause important endemic disease affecting hosts in all countries. Although pigs play a key role in the ecology of IAVs, the epidemiology of IAVs within swine herds is poorly understood. In this longitudinal study we describe the prevalence of IAVs infection in three subpopulations of pigs in 5 breeding herds in the Midwestern USA. Each herd was sampled monthly for a year and, at each visit, 30 individual nasal swabs were collected from the three subpopulations, namely, a) replacement females, resident on-farm for less than 4 weeks (new gilts), b) replacement females, resident on-farm for more than 4 weeks (gilts), and c) neonatal pigs less than 21 days of age (piglets). Real time reverse transcriptase polymerase chain reaction (RRT-PCR) was used to detect IAVs, and the association between IAVs infection and pig subpopulation was measured using a mixed logistic regression model. Nasal swabs (n = 4,190) were collected from 141 groups of pigs. At least, one IAV-positive nasal swab was found in 19.9% (n = 28) of the sampled groups, and 7.7% (n = 324) of all nasal swabs tested positive. After adjusting by annual quarter and sampling event, the odds of testing IAV positive were 7.9 (95% CI 1.4, 43.9) and 4.4 (95% CI 1.1, 17.1) times higher in groups of new gilts and piglets compared to groups of gilts, respectively. Results indicate that new gilts and piglets had higher odds of testing IAV positive than gilts in swine breeding herds and that season influences IAV infection in pigs. Based on these findings, we recommend that IAV control strategies be aimed at preventing infection before gilts are introduced into the farm, and in pigs prior to weaning. PMID:26076494

  10. Sperm characterization and identification of sperm sub-populations in ejaculates from pampas deer (Ozotoceros bezoarticus).

    PubMed

    Beracochea, F; Gil, J; Sestelo, A; Garde, J J; Santiago-Moreno, J; Fumagalli, F; Ungerfeld, R

    2014-10-01

    Pampas deer (Ozotoceros bezoarticus) is a native endangered species. Knowledge of the basic spermiogram characteristics and the morphometric descriptors is necessary to effectively develop sperm cryopreservation. In other species, sperm sub-population is related to sperm cryo-resistance. The objective was to provide a general description of the sperm, including sperm head morphometric descriptors, its repeatability, and the existence of sperm sub-populations. Sperm were obtained from adult males by electroejaculation during the breeding season. The motility score was 3.4 ± 0.2 (mean ± SEM) and progressive motility was 59.4 ± 3.7%. Ejaculated volume was 413.9 ± 51.0 μl, the total number of sperm ejaculated was 321.2 ± 55.4 × 10(6). Also, 63.3 ± 3.1% of the sperm were morphologically abnormal and 23.7 ± 2.3% had acrosome damage. The sperm head length was 7.6 ± 0.01 μm, width 4.4 ± 0.01 μm, area 28.1 ± 0.07 μm(2) and the perimeter was 21.9 ± 0.04 μm. There was a positive relationship among morphometric descriptors and the motility score, overall motility and progressive motility. Also length (P=0.011), width (P=0.003), area (P=0.006) and perimeter (P=0.009) of sperm head obtained in two different collections were positively related. Overall, the low concentration, volume, overall quality and abnormal morphology, and wide variation of these variables may be a limitation for the development of sperm cryopreserved banks. There were three sperm sub-populations with different morphometric characteristics. The morphometric descriptors are maintained similarly among different collections. PMID:25104472

  11. Culture conditions affect the cholinergic development of an isolated subpopulation of chick mesencephalic neural crest cells.

    PubMed

    Barald, K F

    1989-10-01

    Although neural crest cells are known to be very responsive to environmental cues during their development, recent evidence indicates that at least some subpopulations may be committed to a specific differentiation program prior to migration. Because the neural crest is composed of a heterogeneous mixture of cells that contributes to many vertebrate cell lineages, assessing the properties of specific subpopulations and the effect of the environment on their development has been difficult. To address this problem, we have isolated a pure subpopulation of chick mesencephalic neural crest cells by fluorescence no-flow cytometry after labeling them with monoclonal antibodies (Mabs) to a 75-kDa cell surface antigen that is associated with high affinity choline uptake. When cultures of chick mesencephalic neural crest cells are labeled with these Mabs and a fluorescent second step antibody, approximately 5% of the cells are antigen-positive (A+). After sorting, 100% of the resulting cultured mesencephalic neural crest cells are A+. The Mabs we used also label all of the neurons of the embryonic chick and quail ciliary ganglion in vivo and in vitro. We have compared the effect of various cell culture media on the isolated neural crest subpopulation and the heterogeneous chick mesencephalic neural crest from which it was derived. A+ cells were passaged and grown in a variety of media, each of which differently affected its characteristics and development. A+ cells proliferated in the presence of 15% fetal bovine serum (FBS) and high concentrations (10-15%) of chick embryo extract, but did not differentiate, although they retained basal levels of choline acetyltransferase (ChAT) activity. However, in chick serum and high (25 mM as opposed to 7 mM) K+, and heart-, iris-, or lung-conditioned medium, all of which are known to promote survival and/or cholinergic development of ciliary ganglion neurons, the cells ceased to proliferate and all of the cells in the culture became

  12. THE SPATIAL STRUCTURE OF MONO-ABUNDANCE SUB-POPULATIONS OF THE MILKY WAY DISK

    SciTech Connect

    Bovy, Jo; Rix, Hans-Walter; Liu Chao; Hogg, David W.; Beers, Timothy C.; Lee, Young Sun

    2012-07-10

    The spatial, kinematic, and elemental-abundance structure of the Milky Way's stellar disk is complex, and has been difficult to dissect with local spectroscopic or global photometric data. Here, we develop and apply a rigorous density modeling approach for Galactic spectroscopic surveys that enables investigation of the global spatial structure of stellar sub-populations in narrow bins of [{alpha}/Fe] and [Fe/H], using 23,767 G-type dwarfs from SDSS/SEGUE, which effectively sample 5 kpc < R{sub GC} < 12 kpc and 0.3 kpc {approx}< |Z| {approx}< 3 kpc. We fit models for the number density of each such ([{alpha}/Fe] and [Fe/H]) mono-abundance component, properly accounting for the complex spectroscopic SEGUE sampling of the underlying stellar population, as well as for the metallicity and color distributions of the samples. We find that each mono-abundance sub-population has a simple spatial structure that can be described by a single exponential in both the vertical and radial directions, with continuously increasing scale heights ( Almost-Equal-To 200 pc to 1 kpc) and decreasing scale lengths (>4.5 kpc to 2 kpc) for increasingly older sub-populations, as indicated by their lower metallicities and [{alpha}/Fe] enhancements. That the abundance-selected sub-components with the largest scale heights have the shortest scale lengths is in sharp contrast with purely geometric 'thick-thin disk' decompositions. To the extent that [{alpha}/Fe] is an adequate proxy for age, our results directly show that older disk sub-populations are more centrally concentrated, which implies inside-out formation of galactic disks. The fact that the largest scale-height sub-components are most centrally concentrated in the Milky Way is an almost inevitable consequence of explaining the vertical structure of the disk through internal evolution. Whether the simple spatial structure of the mono-abundance sub-components and the striking correlations between age, scale length, and scale height can

  13. Alterations in the T-lymphocyte subpopulation in patients with rhinoscleroma.

    PubMed Central

    Berron, P; Berron, R; Ortiz-Ortiz, L

    1988-01-01

    T-lymphocyte subpopulations were studied in a group of patients with rhinoscleroma due to Klebsiella rhinoscleromatis. The data demonstrated that these patients had a significantly greater number of T-suppressor/cytotoxic lymphocytes than did clinically healthy individuals. This finding correlated with a diminished response to the T-cell mitogen concanavalin A. The evidence indicated that the T-cell response of these patients was decreased and may reflect the host's response to the bacterial invader, thus explaining the chronicity of the disease. PMID:2968362

  14. Effects of subpopulation structure on probability calculations of DNA profiles from forensic PCR analysis.

    PubMed

    Gallo, J C; Thomas, E; Novick, G E; Herrera, R J

    1997-01-01

    DNA typing for forensic identification is a two-step process. The first step involves determining the profiles of samples collected at the crime scene and comparing them with the profiles obtained from suspects and the victims. In the case of a match that includes the suspect as the potential source of the material collected at the crime scene, the last step in the process is to answer the question, what is the likelihood that someone in addition to the suspect could match the profile of the sample studied? This likelihood is calculated by determining the frequency of the suspect's profile in the relevant population databases. The design of forensic databases and the criteria for comparison has been addressed by the NRC report of 1996 (National Research Council, 1996). However, the fact that geographical proximity, migrational patterns, and even cultural and social practices have effects on subpopulation structure establishes the grounds for further study into its effects on the calculation of probability of occurrence values. The issue becomes more relevant in the case of discrete polymorphic markers that show higher probability of occurrence in the reference populations, where several orders of magnitude difference between the databases may have an impact on the jury. In this study, we calculated G values for all possible pairwise comparisons of allelic frequencies in the different databases from the races or subpopulations examined. In addition, we analyzed a set of 24 unrelated Caucasian, 37 unrelated African-American, and 96 unrelated Sioux/Chippewa individuals for seven polymorphic loci (DQA1, LDLR, GYPA, HBGG, D7S8, GC, and D1S80). All three sets of individuals where sampled from Minnesota. The probability of occurrence for all seven loci were calculated with respect to nine different databases: Caucasian, Arabic, Korean, Sioux/Chippewa, Navajo, Pueblo, African American, Southeastern Hispanic, and Southwestern Hispanic. Analysis of the results demonstrated

  15. Epithelial histogenesis during tooth development.

    PubMed

    Lesot, H; Brook, A H

    2009-12-01

    This paper reviews the current understanding of the progressive changes mediating dental epithelial histogenesis as a basis for future collaborative studies. Tooth development involves morphogenesis, epithelial histogenesis and cell differentiation. The consecutive morphological stages of lamina, bud, cap and bell are also characterized by changes in epithelial histogenesis. Differential cell proliferation rates, apoptosis, and alterations in adhesion and shape lead to the positioning of groups of cells with different functions. During tooth histo-morphogenesis changes occur in basement membrane composition, expression of signalling molecules and the localization of cell surface components. Cell positional identity may be related to cell history. Another important parameter is cell plasticity. Independently of signalling molecules, which play a major role in inducing or modulating specific steps, cell-cell and cell-matrix interactions regulate the plasticity/rigidity of particular domains of the enamel organ. This involves specifying in space the differential growth and influences the progressive tooth morphogenesis by shaping the epithelial-mesenchymal junction. Deposition of a mineralized matrix determines the final shape of the crown. All data reviewed in this paper were investigated in the mouse. PMID:18656852

  16. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells. PMID:17460896

  17. Epithelial-Mesenchymal Transition in Cancer: Parallels Between Normal Development and Tumor Progression

    PubMed Central

    Micalizzi, Douglas S.; Farabaugh, Susan M.

    2010-01-01

    From the earliest stages of embryonic development, cells of epithelial and mesenchymal origin contribute to the structure and function of developing organs. However, these phenotypes are not always permanent, and instead, under the appropriate conditions, epithelial and mesenchymal cells convert between these two phenotypes. These processes, termed Epithelial-Mesenchymal Transition (EMT), or the reverse Mesenchymal-Epithelial Transition (MET), are required for complex body patterning and morphogenesis. In addition, epithelial plasticity and the acquisition of invasive properties without the full commitment to a mesenchymal phenotype are critical in development, particularly during branching morphogenesis in the mammary gland. Recent work in cancer has identified an analogous plasticity of cellular phenotypes whereby epithelial cancer cells acquire mesenchymal features that permit escape from the primary tumor. Because local invasion is thought to be a necessary first step in metastatic dissemination, EMT and epithelial plasticity are hypothesized to contribute to tumor progression. Similarities between developmental and oncogenic EMT have led to the identification of common contributing pathways, suggesting that the reactivation of developmental pathways in breast and other cancers contributes to tumor progression. For example, developmental EMT regulators including Snail/Slug, Twist, Six1, and Cripto, along with developmental signaling pathways including TGF-β and Wnt/β-catenin, are misexpressed in breast cancer and correlate with poor clinical outcomes. This review focuses on the parallels between epithelial plasticity/EMT in the mammary gland and other organs during development, and on a selection of developmental EMT regulators that are misexpressed specifically during breast cancer. PMID:20490631

  18. Neutrophil Interactions with Epithelial Expressed ICAM-1 Enhances Intestinal Mucosal Wound Healing

    PubMed Central

    Sumagin, R; Brazil, JC; Nava, P; Nishio, H; Alam, A; Luissint, AC; Weber, DA; Neish, AS; Nusrat, A; Parkos, CA

    2015-01-01

    A characteristic feature of gastrointestinal tract inflammatory disorders, such as inflammatory bowel disease, is polymorphonuclear neutrophil (PMN) transepithelial migration (TEM) and accumulation in the gut lumen. PMN accumulation within the intestinal mucosa contributes to tissue injury. While epithelial infiltration by large numbers of PMNs results in mucosal injury, we found that PMN interactions with luminal epithelial membrane receptors may also play a role in wound healing. Intercellular adhesion molecule-1 (ICAM-1) is a PMN ligand that is upregulated on apical surfaces of intestinal epithelial cells under inflammatory conditions. In our study, increased expression of ICAM-1 resulted in enhanced PMN binding to the apical epithelium, which was associated with reduced PMN apoptosis. Following TEM, PMN adhesion to ICAM-1 resulted in activation of Akt and β-catenin signaling, increased epithelial-cell proliferation, and wound healing. Such responses were ICAM-1 dependent as engagement of epithelial ICAM-1 by antibody-mediated cross-linking yielded similar results. Furthermore, using an in-vivo biopsy-based, colonic-mucosal-injury model, we demonstrated epithelial ICAM-1 plays an important role in activation of epithelial Akt and β-catenin signaling and wound healing. These findings suggest that post-migrated PMNs within the intestinal lumen can regulate epithelial homeostasis, thereby identifying ICAM-1 as a potential therapeutic target for promoting mucosal wound healing. PMID:26732677

  19. Deletion of Lkb1 in Renal Tubular Epithelial Cells Leads to CKD by Altering Metabolism.

    PubMed

    Han, Seung Hyeok; Malaga-Dieguez, Laura; Chinga, Frank; Kang, Hyun Mi; Tao, Jianling; Reidy, Kimberly; Susztak, Katalin

    2016-02-01

    Renal tubule epithelial cells are high-energy demanding polarized epithelial cells. Liver kinase B1 (LKB1) is a key regulator of polarity, proliferation, and cell metabolism in epithelial cells, but the function of LKB1 in the kidney is unclear. Our unbiased gene expression studies of human control and CKD kidney samples identified lower expression of LKB1 and regulatory proteins in CKD. Mice with distal tubule epithelial-specific Lkb1 deletion (Ksp-Cre/Lkb1(flox/flox)) exhibited progressive kidney disease characterized by flattened dedifferentiated tubule epithelial cells, interstitial matrix accumulation, and dilated cystic-appearing tubules. Expression of epithelial polarity markers β-catenin and E-cadherin was not altered even at later stages. However, expression levels of key regulators of metabolism, AMP-activated protein kinase (Ampk), peroxisome proliferative activated receptor gamma coactivator 1-α (Ppargc1a), and Ppara, were significantly lower than those in controls and correlated with fibrosis development. Loss of Lkb1 in cultured epithelial cells resulted in energy depletion, apoptosis, less fatty acid oxidation and glycolysis, and a profibrotic phenotype. Treatment of Lkb1-deficient cells with an AMP-activated protein kinase (AMPK) agonist (A769662) or a peroxisome proliferative activated receptor alpha agonist (fenofibrate) restored the fatty oxidation defect and reduced apoptosis. In conclusion, we show that loss of LKB1 in renal tubular epithelial cells has an important role in kidney disease development by influencing intracellular metabolism. PMID:26054542

  20. Neutrophil interactions with epithelial-expressed ICAM-1 enhances intestinal mucosal wound healing.

    PubMed

    Sumagin, R; Brazil, J C; Nava, P; Nishio, H; Alam, A; Luissint, A C; Weber, D A; Neish, A S; Nusrat, A; Parkos, C A

    2016-09-01

    A characteristic feature of gastrointestinal tract inflammatory disorders, such as inflammatory bowel disease, is polymorphonuclear neutrophil (PMN) transepithelial migration (TEM) and accumulation in the gut lumen. PMN accumulation within the intestinal mucosa contributes to tissue injury. Although epithelial infiltration by large numbers of PMNs results in mucosal injury, we found that PMN interactions with luminal epithelial membrane receptors may also play a role in wound healing. Intercellular adhesion molecule-1 (ICAM-1) is a PMN ligand that is upregulated on apical surfaces of intestinal epithelial cells under inflammatory conditions. In our study, increased expression of ICAM-1 resulted in enhanced PMN binding to the apical epithelium, which was associated with reduced PMN apoptosis. Following TEM, PMN adhesion to ICAM-1 resulted in activation of Akt and β-catenin signaling, increased epithelial-cell proliferation, and wound healing. Such responses were ICAM-1 dependent as engagement of epithelial ICAM-1 by antibody-mediated cross-linking yielded similar results. Furthermore, using an in-vivo biopsy-based, colonic-mucosal-injury model, we demonstrated epithelial ICAM-1 has an important role in activation of epithelial Akt and β-catenin signaling and wound healing. These findings suggest that post-migrated PMNs within the intestinal lumen can regulate epithelial homeostasis, thereby identifying ICAM-1 as a potential therapeutic target for promoting mucosal wound healing. PMID:26732677

  1. MicroRNAs and Epithelial Immunity

    PubMed Central

    Liu, Jun; Drescher, Kristen M.; Chen, Xian-Ming

    2009-01-01

    MicroRNAs are required for development and maintenance of the epithelial barrier. It is hypothesized that microRNAs are involved in regulating epithelial anti-microbial defenses by targeting key epithelial effector molecules and/or influencing intracellular signaling pathways. Additionally, aberrant microRNA expression has been implicated in the pathogenesis of various diseases at the skin and mucosa. Increased understanding of the role of microRNAs in epithelial immunoregulation and identification of microRNAs of pathogenetic significance will enhance our understanding of epithelial immunobiology and immunopathology. PMID:19811319

  2. Characterizing Subpopulations within the near-Earth Objects with NEOWISE: Preliminary Results

    NASA Astrophysics Data System (ADS)

    Mainzer, A.; Grav, T.; Masiero, J.; Bauer, J.; McMillan, R. S.; Giorgini, J.; Spahr, T.; Cutri, R. M.; Tholen, D. J.; Jedicke, R.; Walker, R.; Wright, E.; Nugent, C. R.

    2012-06-01

    We present the preliminary results of an analysis of the sub-populations within the near-Earth asteroids, including the Atens, Apollos, Amors, and those that are considered potentially hazardous using data from the Wide-field Infrared Survey Explorer (WISE). In order to extrapolate the sample of objects detected by WISE to the greater population, we determined the survey biases for asteroids detected by the project's automated moving object processing system (known as NEOWISE) as a function of diameter, visible albedo, and orbital elements. Using this technique, we are able to place constraints on the number of potentially hazardous asteroids larger than 100 m and find that there are ~4700 ± 1450 such objects. As expected, the Atens, Apollos, and Amors are revealed by WISE to have somewhat different albedo distributions, with the Atens being brighter than the Amors. The cumulative size distributions of the various near-Earth object (NEO) subgroups vary slightly between 100 m and 1 km. A comparison of the observed orbital elements of the various sub-populations of the NEOs with the current best model is shown.

  3. Flow Cytometry Analysis Reveals That Only a Subpopulation of Mouse Sperm Undergoes Hyperpolarization During Capacitation1

    PubMed Central

    Escoffier, Jessica; Navarrete, Felipe; Haddad, Doug; Santi, Celia M.; Darszon, Alberto; Visconti, Pablo E.

    2015-01-01

    To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K+, by PKA inhibitors, and by SLO3 inhibitors in CD1 mouse sperm, and undetectable in Slo3 knockout mouse sperm. On the other hand, in sperm incubated in conditions that do not support capacitation, sperm membrane hyperpolarization can be induced by amiloride, high extracellular NaHCO3, and cAMP agonists. Altogether, our observations are consistent with a model in which sperm Em hyperpolarization is downstream of a cAMP-dependent pathway and is mediated by the activation of SLO3 K+ channels. PMID:25855261

  4. Lymphocyte subpopulations and hematologic variables in dogs with congestive heart failure.

    PubMed

    Farabaugh, Andrew E; Freeman, Lisa M; Rush, John E; George, Katherine L

    2004-01-01

    Alterations in lymphocyte subpopulations and in other hematologic variables have been documented in people with heart failure. The purpose of the current study was to compare flow cytometric and hematologic variables in dogs with congestive heart failure (CHF) to healthy controls. CD4+ peripheral blood mononuclear cells (PBMC) and CD8+ lymphocytes were analyzed by flow cytometry, and white blood cell count, platelet count, hematocrit, and hemoglobin were determined by a complete blood count. Twenty-five dogs with CHF (International Small Animal Cardiac Health Council [ISACHC] class 2 [n = 12] and ISACHC class 3a [n = 13]) and 13 healthy controls were enrolled in the study. Compared with the controls, dogs with CHF had markedly lower percentages of CD4+ PBMC, CD8+ lymphocytes, hematocrit, and hemoglobin, but markedly higher leukocytes, neutrophils, and platelets. There were no differences in these variables between dogs with dilated cardiomyopathy (n = 6) and those with chronic valvular disease (n = 19). Dogs in ISACHC class 3a had a markedly lower total lymphocyte number, CD4+ and CD8+ cells, and hematocrit, but markedly higher leukocyte and neutrophil numbers relative to the control group. CD4+ and CD8+ subpopulations and other blood cell variables are altered in dogs with CHF. Future studies to determine possible clinical implications of these changes are warranted. PMID:15320588

  5. Identification of Distinct Tumor Subpopulations in Lung Adenocarcinoma via Single-Cell RNA-seq

    PubMed Central

    Min, Jae-Woong; Kim, Woo Jin; Han, Jeong A.; Jung, Yu-Jin; Kim, Kyu-Tae; Park, Woong-Yang; Lee, Hae-Ock; Choi, Sun Shim

    2015-01-01

    Single-cell sequencing, which is used to detect clinically important tumor subpopulations, is necessary for understanding tumor heterogeneity. Here, we analyzed transcriptomic data obtained from 34 single cells from human lung adenocarcinoma (LADC) patient-derived xenografts (PDXs). To focus on the intrinsic transcriptomic signatures of these tumors, we filtered out genes that displayed extensive expression changes following xenografting and cell culture. Then, we performed clustering analysis using co-regulated gene modules rather than individual genes to minimize read drop-out errors associated with single-cell sequencing. This combined approach revealed two distinct intra-tumoral subgroups that were primarily distinguished by the gene module G64. The G64 module was predominantly composed of cell-cycle genes. E2F1 was found to be the transcription factor that most likely mediates the expression of the G64 module in single LADC cells. Interestingly, the G64 module also indicated inter-tumoral heterogeneity based on its association with patient survival and other clinical variables such as smoking status and tumor stage. Taken together, these results demonstrate the feasibility of single-cell RNA sequencing and the strength of our analytical pipeline for the identification of tumor subpopulations. PMID:26305796

  6. Root canal anatomy of mandibular first premolars in an Emirati subpopulation: A laboratory study

    PubMed Central

    Abraham, Sheela Balu; Gopinath, Vellore Kannan

    2015-01-01

    Objective: The purpose of this study was to determine the root canal anatomy of mandibular first premolar teeth in an Emirati subpopulation using a decalcification and clearing method. Materials and Methods: One hundred permanent mandibular first premolar teeth extracted for orthodontic purposes from an Emirati subpopulation from the United Arab Emirates were used for this study. They were subjected to decalcification and clearing. The tooth length, the canal orifice shape, mesial invagination, canal pattern, the location of apex, presence of lateral canals, and intercanal communications were determined. Results: The most common canal pattern was the Vertucci Type I (65%) followed by Type V (14%) and Type IV (13%). The most common type of canal orifice seen was the oval shape (36%) followed by the round shape (25%). Mesial invaginations were seen in 44% of the teeth. The mean tooth length was 19.9 mm, and apical deltas were seen in 24% of teeth. Conclusion: The Vertucci Type I canal pattern was the most prevalent in the mandibular first premolars while the occurrence of multiple canals was noted in 35% of this population. PMID:26929684

  7. CD36 is expressed in a defined subpopulation of neurons in the olfactory epithelium.

    PubMed

    Xavier, André Machado; Ludwig, Raissa Guimarães; Nagai, Maíra Harume; de Almeida, Tiago Jonas; Watanabe, Hebe Mizuno; Hirata, Marcio Yukio; Rosenstock, Tatiana Rosado; Papes, Fabio; Malnic, Bettina; Glezer, Isaias

    2016-01-01

    The sensory neurons in the olfactory epithelium (OSNs) are equipped with a large repertoire of olfactory receptors and the associated signal transduction machinery. In addition to the canonical OSNs, which express odorant receptors (ORs), the epithelium contains specialized subpopulations of sensory neurons that can detect specific information from environmental cues and relay it to relevant neuronal circuitries. Here we describe a subpopulation of mature OSNs in the main olfactory epithelium (MOE) which expresses CD36, a multifunctional receptor involved in a series of biological processes, including sensory perception of lipid ligands. The Cd36 expressing neurons coexpress markers of mature OSNs and are dispersed throughout the MOE. Unlike several ORs analyzed in our study, we found frequent coexpression of the OR Olfr287 in these neurons, suggesting that only a specific set of ORs may be coexpressed with CD36 in OSNs. We also show that CD36 is expressed in the cilia of OSNs, indicating a possible role in odorant detection. CD36-deficient mice display no signs of gross changes in the organization of the olfactory epithelium, but show impaired preference for a lipid mixture odor. Our results show that CD36-expressing neurons represent a distinct population of OSNs, which may have specific functions in olfaction. PMID:27145700

  8. CD36 is expressed in a defined subpopulation of neurons in the olfactory epithelium

    PubMed Central

    Xavier, André Machado; Ludwig, Raissa Guimarães; Nagai, Maíra Harume; de Almeida, Tiago Jonas; Watanabe, Hebe Mizuno; Hirata, Marcio Yukio; Rosenstock, Tatiana Rosado; Papes, Fabio; Malnic, Bettina; Glezer, Isaias

    2016-01-01

    The sensory neurons in the olfactory epithelium (OSNs) are equipped with a large repertoire of olfactory receptors and the associated signal transduction machinery. In addition to the canonical OSNs, which express odorant receptors (ORs), the epithelium contains specialized subpopulations of sensory neurons that can detect specific information from environmental cues and relay it to relevant neuronal circuitries. Here we describe a subpopulation of mature OSNs in the main olfactory epithelium (MOE) which expresses CD36, a multifunctional receptor involved in a series of biological processes, including sensory perception of lipid ligands. The Cd36 expressing neurons coexpress markers of mature OSNs and are dispersed throughout the MOE. Unlike several ORs analyzed in our study, we found frequent coexpression of the OR Olfr287 in these neurons, suggesting that only a specific set of ORs may be coexpressed with CD36 in OSNs. We also show that CD36 is expressed in the cilia of OSNs, indicating a possible role in odorant detection. CD36-deficient mice display no signs of gross changes in the organization of the olfactory epithelium, but show impaired preference for a lipid mixture odor. Our results show that CD36-expressing neurons represent a distinct population of OSNs, which may have specific functions in olfaction. PMID:27145700

  9. PAX4 Defines an Expandable β-Cell Subpopulation in the Adult Pancreatic Islet

    PubMed Central

    Lorenzo, Petra I.; Fuente-Martín, Esther; Brun, Thierry; Cobo-Vuilleumier, Nadia; Jimenez-Moreno, Carmen María; G. Herrera Gomez, Irene; López Noriega, Livia; Mellado-Gil, José Manuel; Martin-Montalvo, Alejandro; Soria, Bernat; Gauthier, Benoit R.

    2015-01-01

    PAX4 is a key regulator of pancreatic islet development whilst in adult acute overexpression protects β-cells against stress-induced apoptosis and stimulates proliferation. Nonetheless, sustained PAX4 expression promotes β-cell dedifferentiation and hyperglycemia, mimicking β-cell failure in diabetic patients. Herein, we study mechanisms that allow stringent PAX4 regulation endowing favorable β-cell adaptation in response to changing environment without loss of identity. To this end, PAX4 expression was monitored using a mouse bearing the enhanced green fluorescent protein (GFP) and cre recombinase construct under the control of the islet specific pax4 promoter. GFP was detected in 30% of islet cells predominantly composed of PAX4-enriched β-cells that responded to glucose-induced insulin secretion. Lineage tracing demonstrated that all islet cells were derived from PAX4+ progenitor cells but that GFP expression was confined to a subpopulation at birth which declined with age correlating with reduced replication. However, this GFP+ subpopulation expanded during pregnancy, a state of active β-cell replication. Accordingly, enhanced proliferation was exclusively detected in GFP+ cells consistent with cell cycle genes being stimulated in PAX4-overexpressing islets. Under stress conditions, GFP+ cells were more resistant to apoptosis than their GFP- counterparts. Our data suggest PAX4 defines an expandable β-cell sub population within adult islets. PMID:26503027

  10. Surface-Micromachined Microfiltration Membranes for Efficient Isolation and Functional Immunophenotyping of Subpopulations of Immune Cells

    PubMed Central

    Oh, Boram; Lam, Raymond H. W.; Fan, Rong; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

    2015-01-01

    An accurate measurement of the immune status in patients with immune system disorders is critical in evaluating the stage of diseases and tailoring drug treatments. The functional cellular immunity test is a promising method to establish the diagnosis of immune dysfunctions. The conventional functional cellular immunity test involves measurements of the capacity of peripheral blood mononuclear cells to produce pro-inflammatory cytokines when stimulated ex vivo. However, this “bulk” assay measures the overall reactivity of a population of lymphocytes and monocytes, making it difficult to pinpoint the phenotype or real identity of the reactive immune cells involved. In this research, we develop a large surface micromachined polydimethylsiloxane (PDMS) microfiltration membrane (PMM) with high porosity, which is integrated in a microfluidic microfiltration platform. Using the PMM with functionalized microbeads conjugated with antibodies against specific cell surface proteins, we demonstrated rapid, efficient and high-throughput on-chip isolation, enrichment, and stimulation of subpopulations of immune cells from blood specimens. Furthermore, the PMM-integrated microfiltration platform, coupled with a no-wash homogeneous chemiluminescence assay (“AlphaLISA”), enables us to demonstrate rapid and sensitive on-chip immunophenotyping assays for subpopulations of immune cells isolated directly from minute quantities of blood samples. PMID:23335389

  11. CHARACTERIZING SUBPOPULATIONS WITHIN THE NEAR-EARTH OBJECTS WITH NEOWISE: PRELIMINARY RESULTS

    SciTech Connect

    Mainzer, A.; Masiero, J.; Bauer, J.; Giorgini, J.; Grav, T.; McMillan, R. S.; Spahr, T.; Cutri, R. M.; Tholen, D. J.; Jedicke, R.; Walker, R.; Wright, E.; Nugent, C. R.

    2012-06-20

    We present the preliminary results of an analysis of the sub-populations within the near-Earth asteroids, including the Atens, Apollos, Amors, and those that are considered potentially hazardous using data from the Wide-field Infrared Survey Explorer (WISE). In order to extrapolate the sample of objects detected by WISE to the greater population, we determined the survey biases for asteroids detected by the project's automated moving object processing system (known as NEOWISE) as a function of diameter, visible albedo, and orbital elements. Using this technique, we are able to place constraints on the number of potentially hazardous asteroids larger than 100 m and find that there are {approx}4700 {+-} 1450 such objects. As expected, the Atens, Apollos, and Amors are revealed by WISE to have somewhat different albedo distributions, with the Atens being brighter than the Amors. The cumulative size distributions of the various near-Earth object (NEO) subgroups vary slightly between 100 m and 1 km. A comparison of the observed orbital elements of the various sub-populations of the NEOs with the current best model is shown.

  12. A Subpopulation of Serotonergic Neurons That Do Not Express the 5-HT1A Autoreceptor

    PubMed Central

    2012-01-01

    5-HT neurons are topographically organized in the hindbrain, and have been implicated in the etiology and treatment of psychiatric diseases such as depression and anxiety. Early studies suggested that the raphe 5-HT neurons were a homogeneous population showing similar electrical properties, and feedback inhibition mediated by 5-HT1A autoreceptors. We utilized histochemistry techniques in ePet1-eGFP and 5-HT1A-iCre/R26R mice to show that a subpopulation of 5-HT neurons do not express the somatodendritic 5-HT1A autoreceptor mRNA. In addition, we performed patch-clamp recordings followed by single-cell PCR in ePet1-eGFP mice. From 134 recorded 5-HT neurons located in the dorsal, lateral, and median raphe, we found lack of 5-HT1A mRNA expression in 22 cells, evenly distributed across raphe subfields. We compared the cellular characteristics of these neuronal types and found no difference in passive membrane properties and general excitability. However, when injected with large depolarizing current, 5-HT1A-negative neurons fired more action potentials, suggesting a lack of autoinhibitory action of local 5-HT release. Our results support the hypothesis that the 5-HT system is composed of subpopulations of serotonergic neurons with different capacity for adaptation. PMID:23336048

  13. Near-Optimal Decoding of Transient Stimuli from Coupled Neuronal Subpopulations

    PubMed Central

    Trousdale, James; Carroll, Samuel R.; Gabbiani, Fabrizio

    2014-01-01

    Coupling between sensory neurons impacts their tuning properties and correlations in their responses. How such coupling affects sensory representations and ultimately behavior remains unclear. We investigated the role of neuronal coupling during visual processing using a realistic biophysical model of the vertical system (VS) cell network in the blow fly. These neurons are thought to encode the horizontal rotation axis during rapid free-flight maneuvers. Experimental findings suggest that neurons of the VS are strongly electrically coupled, and that several downstream neurons driving motor responses to ego-rotation receive inputs primarily from a small subset of VS cells. These downstream neurons must decode information about the axis of rotation from a partial readout of the VS population response. To investigate the role of coupling, we simulated the VS response to a variety of rotating visual scenes and computed optimal Bayesian estimates from the relevant subset of VS cells. Our analysis shows that coupling leads to near-optimal estimates from a subpopulation readout. In contrast, coupling between VS cells has no impact on the quality of encoding in the response of the full population. We conclude that coupling at one level of the fly visual system allows for near-optimal decoding from partial information at the subsequent, premotor level. Thus, electrical coupling may provide a general mechanism to achieve near-optimal information transfer from neuronal subpopulations across organisms and modalities. PMID:25186763

  14. Identification of Distinct Tumor Subpopulations in Lung Adenocarcinoma via Single-Cell RNA-seq.

    PubMed

    Min, Jae-Woong; Kim, Woo Jin; Han, Jeong A; Jung, Yu-Jin; Kim, Kyu-Tae; Park, Woong-Yang; Lee, Hae-Ock; Choi, Sun Shim

    2015-01-01

    Single-cell sequencing, which is used to detect clinically important tumor subpopulations, is necessary for understanding tumor heterogeneity. Here, we analyzed transcriptomic data obtained from 34 single cells from human lung adenocarcinoma (LADC) patient-derived xenografts (PDXs). To focus on the intrinsic transcriptomic signatures of these tumors, we filtered out genes that displayed extensive expression changes following xenografting and cell culture. Then, we performed clustering analysis using co-regulated gene modules rather than individual genes to minimize read drop-out errors associated with single-cell sequencing. This combined approach revealed two distinct intra-tumoral subgroups that were primarily distinguished by the gene module G64. The G64 module was predominantly composed of cell-cycle genes. E2F1 was found to be the transcription factor that most likely mediates the expression of the G64 module in single LADC cells. Interestingly, the G64 module also indicated inter-tumoral heterogeneity based on its association with patient survival and other clinical variables such as smoking status and tumor stage. Taken together, these results demonstrate the feasibility of single-cell RNA sequencing and the strength of our analytical pipeline for the identification of tumor subpopulations. PMID:26305796

  15. Birth season and environmental influences on blood leucocyte and lymphocyte subpopulations in rural Gambian infants

    PubMed Central

    Collinson, Andrew C; Ngom, Pa Tamba; Moore, Sophie E; Morgan, Gareth; Prentice, Andrew M

    2008-01-01

    Background In rural Gambia, birth season predicts infection-related adult mortality, providing evidence that seasonal factors in early life may programme immune development. This study tested whether lymphocyte subpopulations assessed by automated full blood count and flow cytometry in cord blood and at 8, 16 and 52 weeks in rural Gambian infants (N = 138) are affected by birth season (DRY = Jan-Jun, harvest season, few infections; WET = Jul-Dec, hungry season, many infections), birth size or micronutrient status. Results Geometric mean cord and postnatal counts were higher in births occurring in the WET season with both season of birth and season of sampling effects. Absolute CD3+, CD8+, and CD56+ counts, were higher in WET season births, but absolute CD4+ counts were unaffected and percentage CD4+ counts were therefore lower. CD19+ counts showed no association with birth season but were associated with concurrent plasma zinc status. There were no other associations between subpopulation counts and micronutrient or anthropometric status. Conclusion These results demonstrate a seasonal influence on cell counts with a disproportionate effect on CD8+ and CD56+ relative to CD4+ cells. This seasonal difference was seen in cord blood (indicating an effect in utero) and subsequent samples, and is not explained by nutritional status. These findings are consistent with the hypothesis than an early environmental exposure can programme human immune development. PMID:18462487

  16. Monocyte-derived dendritic cell subpopulations use different types of matrix metalloproteinases inhibited by GM6001.

    PubMed

    Kis-Toth, Katalin; Bacskai, Ildiko; Gogolak, Peter; Mazlo, Anett; Szatmari, Istvan; Rajnavolgyi, Eva

    2013-11-01

    Matrix metalloproteinases (MMPs) are endopeptidases with the potential to cleave extracellular matrix, support tissue renewal and regulate cell migration. Functional activities of MMPs are regulated by tissue inhibitors of MMPs (TIMPs) and disruption of the MMP-TIMP balance has pathological consequences. Here we studied the expression and secretion of MMPs and TIMPs in CD1a(-) and CD1a(+) monocyte-derived dendritic cell (DC) subpopulations. Our results showed that monocytes express TIMPs but lack MMPs, whereas upon differentiation to moDCs and in response to activation signals the expression of MMPs is increased and that of TIMPs is decreased. MMP-9 is expressed dominantly in the CD1a(-) subpopulation, while MMP-12 is preferentially expressed in CD1a(+) cells. Experiments performed with the synthetic MMP inhibitor GM6001 revealed that this drug efficiently inhibits the migration of moDCs through inactivation of MMPs. We conclude that modulation of MMP activity by GM6001 emerges as a novel approach to manipulate DC migration under inflammatory conditions. PMID:23870824

  17. Heterogeneity of Membrane Properties in Sympathetic Preganglionic Neurons of Neonatal Mice: Evidence of Four Subpopulations in the Intermediolateral Nucleus

    PubMed Central

    Zimmerman, Amanda

    2010-01-01

    Spinal cord sympathetic preganglionic neurons (SPNs) integrate activity from descending and sensory systems to determine the final central output of the sympathetic nervous system. The intermediolateral column (IML) has the highest number and density of SPNs and, within this region, SPN somas are found in distinct clusters within thoracic and upper lumbar spinal segments. Whereas SPNs exhibit a rostrocaudal gradient of end-target projections, individual clusters contain SPNs with diverse functional roles. Here we explored diversity in the electrophysiological properties observed in Hb9-eGFP–identified SPNs in the IML of neonatal mice. Overall, mouse SPN intrinsic membrane properties were comparable with those seen in other species. A wide range of values was obtained for all measured properties (up to a 10-fold difference), suggesting that IML neurons are highly differentiated. Using linear regression we found strong correlations between many cellular properties, including input resistance, rheobase, time constant, action potential shape, and degree of spike accommodation. The best predictor of cell function was rheobase, which correlated well with firing frequency–injected current (f–I) slopes as well as other passive and active membrane properties. The range in rheobase suggests that IML neurons have a recruitment order with stronger synaptic drives required for maximal recruitment. Using cluster analysis, we identified at least four subpopulations of SPNs, including one with a long time constant, low rheobase, and high f–I gain. We thus propose that the IML contains populations of neurons that are differentiable by their membrane properties and hypothesize they represent diverse functional classes. PMID:19923248

  18. MICAL2 is a novel human cancer gene controlling mesenchymal to epithelial transition involved in cancer growth and invasion.

    PubMed

    Mariotti, Sara; Barravecchia, Ivana; Vindigni, Carla; Pucci, Angela; Balsamo, Michele; Libro, Rosaliana; Senchenko, Vera; Dmitriev, Alexey; Jacchetti, Emanuela; Cecchini, Marco; Roviello, Franco; Lai, Michele; Broccoli, Vania; Andreazzoli, Massimiliano; Mazzanti, Chiara M; Angeloni, Debora

    2016-01-12

    The MICAL (Molecules Interacting with CasL) proteins catalyze actin oxidation-reduction reactions destabilizing F-actin in cytoskeletal dynamics. Here we show for the first time that MICAL2 mRNA is significantly over-expressed in aggressive, poorly differentiated/undifferentiated, primary human epithelial cancers (gastric and renal). Immunohistochemistry showed MICAL2-positive cells on the cancer invasive front and in metastasizing cancer cells inside emboli, but not at sites of metastasis, suggesting MICAL2 expression was 'on' in a subpopulation of primary cancer cells seemingly detaching from the tissue of origin, enter emboli and travel to distant sites, and was turned 'off' upon homing at metastatic sites. In vitro, MICAL2 knock-down resulted in mesenchymal to epithelial transition, reduction of viability, and loss of motility and invasion properties of human cancer cells. Moreover, expression of MICAL2 cDNA in MICAL2-depleted cells induced epithelial to mesenchymal transition. Altogether our data indicate that MICAL2 over-expression is associated with cancer progression and metastatic disease. MICAL2 might be an important regulator of epithelial to mesenchymal transition and therefore a promising target for anti-metastatic therapy. PMID:26689989

  19. MICAL2 is a novel human cancer gene controlling mesenchymal to epithelial transition involved in cancer growth and invasion

    PubMed Central

    Vindigni, Carla; Pucci, Angela; Balsamo, Michele; Libro, Rosaliana; Senchenko, Vera; Dmitriev, Alexey; Jacchetti, Emanuela; Cecchini, Marco; Roviello, Franco; Lai, Michele; Broccoli, Vania; Andreazzoli, Massimiliano; Mazzanti, Chiara M.; Angeloni, Debora

    2016-01-01

    The MICAL (Molecules Interacting with CasL) proteins catalyze actin oxidation-reduction reactions destabilizing F-actin in cytoskeletal dynamics. Here we show for the first time that MICAL2 mRNA is significantly over-expressed in aggressive, poorly differentiated/undifferentiated, primary human epithelial cancers (gastric and renal). Immunohistochemistry showed MICAL2-positive cells on the cancer invasive front and in metastasizing cancer cells inside emboli, but not at sites of metastasis, suggesting MICAL2 expression was 'on' in a subpopulation of primary cancer cells seemingly detaching from the tissue of origin, enter emboli and travel to distant sites, and was turned 'off' upon homing at metastatic sites. In vitro, MICAL2 knock-down resulted in mesenchymal to epithelial transition, reduction of viability, and loss of motility and invasion properties of human cancer cells. Moreover, expression of MICAL2 cDNA in MICAL2-depleted cells induced epithelial to mesenchymal transition. Altogether our data indicate that MICAL2 over-expression is associated with cancer progression and metastatic disease. MICAL2 might be an important regulator of epithelial to mesenchymal transition and therefore a promising target for anti-metastatic therapy. PMID:26689989

  20. Epithelial TRPV1 signaling accelerates gingival epithelial cell proliferation.

    PubMed

    Takahashi, N; Matsuda, Y; Yamada, H; Tabeta, K; Nakajima, T; Murakami, S; Yamazaki, K

    2014-11-01

    Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the calcium-permeable thermosensitive transient receptor potential superfamily, is a sensor of thermal and chemical stimuli. TRPV1 is activated by noxious heat (> 43°C), acidic conditions (pH < 6.6), capsaicin, and endovanilloids. This pain receptor was discovered on nociceptive fibers in the peripheral nervous system. TRPV1 was recently found to be expressed by non-neuronal cells, such as epithelial cells. The oral gingival epithelium is exposed to multiple noxious stimuli, including heat and acids derived from endogenous and exogenous substances; however, whether gingival epithelial cells (GECs) express TRPV1 is unknown. We show that both TRPV1 mRNA and protein are expressed by GECs. Capsaicin, a TRPV1 agonist, elevated intracellular Ca(2+) levels in the gingival epithelial cell line, epi 4. Moreover, TRPV1 activation in epi 4 cells accelerated proliferation. These responses to capsaicin were inhibited by a specific TRPV1 antagonist, SB-366791. We also observed GEC proliferation in capsaicin-treated mice in vivo. No effects were observed on GEC apoptosis by epithelial TRPV1 signaling. To examine the molecular mechanisms underlying this proliferative effect, we performed complementary (c)DNA microarray analysis of capsaicin-stimulated epi 4 cells. Compared with control conditions, 227 genes were up-regulated and 232 genes were down-regulated following capsaicin stimulation. Several proliferation-related genes were validated by independent experiments. Among them, fibroblast growth factor-17 and neuregulin 2 were significantly up-regulated in capsaicin-treated epi 4 cells. Our results suggest that functional TRPV1 is expressed by GECs and contributes to the regulation of cell proliferation. PMID:25266715

  1. Accessibility to intracellular antigens within nutritionally deprived human mammary epithelial cells

    SciTech Connect

    Dairkee, S.H.; Puett, L.; Counelis, A.M.; Hackett, A.J. )

    1991-01-01

    The authors have previously demonstrated immunolocalization of antikeratin antibodies in apparently random subpopulations of malignant cells in fresh surgical specimens of breast carcinoma. The goal of the present study was to determine whether deficiencies in essential nutrients contribute toward cellular alterations in membrane integrity, consequently allowing antikeratin to bind to the cytoskeleton within live, unfixed cells. They have demonstrated here that in an in vitro model in which human mammary epithelial cells are subjected to an oxygen-glucose gradient, immunolocalization of antikeratin within the cells is observed in a dose-dependent manner in the depleted regions of the gradient, even though the cell appear to be morphologically unaltered. The potential use of antibodies to intracellular antigens for immunotargeting solid tumors and the use of this method in anti-body-loading studies toward understanding functional aspects of specific cellular antigens, as well as determining differential response of various cell types under these culture conditions, are discussed.

  2. Airway Epithelial miRNA Expression Is Altered in Asthma

    PubMed Central

    Solberg, Owen D.; Ostrin, Edwin J.; Love, Michael I.; Peng, Jeffrey C.; Bhakta, Nirav R.; Nguyen, Christine; Solon, Margaret; Nguyen, Cindy; Barczak, Andrea J.; Zlock, Lorna T.; Blagev, Denitza P.; Finkbeiner, Walter E.; Ansel, K. Mark; Arron, Joseph R.; Erle, David J.

    2012-01-01

    Rationale: Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. Objectives: To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13–regulated miRNAs. Methods: We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. Measurements and Main Results: Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate < 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate < 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. Conclusions: Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway

  3. Pre-existing Epithelial Diversity in Normal Human Livers: A Tissue-tethered Cytometric Analysis in Portal/Periportal Epithelial Cells

    PubMed Central

    Isse, Kumiko; Lesniak, Andrew; Grama, Kedar; Maier, John; Specht, Susan; Castillo-Rama, Marcela; Lunz, John; Roysam, Badrinath; Michalopoulos, George; Demetris, Anthony J.

    2012-01-01

    Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BEC). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the Canals of Hering and/or metaplasia of pre-existing mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high resolution whole slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes pre-existed in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. “Virtually digested” WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g. scatter plots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically-associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. Results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bi-potential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. Conclusion Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable pre-existent hybrid epithelial diversity in normal human liver. This computationally-enabled tissue analysis approach offers much broader potential beyond the results presented here. PMID:23150208

  4. T-cell differentation in lethally irradiated and reconstituted mice: functional recovery of PNA-fractionated subpopulations. [X ray

    SciTech Connect

    Kraal, G.; Groeneveld, P.H.P.; Boden, D.; Kors, N.

    1981-07-15

    In mammals T lymphocytes are generated in the thymus from hemopoietic precursor cells. The thymus is able to instruct the T cells during their differentiation resulting in functionally different subpopulations which can leave the thymus for the peripheral lymphoid organs. These functionally different T-cell subpopulations have been described using the alloantigens Ly-1, 2, and 3. Another probe to study thymus subpopulations is the use of the nonmitogenic lectin peanut agglutinin (PNA). After lethal irradiation and reconsitution (IR) there is a 6-day lag before thymus cells start to proliferate. In the first 6 days there is a relative enrichment of PNA-negative cells, which are Ly-1/sup +/ and radioresistant. After IR the mitogenic responses of the PNA-negative cells recover earlier than those of the PNA-positive cells; the implications of this finding for different T-cell lineages are discussed.

  5. [Epithelial hepatoblastomas in the adult].

    PubMed

    Mondragón Sánchez, R; Bernal Maldonado, R; Sada Navarro, L A; Hernández, A I; Hurtado Andrade, H; Cortés Espinoza, T; Sánchez Cisneros, R

    1994-01-01

    Hepatoblastoma is the most frequent primary malignant liver neoplasm in childhood; in adults it is extremely rare and only 27 cases have been published. The prognosis of this neoplasm is poor because it is usually discovered late. Surgery, chemotherapy and liver transplantation have been tried with poor results. We present two adult patients who were diagnosed with an epithelial hepatoblastoma. The pathogenesis, histologic features and current management is reviewed. PMID:7716366

  6. Differential influence of organ site on three subpopulations of a single mouse mammary tumor at two distinct steps in metastasis.

    PubMed

    Aslakson, C J; Rak, J W; Miller, B E; Miller, F R

    1991-02-01

    Tumor subpopulations 66c14, 168FARN, and 4T07 are drug-resistant variants selected from sister subpopulations derived from a single mouse mammary tumor. These subpopulations are heterogeneous in their capacities to form experimental metastatic growth in the lungs and liver. Initial survival kinetics of arrested cells, determined by the clearance of 125IUdR-labelled cells, and subsequent growth rates, determined by sequential recovery of clonogenic tumor cells from occult metastases, both correlated with organ-colonizing potential as determined by necropsy. The growth rates of these 3 subpopulations were determined in vitro in monolayer and in situ in the subcutis, in the liver following intrasplenic injection, and in the lung following intravenous injection. Clonogenic potential of all 3 lines was similar in vitro (54-59%). Growth rates in vitro (population doubling times 16.5-21 hr) and in the subcutis (tumor volume doubling times 5.2-7.4 days) were similar for the 3 subpopulations, but differed significantly in the liver and lungs. For line 4T07, the most metastatic line to both lung and liver, population doubling times in vitro and in the lung and liver were similar, ranging from 17 to 26 hr. For lines 66c14 and 168FARN, the growth rates in lungs and livers were much slower than in vitro. Line 66c14, which is relatively more metastatic to the lungs, grew much faster in the lung (39 hours) than in the liver (91 hr), but line 168FARN, which is relatively more metastatic to the liver, grew at a faster rate in the liver (37 hr) than in the lung (63 hr). Thus, 3 tumor subpopulations (seeds) derived from a single tumor were differentially affected by host organ factors (soil) at 2 distinct stages in the metastatic process. PMID:1993557

  7. Particle size-density relationships in pyroclastic deposits: using component subpopulations to elucidate depositional conditions

    NASA Astrophysics Data System (ADS)

    Mackaman-Lofland, C. A.; Brand, B. D.; Taddeucci, J.

    2012-12-01

    Pyroclastic Density Currents (PDCs) are ground-hugging currents of hot gas, ash, and rock that travel at velocities up to 150 m/s down the flanks of volcanoes. PDCs are the most dangerous hazard associated with explosive volcanic eruptions, but because of current opacity and the risk inherent to observing PDCs in real time, their processes are poorly understood. Geologists rely on depositional relationships to lend insight into PDC transport and depositional processes. Outcrop exposure is typically incomplete, however, and the extent to which outcrop-scale depositional characteristics are representative of the parent current is still uncertain. The May 18th, 1980 eruption of Mt. St. Helens (MSH) produced multiple PDCs, burying the area north of the crater under 10s of meters of PDC deposits. Deep drainage erosion over the past 30 years has exposed these deposits in three dimensions, allowing a detailed study of deposit structures to be conducted for a variety of locations and depositional regimes with distance from source. We examine the grain size distribution and density characteristics of the discrete component subpopulations that make up the solids fraction of PDC deposits, focusing on changes associated with lateral facies variation, distance from source, and degree of topographic roughness. We analyze the grain size and density relationships of the component subpopulations using sequential fragmentation / transport theory (SFT), and use crystal morphoscopy to determine how different regional transport systems effect feldspar and hornblende crystal shape following the methods of Taddeucci and Palladino ((2002) Particle size-density relationships in pyroclastic deposits: inferences for emplacement processes. Bull Volcanol 64:273-284). Calculations of representative proximal and distal samples indicate juvenile pumice densities of ~1.3g/mL, accidental lithic densities of ~2.7g/mL, and crystal densities of ~2.6g/mL. We observe a general decrease in grain size and

  8. Differential down-regulation of HLA-DR on monocyte subpopulations during systemic inflammation

    PubMed Central

    2010-01-01

    Introduction Decreased expression of human leukocyte antigen class II (HLA-DR) on monocytes is a hallmark of altered immune status in patients with a systemic inflammatory response syndrome (SIRS). So far, the analyses were mainly performed without taking into account monocytes subpopulations. Methods We studied this modification on CD14HIGH and CD14LOW monocytes of 20 SIRS patients undergoing abdominal aortic surgery (AAS), 20 patients undergoing carotid artery surgery (CAS), and 9 healthy controls, and we investigated mediators and intracellular molecules that may be involved in this process. Results HLA-DR on CD14HIGH monocytes started to decrease during surgery, after blood reperfusion, and was further reduced post-surgery. In contrast, HLA-DR expression on CD14LOW cells only decreased after surgery, and to a lesser extent than on CD14HIGH monocytes. Negative correlations were found between the reduction of HLA-DR expression and the change in cortisol levels for both subpopulations, whereas a negative correlation between interleukin-10 (IL-10) levels and HLA-DR modulation was only observed for CD14HIGH cells. In accordance with these ex vivo results, HLA-DR on CD14HIGH and CD14LOW monocytes of healthy donors was reduced following incubation with hydrocortisone, whereas IL-10 only acted on CD14HIGH subpopulation. Furthermore, flow cytometry revealed that the expression of IL-10 receptor was higher on CD14HIGH versus CD14LOW monocytes. In addition, hydrocortisone, and to a lesser extent IL-10, reversed the up-regulation of HLA-DR induced by bacterial products. Finally, membrane-associated RING-CH-1 protein (MARCH1) mRNA, a negative regulator of MHC class II, was up-regulated in monocytes of AAS patients on Day 1 post-surgery, and in those of healthy subjects exposed to hydrocortisone. Conclusions This study reveals that HLA-DR expression is modulated differently on CD14HIGH (classical) versus CD14LOW (inflammatory) monocytes after systemic inflammation. PMID

  9. The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

    PubMed

    Helley, M P; Abate, W; Jackson, S K; Bennett, J H; Thompson, S W N

    2015-12-01

    The recent discovery that mammalian nociceptors express Toll-like receptors (TLRs) has raised the possibility that these cells directly detect and respond to pathogens with implications for either direct nociceptor activation or sensitization. A range of neuronal TLRs have been identified, however a detailed description regarding the distribution of expression of these receptors within sub-populations of sensory neurons is lacking. There is also some debate as to the composition of the TLR4 receptor complex on sensory neurons. Here we use a range of techniques to quantify the expression of TLR4, TLR7 and some associated molecules within neurochemically-identified sub-populations of trigeminal (TG) and dorsal root (DRG) ganglion sensory neurons. We also detail the pattern of expression and co-expression of two isoforms of lysophosphatidylcholine acyltransferase (LPCAT), a phospholipid remodeling enzyme previously shown to be involved in the lipopolysaccharide-dependent TLR4 response in monocytes, within sensory ganglia. Immunohistochemistry shows that both TLR4 and TLR7 preferentially co-localize with transient receptor potential vallinoid 1 (TRPV1) and purinergic receptor P2X ligand-gated ion channel 3 (P2X3), markers of nociceptor populations, within both TG and DRG. A gene expression profile shows that TG sensory neurons express a range of TLR-associated molecules. LPCAT1 is expressed by a proportion of both nociceptors and non-nociceptive neurons. LPCAT2 immunostaining is absent from neuronal profiles within both TG and DRG and is confined to non-neuronal cell types under naïve conditions. Together, our results show that nociceptors express the molecular machinery required to directly respond to pathogenic challenge independently from the innate immune system. PMID:26434622

  10. TTC7A mutations disrupt intestinal epithelial apicobasal polarity

    PubMed Central

    Bigorgne, Amélie E.; Farin, Henner F.; Lemoine, Roxane; Mahlaoui, Nizar; Lambert, Nathalie; Gil, Marine; Schulz, Ansgar; Philippet, Pierre; Schlesser, Patrick; Abrahamsen, Tore G.; Oymar, Knut; Davies, E. Graham; Ellingsen, Christian Lycke; Leteurtre, Emmanuelle; Moreau-Massart, Brigitte; Berrebi, Dominique; Bole-Feysot, Christine; Nischke, Patrick; Brousse, Nicole; Fischer, Alain; Clevers, Hans; de Saint Basile, Geneviève

    2013-01-01

    Multiple intestinal atresia (MIA) is a rare cause of bowel obstruction that is sometimes associated with a combined immunodeficiency (CID), leading to increased susceptibility to infections. The factors underlying this rare disease are poorly understood. We characterized the immunological and intestinal features of 6 unrelated MIA-CID patients. All patients displayed a profound, generalized lymphocytopenia, with few lymphocytes present in the lymph nodes. The thymus was hypoplastic and exhibited an abnormal distribution of epithelial cells. Patients also had profound disruption of the epithelial barrier along the entire gastrointestinal tract. Using linkage analysis and whole-exome sequencing, we identified 10 mutations in tetratricopeptide repeat domain–7A (TTC7A), all of which potentially abrogate TTC7A expression. Intestinal organoid cultures from patient biopsies displayed an inversion of apicobasal polarity of the epithelial cells that was normalized by pharmacological inhibition of Rho kinase. Our data indicate that TTC7A deficiency results in increased Rho kinase activity, which disrupts polarity, growth, and differentiation of intestinal epithelial cells, and which impairs immune cell homeostasis, thereby promoting MIA-CID development. PMID:24292712

  11. CaMKII regulates the strength of the epithelial barrier

    PubMed Central

    Shiomi, Ryo; Shigetomi, Kenta; Inai, Tetsuichiro; Sakai, Masami; Ikenouchi, Junichi

    2015-01-01

    Epithelial cells define the boundary between the outside and the inside of our body by constructing the diffusion barrier. Tight junctions (TJs) of epithelial cells function as barriers against invasion of harmful microorganisms into the human body and free diffusion of water or ions from the body. Therefore, formation of TJs has to be strictly controlled in epithelial cells. However, the molecular mechanisms governing this regulation are largely unknown. In this study, we identified Ca2+/calmodulin-dependent protein kinase II (CaMKII) as a regulator of the barrier function of TJs. CaMKII inhibition led to enlargement of TJ-areas and up-regulation of the barrier function. CaMKII inhibition induced excess TJ formation in part by the activation of AMP-activated protein kinase (AMPK) and subsequent phosphorylation of claudin-1. As up-regulation of epithelial barriers is essential for the prevention of chronic inflammatory diseases, the identification of CaMKII as a modulator of TJ function paves the way for the development of new drugs to treat these diseases. PMID:26281891

  12. Epithelial junctions and Rho family GTPases: the zonular signalosome

    PubMed Central

    Citi, Sandra; Guerrera, Diego; Spadaro, Domenica; Shah, Jimit

    2014-01-01

    The establishment and maintenance of epithelial cell-cell junctions is crucially important to regulate adhesion, apico-basal polarity and motility of epithelial cells, and ultimately controls the architecture and physiology of epithelial organs. Junctions are supported, shaped and regulated by cytoskeletal filaments, whose dynamic organization and contractility are finely tuned by GTPases of the Rho family, primarily RhoA, Rac1 and Cdc42. Recent research has identified new molecular mechanisms underlying the cross-talk between these GTPases and epithelial junctions. Here we briefly summarize the current knowledge about the organization, molecular evolution and cytoskeletal anchoring of cell-cell junctions, and we comment on the most recent advances in the characterization of the interactions between Rho GTPases and junctional proteins, and their consequences with regards to junction assembly and regulation of cell behavior in vertebrate model systems. The concept of “zonular signalosome” is proposed, which highlights the close functional relationship between proteins of zonular junctions (zonulae occludentes and adhaerentes) and the control of cytoskeletal organization and signaling through Rho GTPases, transcription factors, and their effectors. PMID:25483301

  13. CaMKII regulates the strength of the epithelial barrier.

    PubMed

    Shiomi, Ryo; Shigetomi, Kenta; Inai, Tetsuichiro; Sakai, Masami; Ikenouchi, Junichi

    2015-01-01

    Epithelial cells define the boundary between the outside and the inside of our body by constructing the diffusion barrier. Tight junctions (TJs) of epithelial cells function as barriers against invasion of harmful microorganisms into the human body and free diffusion of water or ions from the body. Therefore, formation of TJs has to be strictly controlled in epithelial cells. However, the molecular mechanisms governing this regulation are largely unknown. In this study, we identified Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a regulator of the barrier function of TJs. CaMKII inhibition led to enlargement of TJ-areas and up-regulation of the barrier function. CaMKII inhibition induced excess TJ formation in part by the activation of AMP-activated protein kinase (AMPK) and subsequent phosphorylation of claudin-1. As up-regulation of epithelial barriers is essential for the prevention of chronic inflammatory diseases, the identification of CaMKII as a modulator of TJ function paves the way for the development of new drugs to treat these diseases. PMID:26281891

  14. Sp3 regulates fas expression in lung epithelial cells.

    PubMed Central

    Pang, H; Miranda, K; Fine, A

    1998-01-01

    By transducing an apoptotic signal in immune effector cells, Fas has been directly implicated in the control of immunological activity. Expression and functional results, however, have also suggested a role for Fas in regulating cell turnover in specific epithelial populations. To characterize factors responsible for Fas expression in epithelial cells, approximately 3 kb of the 5' flanking region of the mouse Fas gene was isolated. By rapid amplification of cDNA ends and primer extension, transcriptional start sites were identified within 50 bp upstream of the translation start site. Transient transfection of promoter-luciferase constructs in a mouse lung epithelial cell line, MLE-15, localized promoter activity to the first 77 bp of upstream sequence. By using a 60 bp DNA probe (-18 to -77) in electrophoretic mobility-shift assays, three shifted complexes were found. Incubation with excess cold Sp1 oligonucleotide or an anti-Sp3 antibody inhibited complex formation. Site-directed mutagenesis of the Sp1 site resulted in 60-70% loss of promoter activity. In Drosophila SL-2 cells, promoter activity was markedly increased by co-transfection of an Sp3 expression construct. These results show that the Sp3 protein is involved in regulating Fas gene expression in lung epithelial cells. PMID:9639581

  15. An innate antiviral pathway acting before interferons at epithelial surfaces.

    PubMed

    Iversen, Marie B; Reinert, Line S; Thomsen, Martin K; Bagdonaite, Ieva; Nandakumar, Ramya; Cheshenko, Natalia; Prabakaran, Thaneas; Vakhrushev, Sergey Y; Krzyzowska, Malgosha; Kratholm, Sine K; Ruiz-Perez, Fernando; Petersen, Steen V; Goriely, Stanislas; Bibby, Bo Martin; Eriksson, Kristina; Ruland, Jürgen; Thomsen, Allan R; Herold, Betsy C; Wandall, Hans H; Frische, Sebastian; Holm, Christian K; Paludan, Søren R

    2016-02-01

    Mucosal surfaces are exposed to environmental substances and represent a major portal of entry for microorganisms. The innate immune system is responsible for early defense against infections and it is believed that the interferons (IFNs) constitute the first line of defense against viruses. Here we identify an innate antiviral pathway that works at epithelial surfaces before the IFNs. The pathway is activated independently of known innate sensors of viral infections through a mechanism dependent on viral O-linked glycans, which induce CXCR3 chemokines and stimulate antiviral activity in a manner dependent on neutrophils. This study therefore identifies a previously unknown layer of antiviral defense that exerts its action on epithelial surfaces before the classical IFN response is operative. PMID:26595890

  16. Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

    PubMed

    Pickard, Adam; Cichon, Ann-Christin; Barry, Anna; Kieran, Declan; Patel, Daksha; Hamilton, Peter; Salto-Tellez, Manuel; James, Jacqueline; McCance, Dennis J

    2012-07-18

    Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions. PMID:22643222

  17. Tendon Progenitor Cells in Injured Tendons Have Strong Chondrogenic Potential: The CD105-Negative Subpopulation Induces Chondrogenic Degeneration

    PubMed Central

    Asai, Shuji; Otsuru, Satoru; Candela, Maria Elena; Cantley, Leslie; Uchibe, Kenta; Hofmann, Ted J.; Zhang, Kairui; Wapner, Keith L.; Soslowsky, Louis J; Horwitz, Edwin M.; Enomoto-Iwamoto, Motomi

    2014-01-01

    To study the cellular mechanism of the tendon repair process, we used a mouse Achilles tendon injury model to focus on the cells recruited to the injured site. The cells isolated from injured tendon 1 week after the surgery and uninjured tendons contained the connective tissue progenitor populations as determined by colony-forming capacity, cell surface markers and multipotency. When the injured tendon-derived progenitor cells (inTPCs) were transplanted into injured Achilles tendons, they were not only integrated in the regenerating area expressing tenogenic phenotype but also trans-differentiated into chondrogenic cells in the degenerative lesion that underwent ectopic endochondral ossification. Surprisingly, the micromass culture of the inTPCs rapidly underwent chondrogenic differentiation even in the absence of exogenous BMPs or TGFβs. The cells isolated from human ruptured tendon tissues also showed connective tissue progenitor properties and exhibited stronger chondrogenic ability than bone marrow stromal cells. The mouse inTPCs contained two subpopulations one positive and one negative for CD105, a co-receptor of the TGFβ superfamily. The CD105-negative cells showed superior chondrogenic potential in vitro and induced larger chondroid degenerative lesions in mice as compared to the CD105-positive cells. These findings indicate that tendon progenitor cells are recruited to the injured site of tendons and have a strong chondrogenic potential and that the CD105-negative population of these cells would be the cause for chondroid degeneration in injured tendons. The newly identified cells recruited to the injured tendon may provide novel targets to develop therapeutic strategies to facilitate tendon repair. PMID:25220576

  18. Emergence of a novel subpopulation of CC398 Staphylococcus aureus infecting animals is a serious hazard for humans

    PubMed Central

    van der Mee-Marquet, Nathalie L.; Corvaglia, Anna; Haenni, Marisa; Bertrand, Xavier; Franck, Jean-Baptiste; Kluytmans, Jan; Girard, Myriam; Quentin, Roland; François, Patrice

    2014-01-01

    Until recently, Staphylococcus aureus from clonal complex (CC)398 were mostly described as colonizing asymptomatic raised pigs and pig-farmers. Currently, the epidemiology of the CC398 lineage is becoming more complex. CC398 human-adapted isolates are increasingly being identified in bloodstream infections in humans living in animal-free environments. In addition, CC398 isolates are increasingly responsible for invasive infections in various animals. CC398 isolates that colonize asymptomatic pigs and the isolates that infect humans living in animal-free environments (human-adapted isolates) both lack several clinically important S. aureus–associated virulence factors but differ on the basis of their prophage content. Recent findings have provided insight into the influence of a φMR11-like helper prophage on the ability of CC398 isolates to infect humans. To assess the recent spread of the CC398 lineage to various animal species and to investigate the links between the φMR11-like prophage and the emergence of CC398 isolates infecting animals, we studied 277 isolates causing infections in unrelated animals. The prevalence of CC398 isolates increased significantly between 2007 and 2013 (p < 0.001); 31.8% of the animal isolates harbored the φMR11-like prophage. High-density DNA microarray experiments with 37 representative infected-animal isolates positive for φMR11-like DNA established that most infected-animal isolates carried many genetic elements related to antimicrobial resistance and virulence genes, and a φ3 prophage encoding immune-modulating proteins and associated with animal-to-human jumps. Our findings suggest recent clonal expansion and dissemination of a new subpopulation of CC398 isolates, responsible for invasive infections in various animals, with a considerable potential to colonize and infect humans, probably greater than that of human-adapted CC398 isolates, justifying active surveillance. PMID:25538688

  19. Oligopeptides as Biomarkers of Cyanobacterial Subpopulations. Toward an Understanding of Their Biological Role

    PubMed Central

    Agha, Ramsy; Quesada, Antonio

    2014-01-01

    Cyanobacterial oligopeptides comprise a wide range of bioactive and/or toxic compounds. While current research is strongly focused on exploring new oligopeptide variants and their bioactive properties, the biological role of these compounds remains elusive. Oligopeptides production abilities show a remarkably patchy distribution among conspecific strains. This observation has prompted alternative approaches to unveil their adaptive value, based on the use of cellular oligopeptide compositions as biomarkers of intraspecific subpopulations or chemotypes in freshwater cyanobacteria. Studies addressing the diversity, distribution, and dynamics of chemotypes in natural systems have provided important insights into the structure and ecology of cyanobacterial populations and the adaptive value of oligopeptides. This review presents an overview of the fundamentals of this emerging approach and its most relevant findings, and discusses our current understanding of the role of oligopeptides in the ecology of cyanobacteria. PMID:24960202

  20. LKB1/STK11 Inactivation Leads to Expansion of a Prometastatic Tumor Subpopulation in Melanoma

    PubMed Central

    Liu, Wenjin; Monahan, Kimberly B.; Pfefferle, Adam D.; Shimamura, Takeshi; Sorrentino, Jessica; Chan, Keefe T.; Roadcap, David W.; Ollila, David W.; Thomas, Nancy E.; Castrillon, Diego H.; Miller, C. Ryan; Perou, Charles M.; Wong, Kwok-Kin; Bear, James E.

    2013-01-01

    SUMMARY Germline mutations in LKB1 (STK11) are associated with the Peutz-Jeghers syndrome (PJS), which includes aberrant mucocutaneous pigmentation, and somatic LKB1 mutations occur in 10% of cutaneous melanoma. By somatically inactivating Lkb1 with K-Ras activation (±p53 loss) in murine melanocytes, we observed variably pigmented and highly metastatic melanoma with 100% penetrance. LKB1 deficiency resulted in increased phosphorylation of the SRC family kinase (SFK) YES, increased expression of WNT target genes, and expansion of a CD24+ cell population, which showed increased metastatic behavior in vitro and in vivo relative to isogenic CD24− cells. These results suggest that LKB1 inactivation in the context of RAS activation facilitates metastasis by inducing an SFK-dependent expansion of a prometastatic, CD24+ tumor subpopulation. PMID:22698401

  1. KRT14 marks a subpopulation of bladder basal cells with pivotal role in regeneration and tumorigenesis

    PubMed Central

    Papafotiou, George; Paraskevopoulou, Varvara; Vasilaki, Eleni; Kanaki, Zoi; Paschalidis, Nikolaos; Klinakis, Apostolos

    2016-01-01

    The urothelium is a specialized epithelium that lines the urinary tract. It consists of three different cell types, namely, basal, intermediate and superficial cells arranged in relatively distinct cell layers. Normally, quiescent, it regenerates fast upon injury, but the regeneration process is not fully understood. Although several reports have indicated the existence of progenitors, their identity and exact topology, as well as their role in key processes such as tissue regeneration and carcinogenesis have not been clarified. Here we show that a minor subpopulation of basal cells, characterized by the expression of keratin 14, possesses self-renewal capacity and also gives rise to all cell types of the urothelium during natural and injury-induced regeneration. Moreover, these cells represent cells of origin of urothelial cancer. Our findings support the hypothesis of basally located progenitors with profound roles in urothelial homoeostasis. PMID:27320313

  2. Human colonic goblet cells. Demonstration of distinct subpopulations defined by mucin-specific monoclonal antibodies.

    PubMed Central

    Podolsky, D K; Fournier, D A; Lynch, K E

    1986-01-01

    We studied glycoprotein content of human colonic goblet cells, using a library of monoclonal antibodies (MAbs) directed against purified human colonic mucin (HCM). Using indirect immunofluorescence (IIF), we found that 17 of 23 anti-HCM MAbs stained some or all goblet cells of normal human colonic mucosa. We observed a variety of cellular staining patterns, including (a) diffuse (homogeneous) staining of intracellular mucin, (b) speckled (inhomogeneous) staining of mucin droplets, (c) peripheral staining of intracellular droplets, (d) cytoplasmic staining of goblet cells, and (e) apical (luminal) surface staining. Staining patterns were not associated with particular HCM species. In addition to variable patterns of IIF within individual cells, anti-HCM MAbs varied in the proportion of goblet cells stained. Some MAbs stained all goblet cells, while others stained a limited number of goblet cells. Although each goblet cell contained more than one type mucin, HCM species III, and IV and V appeared to exist in mutually exclusive goblet cell populations and it was possible to define at least seven subpopulations of goblet cells in colonic mucosa by their content of various combinations of HCM species. Anti-HCM MAbs stained goblet cells from other sites within the gastrointestinal tract to a varying extent. Anti-HCM MAbs also showed extensive cross-reactivity with rodent, rabbit, and monkey colonic mucosa. However, several anti-HCM MAbs stained only human colonic mucosa. These data show that human colonic mucosa contains discrete subpopulations of goblet cells that produce distinctive combinations of specific mucin glycoprotein species. Images PMID:2420829

  3. Population-level differences in revascularization treatment and outcomes among various United States subpopulations

    PubMed Central

    Graham, Garth; Xiao, Yang-Yu Karen; Rappoport, Dan; Siddiqi, Saima

    2016-01-01

    Despite recent general improvements in health care, significant disparities persist in the cardiovascular care of women and racial/ethnic minorities. This is true even when income, education level, and site of care are taken into consideration. Possible explanations for these disparities include socioeconomic considerations, elements of discrimination and racism that affect socioeconomic status, and access to adequate medical care. Coronary revascularization has become the accepted and recommended treatment for myocardial infarction (MI) today and is one of the most common major medical interventions in the United States, with more than 1 million procedures each year. This review discusses recent data on disparities in co-morbidities and presentation symptoms, care and access to medical resources, and outcomes in revascularization as treatment for acute coronary syndrome, looking especially at women and minority populations in the United States. The data show that revascularization is used less in both female and minority patients. We summarize recent data on disparities in co-morbidities and presentation symptoms related to MI; access to care, medical resources, and treatments; and outcomes in women, blacks, and Hispanics. The picture is complicated among the last group by the many Hispanic/Latino subgroups in the United States. Some differences in outcomes are partially explained by presentation symptoms and co-morbidities and external conditions such as local hospital capacity. Of particular note is the striking differential in both presentation co-morbidities and mortality rates seen in women, compared to men, especially in women ≤ 55 years of age. Surveillance data on other groups in the United States such as American Indians/Alaska Natives and the many Asian subpopulations show disparities in risk factors and co-morbidities, but revascularization as treatment for MI in these populations has not been adequately studied. Significant research is required to

  4. A Cone-Beam Computed Tomographic Study on Mandibular First Molars in a Chinese Subpopulation

    PubMed Central

    Ma, Yue; Han, Ting; Chen, Xinyu; Wan, Fang; Lu, Yating; Yan, Songhe; Wang, Yan

    2015-01-01

    The purpose of this study was to conduct a cone-beam computed tomographic (CBCT) investigation on the root and canal configuration of the mandibular first molars, especially the morphology of the disto-lingual (DL) root, in a Chinese subpopulation. A total of 910 CBCT images of the mandibular first molars were collected from 455 patients who underwent CBCT examinations as a preoperative assessment for implants or orthodontic treatment. The following information was analyzed and evaluated: tooth position, gender, root and root canal number per tooth, root canal type of the mesial root(s) and distal root(s), angle of the DL root canal curvature, distance between two distal canal orifices in the teeth with DL root, and angle of disto-buccal canal orifice–disto-lingual canal orifice–mesio-lingual canal orifice (DB-DL-ML). Most of the mandibular first molars (64.9%, n = 591) had two roots with three root canals, and most of the mesial root canals (87.7%, n = 798) were type VI. The prevalence of the DL root was 22.1% (n = 201). The right side had a higher prevalence of DL root than the left side (p<0.05). Additionally, the curvature of the DL root canal were greater in the bucco-lingual (BL) orientation (30.10°±14.02°) than in the mesio-distal (MD) orientation (14.03°± 8.56°) (p<0.05). Overall there was a high prevalence of DL root in the mandibular first molars, and most of the DL roots were curved in different degrees. This study provided detailed information about the root canal morphology of the mandibular first molars in a Chinese subpopulation. PMID:26241480

  5. Subpopulations of liver coated vesicles resolved by preparative agarose gel electrophoresis

    SciTech Connect

    Kedersha, N.L.; Hill, D.F.; Kronquist, K.E.; Rome, L.H.

    1986-01-01

    Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis, the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from difference populations appeared identical on SDS PAGE, and triskelions stripped from the different populations showed the same mobility on the agarose gel, suggesting that the mobility differences observed in intact CVs were due to differences in the surface charge of underlying vesicles rather than to variations in their clathrin coats. Stripped CVs exhibited considerable heterogeneity when analyzed by Western blotting: the fast-migrating population was enriched in the mannose 6-phosphate receptor, secretory acetyl-choline esterase, and an M/sub r/ 195,000 glycoprotein. The slow-migrating population of CVs was enriched in the asialoglycoprotein receptor, and it appeared to contain all detectable concanavalin A-binding polypeptides as well as the bulk of detectable WGA-binding proteins. When CVs were prepared from /sup 125/I-asialoorosomucoid-perfused rat liver, ligand was found in the slow-migrating CVs, suggesting that these were endocytic in origin. Morphological differences were also observed: the fast-migrating population was enriched in smaller CVs, whereas the slow-migrating population exhibited an enrichment in larger CVs. As liver consists largely of hepatocytes, these subpopulations appear to originate from the same cell type and probably represent CVs of different intracellular origin and destination.

  6. Growth of Streptococcus mutans in Biofilms Alters Peptide Signaling at the Sub-population Level

    PubMed Central

    Shields, Robert C.; Burne, Robert A.

    2016-01-01

    Streptococcus mutans activates multiple cellular processes in response to the formation of a complex between comX-inducing peptide (XIP) and the ComR transcriptional regulator. Bulk phase and microfluidic experiments previously revealed that ComR-dependent activation of comX is altered by pH and by carbohydrate source. Biofilm formation is a major factor in bacterial survival and virulence in the oral cavity. Here, we sought to determine the response of S. mutans biofilm cells to XIP during different stages of biofilm maturation. Using flow cytometry and confocal microscopy, we showed that exogenous addition of XIP to early biofilms resulted in robust comX activation. However, as the biofilms matured, increasing amounts of XIP were required to activate comX expression. Single-cell analysis demonstrated that the entire population was responding to XIP with activation of comX in early biofilms, but only a sub-population was responding in mature biofilms. The sub-population response of mature biofilms was retained when the cells were dispersed and then treated with XIP. The proportion and intensity of the bi-modal response of mature biofilm cells was altered in mutants lacking the Type II toxins MazF and RelE, or in a strain lacking the (p)ppGpp synthase/hydrolase RelA. Thus, competence signaling is markedly altered in cells growing in mature biofilms, and pathways that control cell death and growth/survival decisions modulate activation of comX expression in these sessile populations. PMID:27471495

  7. A cortical astrocyte subpopulation inhibits axon growth in vitro and in vivo.

    PubMed

    Liu, Rui; Wang, Zhe; Gou, Lin; Xu, Hanpeng

    2015-08-01

    Astrocytes are the most heterogeneous and predominant glial cell type in the central nervous system. However, the functional significance of this heterogeneity remains to be elucidated. Following injury, damaged astrocytes inhibit axonal regeneration in vivo and in vitro. Cultured primary astrocytes are commonly considered good supportive substrates for neuron attachment and axon regeneration. However, it is not known whether different populations of cells in the heterogeneous astrocyte culture affect neuron behavior in the same way. In the present study, the effect of astrocyte heterogeneity on neuronal attachment and neurite outgrowth was examined using an in vitro and in vivo coculture system. In vitro, neonatal cortical astrocytes were co-cultured with purified dorsal root ganglia (DRG) neurons and astrocyte growth morphology, neuron attachment and neurite growth were evaluated. The results demonstrated that the heterogeneous astrocyte cells showed two different types of growth pattern, typical and atypical. Typical astrocytes were supportive to neuron attachment and neurite growth, which was consistent with previous studies, whereas atypical astrocytes inhibited neuron attachment and neurite growth. These inhibitory astrocytes exhibited a special growth pattern with various shapes and sizes, a high cell density, few oligodendrocytes on the top layer and occupied a smaller growth area compared with typical astrocytes. Neurites extended freely on typical supportive astrocyte populations, however, moved away when they reached atypical astrocyte growth pattern. Neurons growing on the atypical astrocyte pattern demonstrated minimal neurite outgrowth and these neurites had a dystrophic appearance, however, neuronal survival was unaffected. Immunocytochemistry studies demonstrated that these atypical inhibitory astrocytes were glial fibrillary acidic protein (GFAP) positive cells. The existence of inhibitory astrocyte subpopulations in normal astrocytes reflects the

  8. Divergence Effect at Alaska's Treelines a Result of Sub-Population Behavior?

    NASA Astrophysics Data System (ADS)

    Wilmking, M.; Singh, J.

    2007-12-01

    The "divergence effect" (off-set between tree-ring based temperature reconstruction and measured temperatures) in northern forest poses a serious question to tree-ring based climate reconstructions, since it seems to violate the uniformitarian principle of dendroclimatology. Several possible reasons emerge, among them false assumptions about 1) climate data (e.g. which climate parameter can be modeled most effectively), 2) tree-ring data (e.g. shift in climate sensitivity of tree growth) or 3) a truly new and unprecedented phenomenon (e.g. rapid climate warming exceeding the adaptive capacity of trees). Here we test, if undetected emergent sub-population behavior at Alaska's treelines might result in a divergence effect. We reanalyzed seven data sets spanning the entire northern treeline in the Brooks Range, Alaska, which showed populations of trees responding positively, non-significant and negatively to recent warming. There is no effect of standardization (i.e. conventional versus RCS) technique on the grouping and we thus used RCS for the following analysis. Without grouping into sub- populations, a clear divergence between tree-ring based temperature reconstruction and actual climate data emerged. However, if only those trees were used for the climate reconstruction, which showed a consistent positive response to the target temperatures (June/July), tree growth modeled climate data extremely well following the rise in temperature during the last decades (actual climate data 0.53°/decade versus tree-ring based reconstruction 0.44°/decade over 1970-2000 period). This result hints at the possibility that some of the reported divergence might be eliminated (at least at Alaska's treelines) through careful test of sub-population behavior.

  9. Interleukin-17 induces an atypical M2-like macrophage subpopulation that regulates intestinal inflammation.

    PubMed

    Nishikawa, Kenichiro; Seo, Naohiro; Torii, Mie; Ma, Nei; Muraoka, Daisuke; Tawara, Isao; Masuya, Masahiro; Tanaka, Kyosuke; Takei, Yoshiyuki; Shiku, Hiroshi; Katayama, Naoyuki; Kato, Takuma

    2014-01-01

    Interleukin 17 (IL-17) is a pleiotropic cytokine that acts on both immune and non-immune cells and is generally implicated in inflammatory and autoimmune diseases. Although IL-17 as well as their source, mainly but not limited to Th17 cells, is also abundant in the inflamed intestine, the role of IL-17 in inflammatory bowel disease remains controversial. In the present study, by using IL-17 knockout (KO) mice, we investigated the role of IL-17 in colitis, with special focus on the macrophage subpopulations. Here we show that IL-17KO mice had increased susceptibility to DSS-induced colitis which was associated with decrease in expression of mRNAs implicated in M2 and/or wound healing macrophages, such as IL-10, IL-1 receptor antagonist, arginase 1, cyclooxygenase 2, and indoleamine 2,3-dioxygenase. Lamina propria leukocytes from inflamed colon of IL-17KO mice contained fewer CD11b+Ly6C+MHC Class II+ macrophages, which were derived, at least partly, from blood monocytes, as compared to those of WT mice. FACS-purified CD11b+ cells from WT mice, which were more abundant in Ly6C+MHC Class II+ cells, expressed increased levels of genes associated M2/wound healing macrophages and also M1/proinflammatory macrophages. Depletion of this population by topical administration of clodronate-liposome in the colon of WT mice resulted in the exacerbation of colitis. These results demonstrate that IL-17 confers protection against the development of severe colitis through the induction of an atypical M2-like macrophage subpopulation. Our findings reveal a previously unappreciated mechanism by which IL-17 exerts a protective function in colitis. PMID:25254662

  10. Interleukin-2 in relation to T cell subpopulations in rheumatic heart disease.

    PubMed Central

    Zedan, M M; el-Shennawy, F A; Abou-Bakr, H M; al-Basousy, A M

    1992-01-01

    Interleukin-2 (IL-2) and T cell subpopulations were evaluated in children with rheumatic heart disease (RHD). Three groups were included: 13 patients with active RHD, 12 with non-active RHD, and 14 control children. Serum IL-2 and T cell subpopulations were measured by radioimmunoassay and monoclonal antibodies respectively. Patients with active RHD showed a significant increase in IL-2 concentrations and helper:suppressor (H:S) ratio compared with controls with a mean (SEM) IL-2 of 3.48 (0.62) v 1.26 (0.16) U/ml and H:S ratio 2.31 (0.14) v 1.66 (0.04). There was a significant decrease in T suppressor (CD8+) and pan T (CD3+) cells compared with controls with a mean (SEM) for CD8+ of 23.75 (1.19) v 32.23 (0.56)% and CD3+ of 79.55 (0.94) v 85.00 (0.11)%. Patients with non-active RHD showed a significant decrease only in the CD3+ cells (78.20 (0.20)%) when compared with controls. A deficiency of CD3+ cells is a constant finding in patients with RHD, whether the disease is active or not. There was a significant increase in IL-2 concentration with a significant decrease in CD8+ cells in patients with active RHD in comparison with the non-active group (mean (SEM) IL-2 of 3.48 (0.62) v 1.85 (0.24) U/ml and CD8+ of 23.75 (1.19) v 28.83 (1.91)%). Thus an increase in IL-2 and a decrease in CD8+ cells may be related to rheumatic activity. T helper (CD4+) cells did not differ significantly between groups. PMID:1471890

  11. Estimating abundance of the Southern Hudson Bay polar bear subpopulation using aerial surveys, 2011 and 2012

    USGS Publications Warehouse

    Obbard, Martyn E.; Middel, Kevin R.; Stapleton, Seth P.; Thibault, Isabelle; Brodeur, Vincent; Jutras, Charles

    2013-01-01

    The Southern Hudson Bay (SH) polar bear subpopulation occurs at the southern extent of the species’ range. Although capture-recapture studies indicate that abundance remained stable between 1986 and 2005, declines in body condition and survival were documented during the period, possibly foreshadowing a future decrease in abundance. To obtain a current estimate of abundance, we conducted a comprehensive line transect aerial survey of SH during 2011–2012. We stratified the study site by anticipated densities and flew coastal contour transects and systematically spaced inland transects in Ontario and on Akimiski Island and large offshore islands in 2011. Data were collected with double observer and distance sampling protocols. We also surveyed small islands in Hudson Bay and James Bay and flew a comprehensive transect along the Québec coastline in 2012. We observed 667 bears in Ontario and on Akimiski Island and nearby islands in 2011, and we sighted 80 bears on offshore islands during 2012. Mark-recapture distance sampling and sightresight models yielded a model-averaged estimate of 868 (SE: 177) for the 2011 study area. Our estimate of abundance for the entire SH subpopulation (951; SE: 177) suggests that abundance has remained unchanged. However, this result should be interpreted cautiously because of the methodological differences between historical studies (physical capture) and this survey. A conservative management approach is warranted given the previous increases in the duration of the ice-free season, which are predicted to continue in the future, and previously documented declines in body condition and vital rates.

  12. Estimating the abundance of the Southern Hudson Bay polar bear subpopulation with aerial surveys

    USGS Publications Warehouse

    Obbard, Martyn E.; Stapleton, Seth P.; Middel, Kevin R.; Thibault, Isabelle; Brodeur, Vincent; Jutras, Charles

    2015-01-01

    The Southern Hudson Bay (SH) polar bear subpopulation occurs at the southern extent of the species’ range. Although capture–recapture studies indicate abundance was likely unchanged between 1986 and 2005, declines in body condition and survival occurred during the period, possibly foreshadowing a future decrease in abundance. To obtain a current estimate of abundance, we conducted a comprehensive line transect aerial survey of SH during 2011–2012. We stratified the study site by anticipated densities and flew coastal contour transects and systematically spaced inland transects in Ontario and on Akimiski Island and large offshore islands in 2011. Data were collected with double-observer and distance sampling protocols. We surveyed small islands in James Bay and eastern Hudson Bay and flew a comprehensive transect along the Québec coastline in 2012. We observed 667 bears in Ontario and on Akimiski Island and nearby islands in 2011, and we sighted 80 bears on offshore islands during 2012. Mark–recapture distance sampling and sight–resight models yielded an estimate of 860 (SE = 174) for the 2011 study area. Our estimate of abundance for the entire SH subpopulation (943; SE = 174) suggests that abundance is unlikely to have changed significantly since 1986. However, this result should be interpreted cautiously because of the methodological differences between historical studies (physical capture–recapture) and this survey. A conservative management approach is warranted given previous increases in duration of the ice-free season, which are predicted to continue in the future, and previously documented declines in body condition and vital rates.

  13. Population-level differences in revascularization treatment and outcomes among various United States subpopulations.

    PubMed

    Graham, Garth; Xiao, Yang-Yu Karen; Rappoport, Dan; Siddiqi, Saima

    2016-01-26

    Despite recent general improvements in health care, significant disparities persist in the cardiovascular care of women and racial/ethnic minorities. This is true even when income, education level, and site of care are taken into consideration. Possible explanations for these disparities include socioeconomic considerations, elements of discrimination and racism that affect socioeconomic status, and access to adequate medical care. Coronary revascularization has become the accepted and recommended treatment for myocardial infarction (MI) today and is one of the most common major medical interventions in the United States, with more than 1 million procedures each year. This review discusses recent data on disparities in co-morbidities and presentation symptoms, care and access to medical resources, and outcomes in revascularization as treatment for acute coronary syndrome, looking especially at women and minority populations in the United States. The data show that revascularization is used less in both female and minority patients. We summarize recent data on disparities in co-morbidities and presentation symptoms related to MI; access to care, medical resources, and treatments; and outcomes in women, blacks, and Hispanics. The picture is complicated among the last group by the many Hispanic/Latino subgroups in the United States. Some differences in outcomes are partially explained by presentation symptoms and co-morbidities and external conditions such as local hospital capacity. Of particular note is the striking differential in both presentation co-morbidities and mortality rates seen in women, compared to men, especially in women ≤ 55 years of age. Surveillance data on other groups in the United States such as American Indians/Alaska Natives and the many Asian subpopulations show disparities in risk factors and co-morbidities, but revascularization as treatment for MI in these populations has not been adequately studied. Significant research is required to

  14. Epithelial cells and Von Gierke's disease.

    PubMed

    Negishi, H; Benke, P J

    1977-08-01

    Epithelial cells and not fibroblasts from human liver and amniotic fluid contain inducible glucose-6-phosphatase (G-6-Pase) activity. The diagnosis of Von Gierke's disease has been made in a patient with hepatomegaly utilizing cultured epithelial cells grown from a liver biopsy. G-6-Pase activity in epithelial cells from this patient could not be induced by dibutyryl cyclic AMP and theophylline. This is the first use of epithelial cells for diagnosis of a metabolic disease. G-6-Pase activity in cloned epithelial cells from amniotic fluid increases 2- to 3-fold after 24-hr exposure to dibutyryl cyclic AMP and theophylline. The prenatal diagnosis of Von Gierke's disease may be possible in a laboratory experienced with these techniques if epithelial cell growth is obtained from amniotic fluid. PMID:196249

  15. Epithelial-Mesenchymal Transition (EMT) Gene Variants and Epithelial Ovarian Cancer (EOC) Risk.

    PubMed

    Amankwah, Ernest K; Lin, Hui-Yi; Tyrer, Jonathan P; Lawrenson, Kate; Dennis, Joe; Chornokur, Ganna; Aben, Katja K H; Anton-Culver, Hoda; Antonenkova, Natalia; Bruinsma, Fiona; Bandera, Elisa V; Bean, Yukie T; Beckmann, Matthias W; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A; Brooks-Wilson, Angela; Bunker, Clareann H; Butzow, Ralf; Campbell, Ian G; Carty, Karen; Chen, Zhihua; Chen, Y Ann; Chang-Claude, Jenny; Cook, Linda S; Cramer, Daniel W; Cunningham, Julie M; Cybulski, Cezary; Dansonka-Mieszkowska, Agnieszka; du Bois, Andreas; Despierre, Evelyn; Dicks, Ed; Doherty, Jennifer A; Dörk, Thilo; Dürst, Matthias; Easton, Douglas F; Eccles, Diana M; Edwards, Robert P; Ekici, Arif B; Fasching, Peter A; Fridley, Brooke L; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G; Glasspool, Rosalind; Goodman, Marc T; Gronwald, Jacek; Harrington, Patricia; Harter, Philipp; Hasmad, Hanis N; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A T; Hillemanns, Peter; Hogdall, Claus K; Hogdall, Estrid; Hosono, Satoyo; Iversen, Edwin S; Jakubowska, Anna; Jensen, Allan; Ji, Bu-Tian; Karlan, Beth Y; Jim, Heather; Kellar, Melissa; Kiemeney, Lambertus A; Krakstad, Camilla; Kjaer, Susanne K; Kupryjanczyk, Jolanta; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D; Lee, Alice W; Lele, Shashi; Leminen, Arto; Lester, Jenny; Levine, Douglas A; Liang, Dong; Lim, Boon Kiong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F A G; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R; McNeish, Ian; Menon, Usha; Milne, Roger L; Modugno, Francesmary; Moysich, Kirsten B; Ness, Roberta B; Nevanlinna, Heli; Eilber, Ursula; Odunsi, Kunle; Olson, Sara H; Orlow, Irene; Orsulic, Sandra; Weber, Rachel Palmieri; Paul, James; Pearce, Celeste L; Pejovic, Tanja; Pelttari, Liisa M; Permuth-Wey, Jennifer; Pike, Malcolm C; Poole, Elizabeth M; Risch, Harvey A; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H; Rudolph, Anja; Runnebaum, Ingo B; Rzepecka, Iwona K; Salvesen, Helga B; Schernhammer, Eva; Schwaab, Ira; Shu, Xiao-Ou; Shvetsov, Yurii B; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C; Spiewankiewicz, Beata; Sucheston-Campbell, Lara; Teo, Soo-Hwang; Terry, Kathryn L; Thompson, Pamela J; Thomsen, Lotte; Tangen, Ingvild L; Tworoger, Shelley S; van Altena, Anne M; Vierkant, Robert A; Vergote, Ignace; Walsh, Christine S; Wang-Gohrke, Shan; Wentzensen, Nicolas; Whittemore, Alice S; Wicklund, Kristine G; Wilkens, Lynne R; Wu, Anna H; Wu, Xifeng; Woo, Yin-Ling; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Kelemen, Linda E; Berchuck, Andrew; Schildkraut, Joellen M; Ramus, Susan J; Goode, Ellen L; Monteiro, Alvaro N A; Gayther, Simon A; Narod, Steven A; Pharoah, Paul D P; Sellers, Thomas A; Phelan, Catherine M

    2015-12-01

    Epithelial-mesenchymal transition (EMT) is a process whereby epithelial cells assume mesenchymal characteristics to facilitate cancer metastasis. However, EMT also contributes to the initiation and development of primary tumors. Prior studies that explored the hypothesis that EMT gene variants contribute to epithelial ovarian carcinoma (EOC) risk have been based on small sample sizes and none have sought replication in an independent population. We screened 15,816 single-nucleotide polymorphisms (SNPs) in 296 genes in a discovery phase using data from a genome-wide association study of EOC among women of European ancestry (1,947 cases and 2,009 controls) and identified 793 variants in 278 EMT-related genes that were nominally (P < 0.05) associated with invasive EOC. These SNPs were then genotyped in a larger study of 14,525 invasive-cancer patients and 23,447 controls. A P-value <0.05 and a false discovery rate (FDR) <0.2 were considered statistically significant. In the larger dataset, GPC6/GPC5 rs17702471 was associated with the endometrioid subtype among Caucasians (odds ratio (OR) = 1.16, 95% CI = 1.07-1.25, P = 0.0003, FDR = 0.19), whereas F8 rs7053448 (OR = 1.69, 95% CI = 1.27-2.24, P = 0.0003, FDR = 0.12), F8 rs7058826 (OR = 1.69, 95% CI = 1.27-2.24, P = 0.0003, FDR = 0.12), and CAPN13 rs1983383 (OR = 0.79, 95% CI = 0.69-0.90, P = 0.0005, FDR = 0.12) were associated with combined invasive EOC among Asians. In silico functional analyses revealed that GPC6/GPC5 rs17702471 coincided with DNA regulatory elements. These results suggest that EMT gene variants do not appear to play a significant role in the susceptibility to EOC. PMID:26399219

  16. Interaction of the pathogenic mold Aspergillus fumigatus with lung epithelial cells

    PubMed Central

    Osherov, Nir

    2012-01-01

    Aspergillus fumigatus is an opportunistic environmental mold that can cause severe allergic responses in atopic individuals and poses a life-threatening risk for severely immunocompromised patients. Infection is caused by inhalation of fungal spores (conidia) into the lungs. The initial point of contact between the fungus and the host is a monolayer of lung epithelial cells. Understanding how these cells react to fungal contact is crucial to elucidating the pathobiology of Aspergillus-related disease states. The experimental systems, both in vitro and in vivo, used to study these interactions, are described. Distinction is made between bronchial and alveolar epithelial cells. The experimental findings suggest that lung epithelial cells are more than just “innocent bystanders” or a purely physical barrier against infection. They can be better described as an active extension of our innate immune system, operating as a surveillance mechanism that can specifically identify fungal spores and activate an offensive response to block infection. This response includes the internalization of adherent conidia and the release of cytokines, antimicrobial peptides, and reactive oxygen species. In the case of allergy, lung epithelial cells can dampen an over-reactive immune response by releasing anti-inflammatory compounds such as kinurenine. This review summarizes our current knowledge regarding the interaction of A. fumigatus with lung epithelial cells. A better understanding of the interactions between A. fumigatus and lung epithelial cells has therapeutic implications, as stimulation or inhibition of the epithelial response may alter disease outcome. PMID:23055997

  17. Energy metabolism in intestinal epithelial cells during maturation along the crypt-villus axis

    PubMed Central

    Yang, Huansheng; Wang, Xiaocheng; Xiong, Xia; Yin, Yulong

    2016-01-01

    Intestinal epithelial cells continuously migrate and mature along crypt-villus axis (CVA), while the changes in energy metabolism during maturation are unclear in neonates. The present study was conducted to test the hypothesis that the energy metabolism in intestinal epithelial cells would be changed during maturation along CVA in neonates. Eight 21-day-old suckling piglets were used. Intestinal epithelial cells were isolated sequentially along CVA, and proteomics was used to analyze the changes in proteins expression in epithelial cells along CVA. The identified differentially expressed proteins were mainly involved in cellular process, metabolic process, biological regulation, pigmentation, multicellular organizational process and so on. The energy metabolism in intestinal epithelial cells of piglets was increased from the bottom of crypt to the top of villi. Moreover, the expression of proteins related to the metabolism of glucose, most of amino acids, and fatty acids was increased in intestinal epithelial cells during maturation along CVA, while the expression of proteins related to glutamine metabolism was decreased from crypt to villus tip. The expression of proteins involved in citrate cycle was also increased intestinal epithelial cells during maturation along CVA. Moreover, dietary supplementation with different energy sources had different effects on intestinal structure of weaned piglets. PMID:27558220

  18. Energy metabolism in intestinal epithelial cells during maturation along the crypt-villus axis.

    PubMed

    Yang, Huansheng; Wang, Xiaocheng; Xiong, Xia; Yin, Yulong

    2016-01-01

    Intestinal epithelial cells continuously migrate and mature along crypt-villus axis (CVA), while the changes in energy metabolism during maturation are unclear in neonates. The present study was conducted to test the hypothesis that the energy metabolism in intestinal epithelial cells would be changed during maturation along CVA in neonates. Eight 21-day-old suckling piglets were used. Intestinal epithelial cells were isolated sequentially along CVA, and proteomics was used to analyze the changes in proteins expression in epithelial cells along CVA. The identified differentially expressed proteins were mainly involved in cellular process, metabolic process, biological regulation, pigmentation, multicellular organizational process and so on. The energy metabolism in intestinal epithelial cells of piglets was increased from the bottom of crypt to the top of villi. Moreover, the expression of proteins related to the metabolism of glucose, most of amino acids, and fatty acids was increased in intestinal epithelial cells during maturation along CVA, while the expression of proteins related to glutamine metabolism was decreased from crypt to villus tip. The expression of proteins involved in citrate cycle was also increased intestinal epithelial cells during maturation along CVA. Moreover, dietary supplementation with different energy sources had different effects on intestinal structure of weaned piglets. PMID:27558220

  19. Topical Administration of Acylated Homoserine Lactone Improves Epithelialization of Cutaneous Wounds in Hyperglycaemic Rats

    PubMed Central

    Kitamura, Aya; Quinetti, Paes C.; Nakagami, Gojiro; Mugita, Yuko; Oe, Makoto; Noguchi, Hiroshi; Mori, Taketoshi; Sanada, Hiromi

    2016-01-01

    Clinicians often experience delayed epithelialization in diabetic patients, for which a high glucose condition is one of the causes. However, the mechanisms underlying delayed wound closure have not been fully elucidated, and effective treatments to enhance epithelialization in patients with hyperglycaemia have not been established. Here we propose a new reagent, acylated homoserine lactone (AHL), to improve the delayed epithelialization due to the disordered formation of a basement membrane of epidermis in hyperglycaemic rats. Acute hyperglycaemia was induced by streptozotocin injection in this experiment. Full thickness wounds were created on the flanks of hyperglycaemic or control rats. Histochemical and immunohistochemical analyses were performed to identify hyperglycaemia-specific abnormalities in epidermal regeneration by comparison between groups. We then examined the effects of AHL on delayed epithelialization in hyperglycaemic rats. Histological analysis showed the significantly shorter epithelializing tissue (P < 0.05), abnormal structure of basement membrane (fragmentation and immaturity), and hypo- and hyperproliferation of basal keratinocytes in hyperglycaemic rats. Treating the wound with AHL resulted in the decreased abnormalities of basement membrane, normal distribution of proliferating epidermal keratinocytes, and significantly promoted epithelialization (P < 0.05) in hyperglycemic rats, suggesting the improving effects of AHL on abnormal epithelialization due to hyperglycemia. PMID:27404587

  20. Topical Administration of Acylated Homoserine Lactone Improves Epithelialization of Cutaneous Wounds in Hyperglycaemic Rats.

    PubMed

    Huang, Lijuan; Minematsu, Takeo; Kitamura, Aya; Quinetti, Paes C; Nakagami, Gojiro; Mugita, Yuko; Oe, Makoto; Noguchi, Hiroshi; Mori, Taketoshi; Sanada, Hiromi

    2016-01-01

    Clinicians often experience delayed epithelialization in diabetic patients, for which a high glucose condition is one of the causes. However, the mechanisms underlying delayed wound closure have not been fully elucidated, and effective treatments to enhance epithelialization in patients with hyperglycaemia have not been established. Here we propose a new reagent, acylated homoserine lactone (AHL), to improve the delayed epithelialization due to the disordered formation of a basement membrane of epidermis in hyperglycaemic rats. Acute hyperglycaemia was induced by streptozotocin injection in this experiment. Full thickness wounds were created on the flanks of hyperglycaemic or control rats. Histochemical and immunohistochemical analyses were performed to identify hyperglycaemia-specific abnormalities in epidermal regeneration by comparison between groups. We then examined the effects of AHL on delayed epithelialization in hyperglycaemic rats. Histological analysis showed the significantly shorter epithelializing tissue (P < 0.05), abnormal structure of basement membrane (fragmentation and immaturity), and hypo- and hyperproliferation of basal keratinocytes in hyperglycaemic rats. Treating the wound with AHL resulted in the decreased abnormalities of basement membrane, normal distribution of proliferating epidermal keratinocytes, and significantly promoted epithelialization (P < 0.05) in hyperglycemic rats, suggesting the improving effects of AHL on abnormal epithelialization due to hyperglycemia. PMID:27404587

  1. [The molecular biology of epithelial ovarian cancer].

    PubMed

    Leary, Alexandra; Pautier, Patricia; Tazi, Youssef; Morice, Philippe; Duvillard, Pierre; Gouy, Sébastien; Uzan, Catherine; Gauthier, Hélène; Balleyguier, Corinne; Lhommé, Catherine

    2012-12-01

    Epithelial ovarian cancer frequently presents at an advanced stage where the cornerstone of management remains surgery and platinum-based chemotherapy. Unfortunately, despite sometimes dramatic initial responses, advanced ovarian cancer almost invariably relapses. Little progress has been made in the identification of effective targeted-therapies for ovarian cancer. The majority of clinical trials investigating novel agents have been negative and the only approved targeted-therapy is bevacizumab, for which reliable predictive biomarkers still elude us. Ovarian cancer is treated as a uniform disease. Yet, biological studies have highlighted the heterogeneity of this malignancy with marked differences in histology, oncogenesis, prognosis, chemo-responsiveness, and molecular profile. Recent high throughput molecular analyses have identified a huge number of genomic/phenotypic alterations. Broadly speaking, high grade serous carcinomas (type II) display significant genomic instability and numerous amplifications and losses; low grade (type I) tumors are genomically stable but display frequent mutations. Importantly, many of these genomic alterations relate to known oncogenes for which targeted-therapies are available or in development. There is today a real potential for personalized medicine in ovarian cancer. We will review the current literature regarding the molecular characterization of epithelial ovarian cancer and discuss the biological rationale for a number of targeted strategies. In order to translate these biological advances into meaningful clinical improvements for our patients, it is imperative to incorporate translational research in ovarian cancer trials, a number of strategies will be proposed such as the acquisition of quality tumor samples, including sequential pre- and post-treatment biopsies, the potential of liquid biopsies, and novel trial designs more adapted to the molecular era of ovarian cancer research. PMID:23238064

  2. Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

    PubMed Central

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J. Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz

    2013-01-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  3. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz; Rockx, Barry

    2013-03-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  4. RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis.

    PubMed

    Dannappel, Marius; Vlantis, Katerina; Kumari, Snehlata; Polykratis, Apostolos; Kim, Chun; Wachsmuth, Laurens; Eftychi, Christina; Lin, Juan; Corona, Teresa; Hermance, Nicole; Zelic, Matija; Kirsch, Petra; Basic, Marijana; Bleich, Andre; Kelliher, Michelle; Pasparakis, Manolis

    2014-09-01

    Necroptosis has emerged as an important pathway of programmed cell death in embryonic development, tissue homeostasis, immunity and inflammation. RIPK1 is implicated in inflammatory and cell death signalling and its kinase activity is believed to drive RIPK3-mediated necroptosis. Here we show that kinase-independent scaffolding RIPK1 functions regulate homeostasis and prevent inflammation in barrier tissues by inhibiting epithelial cell apoptosis and necroptosis. Intestinal epithelial cell (IEC)-specific RIPK1 knockout caused IEC apoptosis, villus atrophy, loss of goblet and Paneth cells and premature death in mice. This pathology developed independently of the microbiota and of MyD88 signalling but was partly rescued by TNFR1 (also known as TNFRSF1A) deficiency. Epithelial FADD ablation inhibited IEC apoptosis and prevented the premature death of mice with IEC-specific RIPK1 knockout. However, mice lacking both RIPK1 and FADD in IECs displayed RIPK3-dependent IEC necroptosis, Paneth cell loss and focal erosive inflammatory lesions in the colon. Moreover, a RIPK1 kinase inactive knock-in delayed but did not prevent inflammation caused by FADD deficiency in IECs or keratinocytes, showing that RIPK3-dependent necroptosis of FADD-deficient epithelial cells only partly requires RIPK1 kinase activity. Epidermis-specific RIPK1 knockout triggered keratinocyte apoptosis and necroptosis and caused severe skin inflammation that was prevented by RIPK3 but not FADD deficiency. These findings revealed that RIPK1 inhibits RIPK3-mediated necroptosis in keratinocytes in vivo and identified necroptosis as a more potent trigger of inflammation compared with apoptosis. Therefore, RIPK1 is a master regulator of epithelial cell survival, homeostasis and inflammation in the intestine and the skin. PMID:25132550

  5. Genetic analysis of scattered populations of the Indian eri silkworm, Samia cynthia ricini Donovan: Differentiation of subpopulations.

    PubMed

    Pradeep, Appukuttannair R; Jingade, Anuradha H; Singh, Choba K; Awasthi, Aravind K; Kumar, Vikas; Rao, Guruprasad C; Prakash, N B Vijaya

    2011-07-01

    Deforestation and exploitation has led to the fragmentation of habitats and scattering of populations of the economically important eri silkworm, Samia cynthia ricini, in north-east India. Genetic analysis of 15 eri populations, using ISSR markers, showed 98% inter-population, and 23% to 58% intra-population polymorphism. Nei's genetic distance between populations increased significantly with altitude (R(2) = 0.71) and geographic distance (R(2) = 0.78). On the dendrogram, the lower and upper Assam populations were clustered separately, with intermediate grouping of those from Barpathar and Chuchuyimlang, consistent with geographical distribution. The Nei's gene diversity index was 0.350 in total populations and 0.121 in subpopulations. The genetic differentiation estimate (Gst) was 0.276 among scattered populations. Neutrality tests showed deviation of 118 loci from Hardy-Weinberg equilibrium. The number of loci that deviated from neutrality increased with altitude (R(2) = 0.63). Test of linkage disequilibrium showed greater contribution of variance among eri subpopulations to total variance. D('2)IS exceeded D('2)ST, showed significant contribution of random genetic drift to the increase in variance of disequilibrium in subpopulations. In the Lakhimpur population, the peripheral part was separated from the core by a genetic distance of 0.260. Patchy habitats promoted low genetic variability, high linkage disequilibrium and colonization by new subpopulations. Increased gene flow and habitat-area expansion are required to maintain higher genetic variability and conservation of the original S. c. ricini gene pool. PMID:21931526

  6. [The character of variations in the relationships of lymphocyte subpopulations in the patients presenting with chronic decompensated tonsillitis].

    PubMed

    Iashan, A I; Gerasimiuk, M I

    2015-01-01

    The palatine tonsils are known to be involved in the formation of cellular and humoral immunity. Apoptosis is believed to be one of the main biological mechanism regulating the quantitative composition of subpopulations of the immunocompetent cells. Therefore, the prevalence of apoptotic and anti-apoptotic processes determines the direction of the immune response. Bearing this in mind, we have undertaken a study with a view to elucidating the relationships between lymphocyte subpopulations in the blood and homogenates of the palatal tonsils in the patients with chronic decompensated tonsillitis and establishing their interdependence with the levels of apoptosis and necrosis. The quantification of apoptosis and necrosis as well as the ratio of these processes in neutrophils and lymphocytes belonging to different subpopulations of palatal tonsil homogenates and peripheral blood was performed by cytofluorometry with the use of the "BecmenCulter Epi XL" apparatus (USA). It has been found that decompensated chronic tonsillitis is associated with the depression of cellular immunity apparent as the 2 or 3-fold reduction of lymphocyte CD3, CD4m and CD8 subsets (as compared with the respective normal levels). The number of CD16 and CD19 lymphocytes remains unaltered witch suggests that humoral immunity is un-affectred. It is concluded that the apoptosis-necrosis index and the relationship between different subpopulations of lymphocytes in the peripheral blood may be the additional indicator to be used for diagnostics of decompensated forms of chronic tonsillitis. PMID:26145740

  7. Circulating immune cell subpopulations in pestivirus persistently infected calves and non-infected calves varying in immune status [Abstract

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The circulating immune cell subpopulations in cattle representing varying stages of immune status categorized as; colostrum deprived (CD), receiving colostrum (COL), colostrum plus vaccination (VAC) and persistently infected with a pestivirus (PI) were compared. The PI calves were infected with a H...

  8. PREDICTIVE ORGANOPHOSPHORUS (OP) PESTICIDE QSARS AND PBPK/PD MODELS FOR RISK ASSESSMENT OF SUSCEPTIBLE SUB-POPULATIONS

    EPA Science Inventory

    Successful use of the Exposure Related Dose Estimating Model (ERDEM) in risk assessment of susceptible human sub-populations, e.g., infants and children, requires input of quality experimental data. In the clear absence of quality data, PBPK models can be developed and possibl...

  9. Structural Equation Analyses of Clinical Subpopulation Differences and Comparative Treatment Outcomes: Characterizing the Daily Lives of Drug Addicts.

    ERIC Educational Resources Information Center

    Aiken, Leona S.; And Others

    1994-01-01

    Used structural equation modeling for comparative treatment outcome research conducted with heterogeneous clinical subpopulations within large multimodality treatment settings. Evaluated effect of early period of treatment on daily lives of 486 clients in 2 drug abuse treatment modalities (methadone maintenance and outpatient counseling).…

  10. Novel strategies to enforce an epithelial phenotype in mesenchymal cells.

    PubMed

    Dragoi, Ana-Maria; Swiss, Rachel; Gao, Beile; Agaisse, Hervé

    2014-07-15

    E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin-dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definition of several known regulators of E-cadherin expression, including ZEB1, HDAC1, and MMP14. We identified three new regulators (FLASH, CASP7, and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. In addition, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a posttranscriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through posttranscriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT. PMID:24845104

  11. Novel strategies to enforce an epithelial phenotype in mesenchymal cells

    PubMed Central

    Dragoi, Ana-Maria; Swiss, Rachel; Gao, Beile; Agaisse, Hervé

    2014-01-01

    E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin-dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definion of several known regulators of E-cadherin expression, including ZEB1, HDAC1 and MMP14. We identified three new regulators (FLASH, CASP7 and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. Additionally, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a post-transcriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through post-transcriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT. PMID:24845104

  12. Elimination of the chemotherapy resistant subpopulation of 4T1 mouse breast cancer by haploidentical NK cells cures the vast majority of mice.

    PubMed

    Frings, Peter W H; Van Elssen, Catharina H M J; Wieten, Lotte; Matos, Catarina; Hupperets, Pierre S J G; Schouten, Harry C; Bos, Gerard M J; van Gelder, Michel

    2011-12-01

    Metastatic breast cancer is currently incurable despite initial responsiveness, assumingly due to the presence of chemoresistant subpopulations that can be characterized as label retaining cells (LRC). In the 4T1 mouse breast cancer model, we previously achieved cure after Cyclophosphamide and Total Body Irradiation (CY + TBI) followed by haploidentical bone marrow and spleen transplantation (BMSPLT). CY + TBI without transplantation induced only transient impaired tumor growth indicating a critical role of donor immune cells. As it remained unknown if the 4T1 model resembles human disease with respect to the presence of subpopulations of chemoresistant LRC, we now demonstrate this is indeed the case. Chemoresistance of 4T1 LRC was demonstrated by in vitro co-incubation of fluorescently labeled 4T1 cells in limiting dilution with cyclophosphamide, doxorubicin or cisplatinum, after which only LRC containing colonies remained. LRC also remain in vivo after treatment with CY + TBI. Succeeding experiments set up to identify the haploidentical effector cell responsible for cure and, therefore, for the elimination of chemoresistant LRC designate donor NK cells crucial for the anti-tumor effect. NK cell depletion of the haploidentical graft fully abrogated the anti-tumor effect. Increased disease-free survival retained after transplantation of haploidentical bone marrow and NK cell-enriched spleen cell grafts, even in the absence of donor T-cells or of donor bone marrow. Tumor growth analysis indicates the anti-tumor effect being immediate (days). Based on these data, it is worthwhile to explore alloreactive adoptive NK cell therapy as consolidation for patients with metastasized breast cancer. PMID:21274621

  13. Quantitative-Trait Loci on Chromosomes 1, 2, 3, 4, 8, 9, 11, 12, and 18 Control Variation in Levels of T and B Lymphocyte Subpopulations

    PubMed Central

    Hall, M. A.; Norman, P. J.; Thiel, B.; Tiwari, H.; Peiffer, A.; Vaughan, R. W.; Prescott, S.; Leppert, M.; Schork, N. J.; Lanchbury, J. S.

    2002-01-01

    Lymphocyte subpopulation levels are used for prognosis and monitoring of a variety of human diseases, especially those with an infectious etiology. As a primary step to defining the major gene variation underlying these phenotypes, we conducted the first whole-genome screen for quantitative variation in lymphocyte count, CD4 T cell, CD8 T cell, B cell, and natural killer cell numbers, as well as CD4:CD8 ratio. The screen was performed in 15 of the CEPH families that form the main human genome genetic project mapping resource. Quantitative-trait loci (QTLs) that account for significant proportions of the phenotypic variance of lymphocyte subpopulations were detected on chromosomes 1, 2, 3, 4, 8, 9, 11, 12, and 18. The most significant QTL found was for CD4 levels on chromosome 8 (empirical P=.00005). Two regions of chromosome 4 showed significant linkage to CD4:CD8 ratio (empirical P=.00007 and P=.003). A QTL for the highly correlated measures of CD4 and CD19 levels colocalized at 18q21 (both P=.003). Similarly, a shared region of chromosome 1 was linked to CD8 and CD19 levels (P=.0001 and P=.002, respectively). Several of the identified chromosome regions are likely to harbor polymorphic candidate genes responsible for these important human phenotypes. Their discovery has important implications for understanding the generation of the immune repertoire and understanding immune-system homeostasis. More generally, these data show the power of an integrated human gene–mapping approach for heritable molecular phenotypes, using large pedigrees that have been extensively genotyped. PMID:11951176

  14. Identification of an ABCB1 (P-glycoprotein)-positive carfilzomib-resistant myeloma subpopulation by the pluripotent stem cell fluorescent dye CDy1

    PubMed Central

    Hawley, Teresa S.; Riz, Irene; Yang, Wenjing; Wakabayashi, Yoshiyuki; DePalma, Louis; Chang, Young-Tae; Peng, Weiqun; Zhu, Jun; Hawley, Robert G.

    2013-01-01

    Multiple myeloma (MM) is characterized by the malignant expansion of differentiated plasma cells. Although many chemotherapeutic agents display cytotoxic activity toward MM cells, patients inevitably succumb to their disease because the tumor cells become resistant to the anticancer drugs. The cancer stem cell hypothesis postulates that a small subpopulation of chemotherapy-resistant cancer cells is responsible for propagation of the tumor. Herein we report that efflux of the pluripotent stem cell dye CDy1 identifies a subpopulation in MM cell lines characterized by increased expression of P-glycoprotein, a member of the ABC (ATP-binding cassette) superfamily of transporters encoded by ABCB1. We also demonstrate that ABCB1-overexpressing MM cells are resistant to the second-generation proteasome inhibitor carfilzomib that recently received accelerated approval for the treatment of therapy-refractive MM by the U.S. Food and Drug Administration. Moreover, increased resistance to carfilzomib in sensitive MM cells following drug selection was associated with upregulation of ABCB1 cell-surface expression which correlated with increased transporter activity as measured by CDy1 efflux. We further show that chemosensitization of MM cells to carfilzomib could be achieved in vitro by cotreatment with vismodegib, a hedgehog pathway antagonist which is currently in MM clinical trials. CDy1 efflux may therefore be a useful assay to determine whether high expression of ABCB1 is predictive of poor clinical responses in MM patients treated with carfilzomib. Our data also suggest that inclusion of vismodegib might be a potential strategy to reverse ABCB1-mediated drug resistance should it occur. PMID:23475625

  15. SPERMATOGENESIS IN THE YELLOW PERCH (PERCA FLAVESCENS) - COMPARISON OF RELATIVE GERM CELL TYPES IN 2 SUBPOPULATIONS OF YOUNG-OF-YEAR FISH

    EPA Science Inventory

    An "accelerated growth rate" (AGR) subpopulation of young-of-year yellow perch (Perca flavescens) finished production of mature spermatozoa at least two weeks earlier than a "normal growth rate" (NGR) subpopulation as assessed by semi-quantitative testis histology methods.

  16. Epithelial ovarian cancer: An overview

    PubMed Central

    Desai, Arpita; Xu, Jingyao; Aysola, Kartik; Qin, Yunlong; Okoli, Chika; Hariprasad, Ravipati; Chinemerem, Ugorji; Gates, Candace; Reddy, Avinash; Danner, Omar; Franklin, Geary; Ngozi, Anachebe; Cantuaria, Guilherme; Singh, Karan; Grizzle, William; Landen, Charles; Partridge, Edward E; Rice, Valerie Montgomery; Reddy, E Shyam P; Rao, Veena N

    2014-01-01

    Ovarian cancer is the second most common gynecological cancer and the leading cause of death in the United States. In this article we review the diagnosis and current management of epithelial ovarian cancer which accounts for over 95 percent of the ovarian malignancies. We will present various theories about the potential origin of ovarian malignancies. We will discuss the genetic anomalies and syndromes that may cause ovarian cancers with emphasis on Breast cancer type 1/2 mutations. The pathology and pathogenesis of ovarian carcinoma will also be presented. Lastly, we provide a comprehensive overview of treatment strategies and staging of ovarian cancer, conclusions and future directions. PMID:25525571

  17. Testing for Local Adaptation to Spawning Habitat in Sympatric Subpopulations of Pike by Reciprocal Translocation of Embryos

    PubMed Central

    Berggren, Hanna; Nordahl, Oscar; Tibblin, Petter; Larsson, Per

    2016-01-01

    We tested for local adaption in early life-history traits by performing a reciprocal translocation experiment with approximately 2,500 embryos of pike (Esox lucius) divided in paired split-family batches. The experiment indicated local adaptation in one of the two subpopulations manifested as enhanced hatching success of eggs in the native habitat, both when compared to siblings transferred to a non-native habitat, and when compared to immigrant genotypes from the other subpopulation. Gene-by-environment effects on viability of eggs and larvae were evident in both subpopulations, showing that there existed genetic variation allowing for evolutionary responses to divergent selection, and indicating a capacity for plastic responses to environmental change. Next, we tested for differences in female life-history traits. Results uncovered that females from one population invested more resources into reproduction and also produced more (but smaller) eggs in relation to their body size compared to females from the other population. We suggest that these females have adjusted their reproductive strategies as a counter-adaptation because a high amount of sedimentation on the eggs in that subpopulations spawning habitat might benefit smaller eggs. Collectively, our findings point to adaptive divergence among sympatric subpopulations that are physically separated only for a short period during reproduction and early development—which is rare. These results illustrate how combinations of translocation experiments and field studies of life-history traits might infer about local adaptation and evolutionary divergence among populations. Local adaptations in subdivided populations are important to consider in management and conservation of biodiversity, because they may otherwise be negatively affected by harvesting, supplementation, and reintroduction efforts targeted at endangered populations. PMID:27139695

  18. Different gDNA Content in the Subpopulations of Prostate Cancer Extracellular Vesicles: Apoptotic Bodies, Microvesicles, and Exosomes

    PubMed Central

    Lázaro-Ibáñez, Elisa; Sanz-Garcia, Andres; Visakorpi, Tapio; Escobedo-Lucea, Carmen; Siljander, Pia; Ayuso-Sacido, Ángel; Yliperttula, Marjo

    2014-01-01

    Background Extracellular vesicles (EVs) are cell-derived membrane vesicles. EVs contain several RNAs such as mRNA, microRNAs, and ncRNAs, but less is known of their genomic DNA (gDNA) content. It is also unknown whether the DNA cargo is randomly sorted or if it is systematically packed into specific EV subpopulations. The aim of this study was to analyze whether different prostate cancer (PCa) cell-derived EV subpopulations (apoptotic bodies, microvesicles, and exosomes) carry different gDNA fragments. Methods EV subpopulations were isolated from three PCa cell lines (LNCaP, PC-3, and RC92a/hTERT) and the plasma of PCa patients and healthy donors, and characterized by transmission electron microscopy, nanoparticle tracking analysis and total protein content. gDNA fragments of different genes were detected by real time quantitative PCR and confirmed by DNA sequencing. Results We report that the concentration of EVs was higher in the cancer patients than in the healthy controls. EV subpopulations differed from each other in terms of total protein and DNA content. Analysis of gDNA fragments of MLH1, PTEN, and TP53 genes from the PCa cell-derived EV subpopulations showed that different EVs carried different gDNA content, which could even harbor specific mutations. Altogether, these results suggest that both nucleic acids and proteins are selectively and cell-dependently packed into the EV subtypes. Conclusions EVs derived from PCa cell lines and human plasma samples contain double-stranded gDNA fragments which could be used to detect specific mutations, making EVs potential biomarkers for cancer diagnostics and prognostics. PMID:25111183

  19. Functional ESCRT machinery is required for constitutive recycling of claudin-1 and maintenance of polarity in vertebrate epithelial cells.

    PubMed

    Dukes, Joseph D; Fish, Laura; Richardson, Judith D; Blaikley, Elizabeth; Burns, Samir; Caunt, Christopher J; Chalmers, Andrew D; Whitley, Paul

    2011-09-01

    Genetic screens in Drosophila have identified regulators of endocytic trafficking as neoplastic tumor suppressor genes. For example, Drosophila endosomal sorting complex required for transport (ESCRT) mutants lose epithelial polarity and show increased cell proliferation, suggesting that ESCRT proteins could function as tumor suppressors. In this study, we show for the for the first time to our knowledge that ESCRT proteins are required to maintain polarity in mammalian epithelial cells. Inhibition of ESCRT function caused the tight junction protein claudin-1 to accumulate in intracellular vesicles. In contrast E-cadherin and occludin localization was unaffected. We investigated the cause of this accumulation and show that claudin-1 is constitutively recycled in kidney, colon, and lung epithelial cells, identifying claudin-1 recycling as a newly described feature of diverse epithelial cell types. This recycling requires ESCRT function, explaining the accumulation of intracellular claudin-1 when ESCRT function is inhibited. We further demonstrate that small interfering RNA knockdown of the ESCRT protein Tsg101 causes epithelial monolayers to lose their polarized organization and interferes with the establishment of a normal epithelial permeability barrier. ESCRT knockdown also reduces the formation of correctly polarized three-dimensional cysts. Thus, in mammalian epithelial cells, ESCRT function is required for claudin-1 trafficking and for epithelial cell polarity, supporting the hypothesis that ESCRT proteins function as tumor suppressors. PMID:21757541

  20. Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

    PubMed Central

    Zhong, Aimei; Wang, Guohua; Yang, Jie; Xu, Qijun; Yuan, Quan; Yang, Yanqing; Xia, Yun; Guo, Ke; Horch, Raymund E; Sun, Jiaming

    2014-01-01

    True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co-culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non-macromastic epithelial cells when co-cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia-derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co-culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co-cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy. PMID:24720804

  1. Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

    PubMed Central

    2014-01-01

    Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

  2. Pten in Stromal Fibroblasts Suppresses Mammary Epithelial Tumors

    PubMed Central

    Trimboli, Anthony J.; Cantemir-Stone, Carmen Z.; Li, Fu; Wallace, Julie A.; Merchant, Anand; Creasap, Nicholas; Thompson, John C.; Caserta, Enrico; Wang, Hui; Chong, Jean-Leon; Naidu, Shan; Wei, Guo; Sharma, Sudarshana M.; Stephens, Julie A.; Fernandez, Soledad A.; Gurcan, Metin N.; Weinstein, Michael B.; Barsky, Sanford H.; Yee, Lisa; Rosol, Thomas J.; Stromberg, Paul C.; Robinson, Michael L.; Pepin, Francois; Hallett, Michael; Park, Morag; Ostrowski, Michael C.; Leone, Gustavo

    2009-01-01

    SUMMARY The tumor stroma is believed to contribute to some of the most malignant characteristics of epithelial tumors. However, signaling between stromal and tumor cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumors. This was associated with the massive remodeling of the extra-cellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumors ameliorated disruption of the tumor microenvironment and was sufficient to decrease tumor growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumor stroma of breast cancer patients. These findings identify the Pten-Ets2 axis as a critical stroma-specific signaling pathway that suppresses mammary epithelial tumors. PMID:19847259

  3. Genetic control of epithelial tube fusion during Drosophila tracheal development.

    PubMed

    Samakovlis, C; Manning, G; Steneberg, P; Hacohen, N; Cantera, R; Krasnow, M A

    1996-11-01

    During development of tubular networks such as the mammalian vascular system, the kidney and the Drosophila tracheal system, epithelial tubes must fuse to each other to form a continuous network. Little is known of the cellular mechanisms or molecular control of epithelial tube fusion. We describe the cellular dynamics of a tracheal fusion event in Drosophila and identify a gene regulatory hierarchy that controls this extraordinary process. A tracheal cell located at the developing fusion point expresses a sequence of specific markers as it grows out and contacts a similar cell from another tube; the two cells adhere and form an intercellular junction, and they become doughnut-shaped cells with the lumen passing through them. The early fusion marker Fusion-1 is identified as the escargot gene. It lies near the top of the regulatory hierarchy, activating the expression of later fusion markers and repressing genes that promote branching. Ectopic expression of escargot activates the fusion process and suppresses branching throughout the tracheal system, leading to ectopic tracheal connections that resemble certain arteriovenous malformations in humans. This establishes a simple genetic system to study fusion of epithelial tubes. PMID:8951068

  4. Ovarian Epithelial-Stromal Interactions: Role of Interleukins 1 and 6

    PubMed Central

    Woolery, Kamisha T.; Kruk, Patricia A.

    2011-01-01

    Ovarian epithelial cancer is the most lethal gynecologic malignancy. The high mortality is attributed to the fact that most cases typically present in late stage when ovarian cancer (OC) has already spread beyond the ovary. Ovarian epithelial cancer cells are shed into intraperitoneal ascites and easily disseminate throughout the peritoneal cavity with preferential metastasis to the omentum, peritoneum, and local organs. Understanding how ovarian epithelial cells interact with and modulate their microenvironment can provide insight into the molecular mechanism(s) involved with malignant transformation and progression which may eventually identify novel diagnostic, prognostic, and therapeutic targets. The objective of this paper is to provide a brief consideration of ovarian surface epithelial-stromal interactions in regard to normal physiological function and tumor progression as influenced by two potentially key interleukins, interleukins-1 (IL-1) and -6 (IL-6), present in the microenvironment. Lastly, we will consider the clinical implications of IL-1 and IL-6 for OC patients. PMID:21765834

  5. Shape Transformations of Epithelial Shells.

    PubMed

    Misra, Mahim; Audoly, Basile; Kevrekidis, Ioannis G; Shvartsman, Stanislav Y

    2016-04-12

    Regulated deformations of epithelial sheets are frequently foreshadowed by patterning of their mechanical properties. The connection between patterns of cell properties and the emerging tissue deformations is studied in multiple experimental systems, but the general principles remain poorly understood. For instance, it is in general unclear what determines the direction in which the patterned sheet is going to bend and whether the resulting shape transformation will be discontinuous or smooth. Here these questions are explored computationally, using vertex models of epithelial shells assembled from prismlike cells. In response to rings and patches of apical cell contractility, model epithelia smoothly deform into invaginated or evaginated shapes similar to those observed in embryos and tissue organoids. Most of the observed effects can be captured by a simpler model with polygonal cells, modified to include the effects of the apicobasal polarity and natural curvature of epithelia. Our models can be readily extended to include the effects of multiple constraints and used to describe a wide range of morphogenetic processes. PMID:27074691

  6. Keratin metaplasia in the epithelial lining of odontogenic cysts

    PubMed Central

    Maheswaran, Thangadurai; Ramesh, Venkatapathy; Oza, Nirima; Panda, Abikshyeet; Balamurali, P. D.

    2014-01-01

    Objective: To find the prevalence of keratin metaplasia and its relation with clinico-pathological profile of the odontogenic cyst. Materials and Methods: Odontogenic cysts were studied histologically with special stains to identify the presence of keratin and compared with various parameters such as underlying connective tissue inflammation, average epithelial thickness, and site of the cyst, type of the cyst, age and the sex of the patient. Results: Of 71 cases of various odontogenic cysts, 26 (36.6%) cases exhibited keratinization in the epithelial lining. In cysts with severe inflammation there is absence of keratinization. Conclusions: This study reveals higher prevalence of keratin metaplasia in the odontogenic cysts. Furthermore, inflammation is found to be one of factor influencing keratin metaplasia. PMID:25210349

  7. Identification of epithelial auto-antigens associated with periodontal disease

    PubMed Central

    Ye, P; Simonian, M; Nadkarni, M A; DeCarlo, A A; Chapple, C C; Hunter, N

    2005-01-01

    We previously reported evidence that patients with periodontitis have serum antibodies to oral Gram positive bacteria that are cross-reactive with epithelial antigens. In the present report cross-reactive epithelial antigens including CD24, lactate dehydrogenase A [LDM-A], antioxidant protein 2 [AOP 2] and nuclear factor of activated T cells 5 [NFAT 5], were identified by screening a cDNA expression library with pooled patient sera. Titres of antibodies to CD24 peptide correlated negatively with indices of periodontal disease severity. Strong expression of CD24 in the reactive periodontal epithelium and inflamed gingival attachment contrasted with low to undetectable expression in the external gingival epithelium. In periodontitis, a local action of these auto-reactive antibodies could modulate the regulatory potential associated with expression of CD24 in this epithelium. PMID:15654832

  8. Culture and immortalization of pancreatic ductal epithelial cells.

    PubMed

    Lawson, Terence; Ouellette, Michel; Kolar, Carol; Hollingsworth, Michael

    2005-01-01

    Some populations of the epithelial cells from the duct and ductular network of the mammalian pancreas have been isolated and maintained in vitro for up to 3 mo. These cells express many of the surface factors that are unique to them in vivo. They also retain significant drug- and carcinogen-metabolizing capacity in vitro. In this chapter we review the progression of the methods for the isolation, culture and maintenance in vitro for these cells from the earliest when only duct/ductular fragments were obtainable to the current ones which provide epithelial cells. The critical steps in the isolation process are identified and strategies are provided to facilitate these steps. These include the selection of tissue digestive enzymes, the importance of extensive mincing before culture and the importance of roles of some co-factors used in the culture medium. PMID:15542901

  9. Induction of stem-like cells with malignant properties by chronic exposure of human lung epithelial cells to single-walled carbon nanotubes

    PubMed Central

    2014-01-01

    Background Carbon nanotubes (CNT) hold great promise to create new and better products for commercial and biomedical applications, but their long-term adverse health effects are a major concern. The objective of this study was to address human lung cancer risks associated with chronic pulmonary exposure to single-walled (SW) CNT through the fundamental understanding of cellular and molecular processes leading to carcinogenesis. We hypothesized that the acquisition of cancer stem cells (CSC), a subpopulation that drive tumor initiation and progression, may contribute to CNT carcinogenesis. Methods Non-tumorigenic human lung epithelial cells were chronically exposed to well-dispersed SWCNT for a period of 6 months at the physiologically relevant concentration of 0.02 μg/cm2 surface area dose. Chronic SWCNT-exposed cells were evaluated for the presence of CSC-like cells under CSC-selective conditions of tumor spheres and side population (SP). CSC-like cells were isolated using fluorescence-activated cell sorting and were assessed for aggressive behaviors, including acquired apoptosis resistance and increased cell migration and invasion in vitro, and tumor-initiating capability in vivo. Non-small cell lung cancer cells served as a positive control. Results We demonstrated for the first time the existence of CSC-like cells in all clones of chronic SWCNT-exposed lung epithelial cells. These CSC-like cells, in contrary to their non-CSC counterpart, possessed all biological features of lung CSC that are central to irreversible malignant transformation, self-renewal, aggressive cancer behaviors, and in vivo tumorigenesis. These cells also displayed aberrant stem cell markers, notably Nanog, SOX-2, SOX-17 and E-cadherin. Restored expression of tumor suppressor p53 abrogated CSC properties of CSC-like cells. Furthermore, we identified specific stem cell surface markers CD24low and CD133high that are associated with SWCNT-induced CSC formation and tumorigenesis. Conclusions

  10. Immune Evasion by Yersinia enterocolitica: Differential Targeting of Dendritic Cell Subpopulations In Vivo

    PubMed Central

    Hiller, Clara; Keller, Birgit; Warnke, Philipp; Köberle, Martin; Bohn, Erwin; Biedermann, Tilo; Bühring, Hans-Jörg; Hämmerling, Günter J.; Rammensee, Hans-Georg; Autenrieth, Ingo B.

    2010-01-01

    CD4+ T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4+ T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4+ T cells was markedly reduced when cultured with splenic CD8α+ DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4+ or CD4−CD8α− DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α+ DCs, but not in CD4+ and CD4−CD8α− DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α+ DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α+ DCs. Three days post infection with Ye the number of splenic CD8α+ and CD4+ DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4+ and CD8α+ DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye. PMID:21124820

  11. Investigating Voluntary Medical Male Circumcision Program Efficiency Gains through Subpopulation Prioritization: Insights from Application to Zambia

    PubMed Central

    Awad, Susanne F.; Sgaier, Sema K.; Tambatamba, Bushimbwa C.; Mohamoud, Yousra A.; Lau, Fiona K.; Reed, Jason B.; Njeuhmeli, Emmanuel; Abu-Raddad, Laith J.

    2015-01-01

    Background Countries in sub-Saharan Africa are scaling-up voluntary male medical circumcision (VMMC) as an HIV intervention. Emerging challenges in these programs call for increased focus on program efficiency (optimizing program impact while minimizing cost). A novel analytic approach was developed to determine how subpopulation prioritization can increase program efficiency using an illustrative application for Zambia. Methods and Findings A population-level mathematical model was constructed describing the heterosexual HIV epidemic and impact of VMMC programs (age-structured mathematical (ASM) model). The model stratified the population according to sex, circumcision status, age group, sexual-risk behavior, HIV status, and stage of infection. A three-level conceptual framework was also developed to determine maximum epidemic impact and program efficiency through subpopulation prioritization, based on age, geography, and risk profile. In the baseline scenario, achieving 80% VMMC coverage by 2017 among males 15–49 year old, 12 VMMCs were needed per HIV infection averted (effectiveness). The cost per infection averted (cost-effectiveness) was USD $1,089 and 306,000 infections were averted. Through age-group prioritization, effectiveness ranged from 11 (20–24 age-group) to 36 (45–49 age-group); cost-effectiveness ranged from $888 (20–24 age-group) to $3,300 (45–49 age-group). Circumcising 10–14, 15–19, or 20–24 year old achieved the largest incidence rate reduction; prioritizing 15–24, 15–29, or 15–34 year old achieved the greatest program efficiency. Through geographic prioritization, effectiveness ranged from 9–12. Prioritizing Lusaka achieved the highest effectiveness. Through risk-group prioritization, prioritizing the highest risk group achieved the highest effectiveness, with only one VMMC needed per infection averted; the lowest risk group required 80 times more VMMCs. Conclusion Epidemic impact and efficiency of VMMC programs can be

  12. Epithelialization in Wound Healing: A Comprehensive Review

    PubMed Central

    Pastar, Irena; Stojadinovic, Olivera; Yin, Natalie C.; Ramirez, Horacio; Nusbaum, Aron G.; Sawaya, Andrew; Patel, Shailee B.; Khalid, Laiqua; Isseroff, Rivkah R.; Tomic-Canic, Marjana

    2014-01-01

    Significance: Keratinocytes, a major cellular component of the epidermis, are responsible for restoring the epidermis after injury through a process termed epithelialization. This review will focus on the pivotal role of keratinocytes in epithelialization, including cellular processes and mechanisms of their regulation during re-epithelialization, and their cross talk with other cell types participating in wound healing. Recent Advances: Discoveries in epidermal stem cells, keratinocyte immune function, and the role of the epidermis as an independent neuroendocrine organ will be reviewed. Novel mechanisms of gene expression regulation important for re-epithelialization, including microRNAs and histone modifications, will also be discussed. Critical Issues: Epithelialization is an essential component of wound healing used as a defining parameter of a successful wound closure. A wound cannot be considered healed in the absence of re-epithelialization. The epithelialization process is impaired in all types of chronic wounds. Future Directions: A comprehensive understanding of the epithelialization process will ultimately lead to the development of novel therapeutic approaches to promote wound closure. PMID:25032064

  13. Microfluidic single-cell transcriptional analysis rationally identifies novel surface marker profiles to enhance cell-based therapies.

    PubMed

    Rennert, Robert C; Januszyk, Michael; Sorkin, Michael; Rodrigues, Melanie; Maan, Zeshaan N; Duscher, Dominik; Whittam, Alexander J; Kosaraju, Revanth; Chung, Michael T; Paik, Kevin; Li, Alexander Y; Findlay, Michael; Glotzbach, Jason P; Butte, Atul J; Gurtner, Geoffrey C

    2016-01-01

    Current progenitor cell therapies have only modest efficacy, which has limited their clinical adoption. This may be the result of a cellular heterogeneity that decreases the number of functional progenitors delivered to diseased tissue, and prevents correction of underlying pathologic cell population disruptions. Here, we develop a high-resolution method of identifying phenotypically distinct progenitor cell subpopulations via single-cell transcriptional analysis and advanced bioinformatics. When combined with high-throughput cell surface marker screening, this approach facilitates the rational selection of surface markers for prospective isolation of cell subpopulations with desired transcriptional profiles. We establish the usefulness of this platform in costly and highly morbid diabetic wounds by identifying a subpopulation of progenitor cells that is dysfunctional in the diabetic state, and normalizes diabetic wound healing rates following allogeneic application. We believe this work presents a logical framework for the development of targeted cell therapies that can be customized to any clinical application. PMID:27324848

  14. Differential Regulation of Gene Expression of Alveolar Epithelial Cell Markers in Human Lung Adenocarcinoma-Derived A549 Clones

    PubMed Central

    Kondo, Hiroshi; Miyoshi, Keiko; Sakiyama, Shoji; Tangoku, Akira; Noma, Takafumi

    2015-01-01

    Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated an in vitro system to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII) cell line, A549. After cloning A549 subpopulations, each clone was classified into five groups according to cell morphology and marker gene expression. Two clones (B7 and H12) were further analyzed. Under serum-free culture conditions, surfactant protein C (SPC), an ATII marker, was upregulated in both H12 and B7. Aquaporin 5 (AQP5), an ATI marker, was upregulated in H12 and significantly induced in B7. When the RAS/MAPK pathway was inhibited, SPC and thyroid transcription factor-1 (TTF-1) expression levels were enhanced. After treatment with dexamethasone (DEX), 8-bromoadenosine 3′5′-cyclic monophosphate (8-Br-cAMP), 3-isobutyl-1-methylxanthine (IBMX), and keratinocyte growth factor (KGF), surfactant protein B and TTF-1 expression levels were enhanced. We found that A549-derived clones have plasticity in gene expression of alveolar epithelial differentiation markers and could be useful in studying ATII maintenance and differentiation. PMID:26167183

  15. Bone Marrow Cells Expressing Clara Cell Secretory Protein Increase Epithelial Repair After Ablation of Pulmonary Clara Cells

    PubMed Central

    Bustos, Martha L; Mura, Marco; Marcus, Paula; Hwang, David; Ludkovski, Olga; Wong, Amy P; Waddell, Thomas K

    2013-01-01

    We have previously reported a subpopulation of bone marrow cells (BMC) that express Clara cell secretory protein (CCSP), generally felt to be specific to lung Clara cells. Ablation of lung Clara cells has been reported using a transgenic mouse that expresses thymidine kinase under control of the CCSP promoter. Treatment with ganciclovir results in permanent elimination of CCSP+ cells, failure of airway regeneration, and death. To determine if transtracheal delivery of wild-type bone marrow CCSP+ cells is beneficial after ablation of lung CCSP+ cells, transgenic mice were treated with ganciclovir followed by transtracheal administration of CCSP+ or CCSP− BMC. Compared with mice administered CCSP− cells, mice treated with CCSP+ cells had more donor cells lining the airway epithelium, where they expressed epithelial markers including CCSP. Although donor CCSP+ cells did not substantially repopulate the airway, their administration resulted in increased host ciliated cells, better preservation of airway epithelium, reduction of inflammatory cells, and an increase in animal survival time. Administration of CCSP+ BMC is beneficial after permanent ablation of lung Clara cells by increasing bronchial epithelial repair. Therefore, CCSP+ BMC could be important for treatment of lung diseases where airways re-epithelialization is compromised. PMID:23609017

  16. Epithelial organization, cell polarity and tumorigenesis.

    PubMed

    McCaffrey, Luke Martin; Macara, Ian G

    2011-12-01

    Epithelial cells comprise the foundation for the majority of organs in the mammalian body, and are the source of approximately 90% of all human cancers. Characteristically, epithelial cells form intercellular adhesions, exhibit apical/basal polarity, and orient their mitotic spindles in the plane of the epithelial sheet. Defects in these attributes result in the tissue disorganization associated with cancer. Epithelia undergo self-renewal from stem cells, which might in some cases be the cell of origin for cancers. The PAR polarity proteins are master regulators of epithelial organization, and are closely linked to signaling pathways such as Hippo, which orchestrate proliferation and apoptosis to control organ size. 3D ex vivo culture systems can now faithfully recapitulate epithelial organ morphogenesis, providing a powerful approach to study both normal development and the initiating events in carcinogenesis. PMID:21782440

  17. [Recent studies on corneal epithelial barrier function].

    PubMed

    Liu, F F; Li, W; Liu, Z G; Chen, W S

    2016-08-01

    Corneal epithelium, the outermost layer of eyeball, is the main route for foreign materials to enter the eye. Under physiological conditions, the corneal epithelial superficial cells form a functionally selective permeability barrier. Integral corneal epithelial barrier function not only ensures the enrolling of nutrients which is required for regular metabolism, but also prevents foreign bodies, or disease-causing microorganism invasion. Recently, a large number of clinical and experimental studies have shown that abnormal corneal epithelial barrier function is the pathological basis for many ocular diseases. In addition, some study found that corneal epithelial barrier constitutes a variety of proteins involved in cell proliferation, differentiation, apoptosis, and a series of physiological and pathological processes. This paper reviewed recent studies specifically on the corneal epithelial barrier, highlights of its structure, function and influence factors. (Chin J Ophthalmol, 2016, 52: 631-635). PMID:27562284

  18. Expression of CD11a in lymphocyte subpopulation in immune thrombocytopenia

    PubMed Central

    Liu, Yan-Xia; Zhang, Feng; Yao, Qing-Min; Yuan, Ting; Xu, Jian; Zhu, Xiao-Juan

    2015-01-01

    Recent research demonstrates that the underlying mechanism in immune thrombocytopenia (ITP) is very complex. Lymphocyte function associated antigen-1 (LFA-1) plays important roles in autoimmune diseases. The purpose of this study was to investigate the expression of CD11a on lymphocytes and explore its possible role in ITP. The expression of CD11a on lymphocyte subpopulations (CD3+ T cells, CD3+CD4+ T cells, CD3+CD4- T cells, CD4+Foxp3+ T regulatory cells and CD19+ B cells) were analyzed by flow cytometry. Specific anti-platelet GPIIb/IIIa and/or GPIb/IX autoantibodies were assayed by modified monoclonal antibody specific immobilization of platelet antigens (MAIPA). The mean fluorescence intensity of CD11a on CD3+ T, CD3+CD4- T and CD19+ B lymphocytes were increased in ITP patients compared to healthy controls. No significant difference of CD11a expression on CD3+CD4+ T cells or CD4+Foxp3+ T regulatory cells was found between ITP patients and controls. Our data indicates the possible role of CD11a in the pathogenesis of ITP. PMID:26884833

  19. Expression of CD11a in lymphocyte subpopulation in immune thrombocytopenia.

    PubMed

    Liu, Yan-Xia; Zhang, Feng; Yao, Qing-Min; Yuan, Ting; Xu, Jian; Zhu, Xiao-Juan

    2015-01-01

    Recent research demonstrates that the underlying mechanism in immune thrombocytopenia (ITP) is very complex. Lymphocyte function associated antigen-1 (LFA-1) plays important roles in autoimmune diseases. The purpose of this study was to investigate the expression of CD11a on lymphocytes and explore its possible role in ITP. The expression of CD11a on lymphocyte subpopulations (CD3(+) T cells, CD3(+)CD4(+) T cells, CD3(+)CD4(-) T cells, CD4(+)Foxp3(+) T regulatory cells and CD19(+) B cells) were analyzed by flow cytometry. Specific anti-platelet GPIIb/IIIa and/or GPIb/IX autoantibodies were assayed by modified monoclonal antibody specific immobilization of platelet antigens (MAIPA). The mean fluorescence intensity of CD11a on CD3(+) T, CD3(+)CD4(-) T and CD19(+) B lymphocytes were increased in ITP patients compared to healthy controls. No significant difference of CD11a expression on CD3(+)CD4(+) T cells or CD4(+)Foxp3(+) T regulatory cells was found between ITP patients and controls. Our data indicates the possible role of CD11a in the pathogenesis of ITP. PMID:26884833

  20. Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient.

    PubMed

    Laupheimer, Michael; Skorska, Anna; Große, Jana; Tiedemann, Gudrun; Steinhoff, Gustav; David, Robert; Lux, Cornelia A

    2014-01-01

    Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes. PMID:25610653

  1. Functional characterization of human thymocyte subpopulations separated by density gradient centrifugation.

    PubMed Central

    Lederman, H M; Lee, J W; Cohen, A; Gelfand, E W

    1984-01-01

    A bovine serum albumin gradient was used to separate two populations of human thymocytes--a minority population (8%) of large thymocytes (LT) and a majority population (92%) of small thymocytes (ST). Fifty per cent of LT cells were in the S, G2 or M phases of the cell cycle compared to 5% of ST cells and 15% of unfractionated thymocytes. LT cells proliferated in response to T cell mitogens and included all of the T colony precursor cells (TCPC). In contrast, ST cells proliferated with mitogens only in the presence of added T cell growth factors and contained none of the thymocyte TCPC. ST cells neither helped nor suppressed the function of LT cells in any assay. This separation technique has provided a rapid method for isolating functionally distinct thymic lymphocyte subpopulations and permitted a further definition of the TCPC in the human thymus. Furthermore it should prove useful in studies of thymocytes at different stages of the cell cycle. PMID:6607793

  2. Hypotonic resistance of boar spermatozoa: sperm subpopulations and relationship with epididymal maturation and fertility.

    PubMed

    Druart, Xavier; Gatti, Jean-Luc; Huet, Sylvie; Dacheux, Jean-Louis; Humblot, Patrice

    2009-02-01

    Hypotonic resistance of boar spermatozoa was investigated by measuring the ratio of live/dead spermatozoa (SYBR-14/propidium iodide) by flow cytometry after hypotonic stress. The survival rate of ejaculated spermatozoa incubated in hypotonic solutions ranging from 3 to 330 mmol/kg followed a sigmoid curve that fitted a simple logistic model. The critical osmolality value (Osm(crit)) at which 50% of spermatozoa died was determined with this model. Hypotonic resistance of spermatozoa increased with temperature between 15 and 39 degrees C and decreased after hydrogen superoxide treatment, but was not modified during 8 days of preservation in Beltsville thawing solution. Hypotonic resistance markedly decreased during epididymal maturation and after ejaculation as Osm(crit) at 15 degrees C was 54.7+/-3.2, 68.5+/-10.6, 116.7+/-2.1 and 194.3+/-3.7 mmol/kg for the caput, corpus, cauda and ejaculated spermatozoa respectively. Hypo-osmotic stress of 100 mmol/kg revealed a sperm subpopulation exhibiting increased hypotonic resistance compared with the whole ejaculate (Osm(crit)=67.8+/-2.1 mmol/kg). Consistent differences were observed between lean and standard breeds (Pietrain versus Large White) and between boars within the same breed. According to data collected by artificial insemination centers during a large-scale field trial, hypotonic resistance of ejaculates was found to be positively correlated with in vivo fertility. PMID:18996973

  3. Dyslipidemia-associated alterations in B cell subpopulation frequency and phenotype during experimental atherosclerosis.

    PubMed

    Rincón-Arévalo, Héctor; Castaño, Diana; Villa-Pulgarín, Janny; Rojas, Mauricio; Vásquez, Gloria; Correa, Luis A; Ramírez-Pineda, José R; Yassin, Lina M

    2016-04-01

    Lymphocytes, the cellular effectors of adaptive immunity, are involved in the chronic inflammatory process known as atherosclerosis. Proatherogenic and atheroprotective properties have been ascribed to B cells. However, information regarding the role of B cells during atherosclerosis is scarce. Both the frequency and the phenotype of B cell subpopulations were studied by flow cytometry in wild type and apolipoprotein-E-deficient (apoE(-/-)) mice fed a high-fat (HFD) or control diet. Whereas the proportion of follicular cells was decreased, transitional 1-like cells were increased in mice with advanced atherosclerotic lesions (apoE(-/-) HFD). B cells in atherosclerotic mice were more activated, indicated by their higher surface expression of CD80, CD86, CD40 and CD95 and increased serum IgG1 levels. In the aorta, a decreased frequency of B cells was observed in mice with advanced atherosclerosis. Low expression of CD19 was observed on B cells from the spleen, aorta and lymph nodes of apoE(-/-) HFD mice. This alteration correlated with serum levels of IgG1 and cholesterol. A reduction in CD19 expression was induced in splenic cells from young apoE(-/-) mice cultured with lipemic serum. These results show that mice with advanced atherosclerosis display a variety of alterations in the frequency and phenotype of B lymphocytes, most of which are associated with dyslipidemia. PMID:26897258

  4. Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations.

    PubMed

    Monette, Jeffrey S; Gómez, Luis A; Moreau, Régis F; Bemer, Brett A; Taylor, Alan W; Hagen, Tory M

    2010-07-23

    Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (approximately 10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (approximately 70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other. PMID:20599536

  5. Characteristics of the Rat Cardiac Sphingolipid Pool in Two Mitochondrial Subpopulations

    PubMed Central

    Monette, Jeffrey S.; Gómez, Luis A.; Moreau, Régis F.; Bemer, Brett A.; Taylor, Alan W.; Hagen, Tory M.

    2010-01-01

    Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary (IFM) and subsarcolemmal (SSM) mitochondria were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (~10,000 pmols/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (~70 pmols/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16 to 24 carbon units in their acyl side-chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmols/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other. PMID:20599536

  6. Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient

    PubMed Central

    Laupheimer, Michael; Skorska, Anna; Große, Jana; Tiedemann, Gudrun; Steinhoff, Gustav; David, Robert; Lux, Cornelia A.

    2014-01-01

    Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes. PMID:25610653

  7. Natural killer cell function and lymphoid subpopulations in acute non-lymphoblastic leukaemia in complete remission.

    PubMed Central

    Pizzolo, G.; Trentin, L.; Vinante, F.; Agostini, C.; Zambello, R.; Masciarelli, M.; Feruglio, C.; Dazzi, F.; Todeschini, G.; Chilosi, M.

    1988-01-01

    A long term follow-up study has been undertaken in 33 patients with acute non-lymphoblastic leukaemia (ANLL) in order to establish whether a correlation exists between the clinical course and the immunologic pattern of lymphoid subpopulations. Peripheral blood lymphoid cells have been investigated longitudinally (each 1 to 4 months) during complete remission (CR), by morphologic, phenotypic and functional analyses. Particular attention has been paid to the evaluation of the natural killer (NK) cell compartment, by the detection of cells expressing an NK-related phenotype and by NK in vitro assay. Among the patients so far evaluable, 20 relapsed (R) and 10 are long survivors in CR 'off therapy' (LS). The most relevant finding was represented by statistically higher values of NK activity observed in LS vs. R patients (P less than 0.01). The removal of adherent cells before the NK assay, performed to investigate the possible inhibitory effect on NK function played by the macrophage component, abolished this difference, due to a selective increase of NK function in the R group. The longitudinal study revealed that NK activity tended to decrease in individual patients who subsequently relapsed. These data suggest a possible role of NK cells in the relapse control of ANLL, although it cannot be excluded that the low level of NK activity observed in the R group is the result of impending relapse rather than its cause. PMID:3179190

  8. Mesenchymal stem cells and their subpopulation, pluripotent muse cells, in basic research and regenerative medicine.

    PubMed

    Kuroda, Yasumasa; Dezawa, Mari

    2014-01-01

    Mesenchymal stem cells (MSCs) have gained a great deal of attention for regenerative medicine because they can be obtained from easy accessible mesenchymal tissues, such as bone marrow, adipose tissue, and the umbilical cord, and have trophic and immunosuppressive effects to protect tissues. The most outstanding property of MSCs is their potential for differentiation into cells of all three germ layers. MSCs belong to the mesodermal lineage, but they are known to cross boundaries from mesodermal to ectodermal and endodermal lineages, and differentiate into a variety of cell types both in vitro and in vivo. Such behavior is exceptional for tissue stem cells. As observed with hematopoietic and neural stem cells, tissue stem cells usually generate cells that belong to the tissue in which they reside, and do not show triploblastic differentiation. However, the scientific basis for the broad multipotent differentiation of MSCs still remains an enigma. This review summarizes the properties of MSCs from representative mesenchymal tissues, including bone marrow, adipose tissue, and the umbilical cord, to demonstrate their similarities and differences. Finally, we introduce a novel type of pluripotent stem cell, multilineage-differentiating stress-enduring (Muse) cells, a small subpopulation of MSCs, which can explain the broad spectrum of differentiation ability in MSCs. PMID:24293378

  9. Genetic diversity and genetic differentiation in Daphnia metapopulations with subpopulations of known age.

    PubMed

    Haag, Christoph R; Riek, Myriam; Hottinger, Jürgen W; Pajunen, V Ilmari; Ebert, Dieter

    2005-08-01

    If colonization of empty habitat patches causes genetic bottlenecks, freshly founded, young populations should be genetically less diverse than older ones that may have experienced successive rounds of immigration. This can be studied in metapopulations with subpopulations of known age. We studied allozyme variation in metapopulations of two species of water fleas (Daphnia) in the skerry archipelago of southern Finland. These populations have been monitored since 1982. Screening 49 populations of D. longispina and 77 populations of D. magna, separated by distances of 1.5-2180 m, we found that local genetic diversity increased with population age whereas pairwise differentiation among pools decreased with population age. These patterns persisted even after controlling for several potentially confounding ecological variables, indicating that extinction and recolonization dynamics decrease local genetic diversity and increase genetic differentiation in these metapopulations by causing genetic bottlenecks during colonization. We suggest that the effect of these bottlenecks may be twofold, namely decreasing genetic diversity by random sampling and leading to population-wide inbreeding. Subsequent immigration then may not only introduce new genetic material, but also lead to the production of noninbred hybrids, selection for which may cause immigrant alleles to increase in frequency, thus leading to increased genetic diversity in older populations. PMID:15937138

  10. Increased radiosensitivity of a subpopulation of T-lymphocyte progenitors from patients with Fanconi's anemia

    SciTech Connect

    Knox, S.J.; Wilson, F.D.; Greenberg, B.R.; Shifrine, M.; Rosenblatt, L.S.; Reeves, J.D.; Misra, H.

    1981-06-01

    In vitro radiation survival of peripheral blood T lymphocytes was studied in 15 clinically normal adults and 4 patients with Fanconi's anemia. Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and a newly developed whole blood T-lymphocyte colony assay were used to measure lymphocyte blastogenesis and colony formation in response to phytohemagglutinin (PHA) or concanavalin-A (Con-A) stimulation. Lymphocyte colony formation was found to be consistently more sensitive than the LST for detection of low-level radiation effects using both normal cells and lymphocytes from Fanconi's anemia patients. Lymphocytes from patients with Fanconi's anemia were significantly more sensitive to in vitro x irradiation than lymphocytes from clinically normal individuals as measured by their ability to divide when stimulated by PHA in the LST and colony formation assay. No significant difference in the radiosensitivity of the Con-A response was observed between the two groups. The PHA-responsive T-lymphocyte subpopulation in Fanconi's anemia patients appears to be intrinsically defective. The nature of this defect, significance in the disease process, and relevancy of these findings to the establishment of radiation protection standards are discussed.

  11. Increased radiosensitivity of a subpopulation ot T-lymphocyte progenitors from patients with Fanconi's anemia

    SciTech Connect

    Knox, S.J.; Wilson, F.D.; Greenberg, B.R.; Shifrine, M.; Rosenblatt, L.S.; Reeves, J.D.; Misra, H.

    1981-06-01

    In vitro radiation survival of peripheral blood T lymphocytes was studied in 15 clinically normal adults and 4 patients with Fanconi's anemia. Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and a newly developed whole blood T-lymphocyte colony assay were used to measure lymphocyte blastogenesis and colony formation in response to phytohemagglutinin (PHA) or concanavalin-A (Con-A) stimulation. Lymphocyte colony formation was found to be consistently more sensitive than the LST for detection of low-level radiation effects using both normal cells and lymphocytes from Fanconi's anemia patients. Lymphocytes from patients with Fanconi's anemia were significantly more sensitive to in vitro x-irradiation than lymphocytes from clinically normal individuals as measured by their ability to divide when stimulated by PHA in the LST (patients, D37 . 198 R; normals, D37 . 309 R, p . 0.057) and colony formation assay (patients, D37 . 53 R; normals, D37 . 109 R, p . 0.016). No significant difference in the radiosensitivity of the Con-A response was observed between the two groups. The PHA-responsive T-lymphocyte subpopulation in Fanconi's anemia patients appears to be intrinsically defective. The nature of this defect, significance in the disease process, and relevancy of these findings to the establishment of radiation protection standards are discussed.

  12. Distinct subpopulations of FOXD1 stroma-derived cells regulate renal erythropoietin.

    PubMed

    Kobayashi, Hanako; Liu, Qingdu; Binns, Thomas C; Urrutia, Andres A; Davidoff, Olena; Kapitsinou, Pinelopi P; Pfaff, Andrew S; Olauson, Hannes; Wernerson, Annika; Fogo, Agnes B; Fong, Guo-Hua; Gross, Kenneth W; Haase, Volker H

    2016-05-01

    Renal peritubular interstitial fibroblast-like cells are critical for adult erythropoiesis, as they are the main source of erythropoietin (EPO). Hypoxia-inducible factor 2 (HIF-2) controls EPO synthesis in the kidney and liver and is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3, which function as cellular oxygen sensors. Renal interstitial cells with EPO-producing capacity are poorly characterized, and the role of the PHD/HIF-2 axis in renal EPO-producing cell (REPC) plasticity is unclear. Here we targeted the PHD/HIF-2/EPO axis in FOXD1 stroma-derived renal interstitial cells and examined the role of individual PHDs in REPC pool size regulation and renal EPO output. Renal interstitial cells with EPO-producing capacity were entirely derived from FOXD1-expressing stroma, and Phd2 inactivation alone induced renal Epo in a limited number of renal interstitial cells. EPO induction was submaximal, as hypoxia or pharmacologic PHD inhibition further increased the REPC fraction among Phd2-/- renal interstitial cells. Moreover, Phd1 and Phd3 were differentially expressed in renal interstitium, and heterozygous deficiency for Phd1 and Phd3 increased REPC numbers in Phd2-/- mice. We propose that FOXD1 lineage renal interstitial cells consist of distinct subpopulations that differ in their responsiveness to Phd2 inactivation and thus regulation of HIF-2 activity and EPO production under hypoxia or conditions of pharmacologic or genetic PHD inactivation. PMID:27088801

  13. Sub-population structure evident in forensic Y-STR profiles from Armenian geographical groups.

    PubMed

    Lowery, Robert K; Herrera, Kristian; Uribe, Gabriel; Reguiero, Maria; Herrera, Rene J

    2013-03-01

    Over the course of its long history, Armenia has acted as both a source of numerous indigenous cultures and as a recipient of foreign invasions. As a result of this complex history among populations, the gene pool of the Armenian population may contain traces of historically well-documented ancient migrations. Furthermore, the regions within the historical boundaries of Armenia possess unique demographic histories, having hosted both autochthonous and specific exogenous genetic influences. In the present study, we analyze the Armenian population sub-struc